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although chemical substances are a very important factor in the realization of current industrialization, because they were used indiscriminately without a full understanding of the damage they could cause, issues have arisen on a continual basis, such as global environment changes like ozone depletion and global warming, and chemical damage cases like minamata disease and itai - itai disease, so that the importance of safe chemical use has emerged. consequently, the major developed countries and international society agreed in 1972, at the united nations (un) conference on the human environment held for safe use and control of chemicals, to the need for a specialized agency affiliated to the un to deal with global environmental problems ; the the un environment programme was thus established through the 27th un general assembly. further efforts to ensure the safer use of commercial chemicals and for reduction in the use of hazardous chemicals have included the stockholm convention on persistent organic pollutants for restriction of volatile organic compounds (vocs) and the rotterdam convention to regulate the international trade in hazardous substances. p. t. anastas of the us environmental protection agency (epa) proposed green chemistry theory in 1998, arguing for a systemic reduction in the use of hazardous chemicals and the safer use and control of chemicals. the theory is composed of 12 principles to reduce or remove the harmfulness of chemicals throughout their entire life - cycle, from raw material step to the waste disposal step, via manufacturing and production to safer use of hazardous chemicals. risk assessment studies of chemicals and research into the development of alternative chemicals have been actively carried out, in recognition of their value as a practical alternative for the establishment of an infrastructure for green chemistry ; such investigations have been led by the major developed countries. in addition, national support for the establishment of an infrastructure for green chemistry has been provided, along with award programs, to encourage the active participation of industry. the green chemistry market of south korea (hereafter korea) is about us$ 96.4 billion, placing it at 13th in the world, after major developed countries such as the us, china, and japan, and accounting for about 1.8% of the entire green chemistry market, alongside canada. the global green chemistry market is expected to grow continuously to about us$3000 billion by 2020, and efforts to monopolize the green chemistry market have swiftly centered on the major developed countries. since it is necessary for korean industries, which occupy only 1.8% of the global green chemistry market, to enter into overseas markets, various environmental changes, along with growth of the green market, may provide a chance but also a crisis for korean industries, so that a response from the korean government is very crucial (table 1). t. anastas, the epa has maintained various policies to spread green chemistry, including environmental assessments of hazardous chemicals by the federal government ; relevant research and education ; support for environment - related industries, such as prevention programs and energy saving ; the presidential green chemistry challenge awards ; and support for the reduction in use and generation of hazardous substances. the epa also developed and distributed a tool to provide information about the results of the policies related to green chemistry, including green chemistry expert system (gces) and green chemistry assistant (gca). gces is composed of five modules that enable industries to identify information for evaluation and prediction, leading to the selection of safer green chemistry alternative chemicals. first, the synthetic methodology assessment for reduction techniques module receives inputs such as information about chemicals, number of reactions, and yield of reactions ; identifies properties of the chemical reaction process ; and estimates and assesses the amounts of products and wastes. second, the green synthetic reaction module makes it possible to identify reaction information to synthesize chemicals in a safer and less hazardous way, with the input of basic information about chemicals. third, the designing safer chemicals module proposes compound designs and analyzes toxic mechanisms, which supports the prediction of structures that can reduce toxicity. fourth, the green solvent / reaction condition module makes it possible to identify information on the physical - chemical properties of about 600 different solvents, as well as the reaction conditions for some alternative chemicals that have been confirmed. fifth, the green chemistry reference module has been designed to search and identify reports and data related to studies on green chemistry. a web - based program developed collaboratively by the epa and st. olaf college, gca has functions to calculate the theoretical yields of reactions, using chemical reaction equations, and the appropriate amounts of chemicals that are required for reactions ; this avoids the need to use chemicals during design. in addition, it provides information on about 60000 chemicals and about 600 solvents through a search function linked with gces database. state governments and a number of colleges in the us have made various attempts to establish an infrastructure for green chemistry, alongside the us federal government. massachusetts state has established and operates the toxics use reduction institute, in collaboration with industries and government agencies, in line with the toxics use reduction act for research and public relations on the reduction in the use of hazardous chemicals. to reduce hazardous chemicals, the institute has been running various environmental protection programs, including continuous education for industry and the general public, reduction technology, funding, a chemical fact sheet provided by the toxic use reduction science advisory board, and evaluation of research on alternatives to hazardous chemicals like hexavalent chrome and diethylhexyl phthalate. the institute has also developed pollution prevention options assessment system, an excel - based program that has the function of calculating the harmfulness scores of chemicals, based on the collected toxicity information, in order to help users to understand objectively and easily and to allow them to evaluate comparatively. massachusetts institute of technology of the us has formed a network of research organizations, academics, and research institutes related to green chemistry. this network has constructed a database focusing on data from major journals and has been running the green alternatives purchasing wizard, which provides information about hazardous chemicals. this is part of an eco - friendly purchasing policy to minimize waste and prevent pollution, in line with the resource conservation and recovery act. the wizard is a web - based program that was developed for cost reduction purposes achieved through reduction in hazardous wastes and environmental loads from research institutes. it also provides information about alternative chemicals, their advantages and disadvantages, and sources ; it is linked to a purchasing system called systems applications and products to facilitate purchases (figure 1). clean production action (cpa) is a project run by the tides center, a non - profit profit organization that financially supports innovative and creative groups in the us. in order to provide green chemistry solutions to the design and production of sustainable eco - friendly products, for the safety of products and materials, and for rapid and effective methods of toxicity evaluation, cpa has produced green screen, composed of four categories environmental fate, eco - toxicity, human health, and physical- chemical properties and 17 detailed evaluation items, presented in four types, as benchmark 1 to 4, based on the evaluation results. the epa of denmark developed a web - based program in 2003 to search for hazardous chemicals and alternatives, based on a case report called a catalogue of example of substitution of hazardous chemicals, which in 1989 provided information on alternatives to 162 hazardous chemicals and actual cases that utilized alternatives. the program provides information, collected from danish businesses, about alternative chemicals and technologies, the occupational health service, and the danish working environment authority. the program provides information on 311 actual cases in 83 companies where alternative chemicals were used and encourage more active use of alternative chemicals in industry. in 2009, the ministry of environment of korea formed a forum composed of representatives from industry, academia, and research institutes in recognition of the strengthening international regulation of hazard and risk controls of chemicals ; the ministry proposed a measure for the advance of chemical management to move toward a green chemistry system in order to improve the chemical management system in korea and to support the chemical industry. accordingly, the green shift to move toward green chemistry has ultimately been pursued in the direction of advancing management of the risks of hazardous chemicals ; of safety management, people s health, and environmental protection from chemicals and chemical products ; of advances in the management of chemical information ; of the construction of mutual communication and cooperation systems between interested parties;and of the attainment of two goals : health and environmental protection and improvement of the competitiveness of the chemical industry. as a part of this project, the ministry of environment of korea has run the next - generation eco - innovation (ei) project since 2011, with the goals of constructing the infrastructure for green chemistry in industrial facilities in korea and of improving foreign trade competitiveness. the project also had the research group on green chemistry management techniques along with four sub - organizations, led by the korea environment corporation, in order to construct the green chemistry policy / application support system. in particular, the project has been developing a search program for related alternative chemicals for small and medium - sized businesses that have difficulty being competitive because of difficulties in accessing information about the regulation of chemicals and the regulation of alternative chemicals in korea and other countries. the program has enabled them to identify and search for information about reliable alternative chemicals and rapidly changing international chemical regulations. the project has collected information about alternative chemicals based on domestic and international journals, research data, and particularly patent data. it has analyzed this information, verified it with the help of experts in the field to secure the reliability of data, and then presented it. in addition, successful cases of the application of alternative chemicals to actual processes were focused on in the process of collection and presented in order to increase the applicability of alternative chemicals. at present, information on a total of 341 alternative chemicals by use and 75 application cases have been collected and provided. the program can be downloaded from the next - generation ei research group homepage and the homepage of to21 co., ltd. ; it is distributed to companies free of charge (figure 2). t. anastas, the epa has maintained various policies to spread green chemistry, including environmental assessments of hazardous chemicals by the federal government ; relevant research and education ; support for environment - related industries, such as prevention programs and energy saving ; the presidential green chemistry challenge awards ; and support for the reduction in use and generation of hazardous substances. the epa also developed and distributed a tool to provide information about the results of the policies related to green chemistry, including green chemistry expert system (gces) and green chemistry assistant (gca). gces is composed of five modules that enable industries to identify information for evaluation and prediction, leading to the selection of safer green chemistry alternative chemicals. first, the synthetic methodology assessment for reduction techniques module receives inputs such as information about chemicals, number of reactions, and yield of reactions ; identifies properties of the chemical reaction process ; and estimates and assesses the amounts of products and wastes. second, the green synthetic reaction module makes it possible to identify reaction information to synthesize chemicals in a safer and less hazardous way, with the input of basic information about chemicals. third, the designing safer chemicals module proposes compound designs and analyzes toxic mechanisms, which supports the prediction of structures that can reduce toxicity. fourth, the green solvent / reaction condition module makes it possible to identify information on the physical - chemical properties of about 600 different solvents, as well as the reaction conditions for some alternative chemicals that have been confirmed. fifth, the green chemistry reference module has been designed to search and identify reports and data related to studies on green chemistry. a web - based program developed collaboratively by the epa and st. olaf college, gca has functions to calculate the theoretical yields of reactions, using chemical reaction equations, and the appropriate amounts of chemicals that are required for reactions ; this avoids the need to use chemicals during design. in addition, it provides information on about 60000 chemicals and about 600 solvents through a search function linked with gces database. state governments and a number of colleges in the us have made various attempts to establish an infrastructure for green chemistry, alongside the us federal government. massachusetts state has established and operates the toxics use reduction institute, in collaboration with industries and government agencies, in line with the toxics use reduction act for research and public relations on the reduction in the use of hazardous chemicals. to reduce hazardous chemicals, the institute has been running various environmental protection programs, including continuous education for industry and the general public, reduction technology, funding, a chemical fact sheet provided by the toxic use reduction science advisory board, and evaluation of research on alternatives to hazardous chemicals like hexavalent chrome and diethylhexyl phthalate. the institute has also developed pollution prevention options assessment system, an excel - based program that has the function of calculating the harmfulness scores of chemicals, based on the collected toxicity information, in order to help users to understand objectively and easily and to allow them to evaluate comparatively. massachusetts institute of technology of the us has formed a network of research organizations, academics, and research institutes related to green chemistry. this network has constructed a database focusing on data from major journals and has been running the green alternatives purchasing wizard, which provides information about hazardous chemicals. this is part of an eco - friendly purchasing policy to minimize waste and prevent pollution, in line with the resource conservation and recovery act. the wizard is a web - based program that was developed for cost reduction purposes achieved through reduction in hazardous wastes and environmental loads from research institutes. it also provides information about alternative chemicals, their advantages and disadvantages, and sources ; it is linked to a purchasing system called systems applications and products to facilitate purchases (figure 1). clean production action (cpa) is a project run by the tides center, a non - profit profit organization that financially supports innovative and creative groups in the us. in order to provide green chemistry solutions to the design and production of sustainable eco - friendly products, for the safety of products and materials, and for rapid and effective methods of toxicity evaluation, cpa has produced green screen, composed of four categories environmental fate, eco - toxicity, human health, and physical- chemical properties and 17 detailed evaluation items, presented in four types, as benchmark 1 to 4, based on the evaluation results. the epa of denmark developed a web - based program in 2003 to search for hazardous chemicals and alternatives, based on a case report called a catalogue of example of substitution of hazardous chemicals, which in 1989 provided information on alternatives to 162 hazardous chemicals and actual cases that utilized alternatives. the program provides information, collected from danish businesses, about alternative chemicals and technologies, the occupational health service, and the danish working environment authority. the program provides information on 311 actual cases in 83 companies where alternative chemicals were used and encourage more active use of alternative chemicals in industry. in 2009, the ministry of environment of korea formed a forum composed of representatives from industry, academia, and research institutes in recognition of the strengthening international regulation of hazard and risk controls of chemicals ; the ministry proposed a measure for the advance of chemical management to move toward a green chemistry system in order to improve the chemical management system in korea and to support the chemical industry. accordingly, the green shift to move toward green chemistry has ultimately been pursued in the direction of advancing management of the risks of hazardous chemicals ; of safety management, people s health, and environmental protection from chemicals and chemical products ; of advances in the management of chemical information ; of the construction of mutual communication and cooperation systems between interested parties;and of the attainment of two goals : health and environmental protection and improvement of the competitiveness of the chemical industry. as a part of this project, the ministry of environment of korea has run the next - generation eco - innovation (ei) project since 2011, with the goals of constructing the infrastructure for green chemistry in industrial facilities in korea and of improving foreign trade competitiveness. the project also had the research group on green chemistry management techniques along with four sub - organizations, led by the korea environment corporation, in order to construct the green chemistry policy / application support system. in particular, the project has been developing a search program for related alternative chemicals for small and medium - sized businesses that have difficulty being competitive because of difficulties in accessing information about the regulation of chemicals and the regulation of alternative chemicals in korea and other countries. the program has enabled them to identify and search for information about reliable alternative chemicals and rapidly changing international chemical regulations. the project has collected information about alternative chemicals based on domestic and international journals, research data, and particularly patent data. it has analyzed this information, verified it with the help of experts in the field to secure the reliability of data, and then presented it. in addition, successful cases of the application of alternative chemicals to actual processes were focused on in the process of collection and presented in order to increase the applicability of alternative chemicals. at present, information on a total of 341 alternative chemicals by use and 75 application cases have been collected and provided. the program can be downloaded from the next - generation ei research group homepage and the homepage of to21 co., ltd. ; it is distributed to companies free of charge (figure 2). in recent international trade, the free trade agreement has promoted an increase in trade between countries by removing or reducing the existing trade barriers, such as customs or import restrictions. as such, major developed countries have utilized technology regulations called technical barriers to trade (tbt) as new non - tariff barriers, including energy saving, a reduction of carbon emissions, and environment - related standards, in order to protect the environment but also the trade of their own countries. according to a report from the world trade organization, the number of tbt notice letters increased from 638 cases in 2004 to 1491 in 2013. in addition, there have been various efforts to develop alternative chemicals and alternative process technologies to meet stronger technology regulations. accordingly, various environment - related projects, including the next - generation ei project, have been implemented in collaboration with industry, academia, and research institutes in korea since 2011, in order to improve the international competitiveness of korean businesses ; through the project, various forms of content were developed and provided to companies. in addition, the existing hazardous chemical control act has been completely changed, becoming the act on the registration and evaluation, etc. of chemical substances and the chemical control act, based on the obtained data, to protect the korean environment and businesses, and through which korea has endeavored more complete chemical control. previously, the chemical industry was evaluated by its ability to synthesize a higher amount of chemicals and to develop new chemicals. however, conversion to green chemistry, which uses chemicals that are safer and less harmful, will be an important force for national growth in the future, and it may provide a good opportunity to secure the competitiveness of korean industrial facilities in the international markets. | objectivesdespite the great contribution made by chemical substances to the development of modern civilization, their indiscriminate use has caused various kinds of damage to the global environment and human beings. accordingly, the major developed countries and international society have tried to ensure the safe use of chemicals and a reduction in the use of hazardous chemicals through the establishment of the united nations environment programme and various international agreements. in this reason, we tried to introduce about green chemistry progress at the present in worldwide and korea.methodswe checked and analyzed relative journals, reports using keyword as like green chemistry, alternative chemicals, eco - friendly etc. and major country s government homepage search.resultsgreen chemistry theory, which argues for the reduction or removal of harmfulness in chemicals throughout their entire life - cycle, has been spreading, and major developed countries, such as the us and denmark, have developed and operate programs to provide reliable chemical information to help replace hazardous chemicals. korea has also been conducting studies as like eco - innovation project. through this project the alternative chemical search program, has been developed, distributed, and operated since 2011 to provide reliable information to small and medium - sized businesses that have difficulties collecting information to ensure conformity to international regulations. the program provides information that includes the regulations of major countries and korea, information on 340 alternative chemicals, 70 application cases, and 1:1 consulting.conclusionsthe alternative chemical search program is expected to contribute to the establishment of response systems for regulation of korean small and medium - sized businesses, and it also will be used to provide basic data for korean hazardous chemical regulation, together with the act on the registration and evaluation, etc. of chemical substances and the chemical control act, making it possible to establish an infrastructure for green chemistry in korea and to increase national competitiveness. |
this randomized controlled study was conducted in a tertiary care teaching institute of rajasthan after taking approval from the institutional ethics committee. data were collected in the approved pro forma after taking written consent of the patient. all the sputum - positive tubercular patients aged between 18 and 65 years of either sex without history of any osteoarthritic condition and intake of any other hyperuricemic drugs were included in the study in whom standard four - drug att was given. patients having hepatic dysfunction, history of renal calculi, pregnant, and lactating females were excluded from this study. serum uric acid level was monitored at 0, 2, 4, 6, and 8 week of att. patients whose uric acid level was found to be increased at 2 week were finally recruited in the study. ninety patients who developed hyperuricemia due to att were divided randomly into three groups (group a, group b, and group c) of thirty patients each. group a was treated with febuxostat and group b with allopurinol at a daily dose of 40 and 300 mg, respectively. mean serum uric acid levels were calculated at all the weeks in all the groups, and serum uric acid levels were compared by applying student 's t - test and anova. a total of ninety patients were recruited in this study who developed hyperuricemia after 2 weeks of starting att, of which sixty patients were male and thirty patients were female. the mean age of the patients was 41.5 years (ranging from 18 to 65 years). mean serum uric acid levels were calculated, and it was found that serum uric acid levels increased sharply at 2 week in all the groups a, b, and c as shown in table 1. mean uric acid levels at various weeks in all the groups mean serum uric acid level decreased from 10.698 mg / dl (at 2 week) to 7.846 mg / dl (at 8 week) in group a and from 11.34 mg / dl (at 2 week) to 7.280 mg / dl (at 8 week) in group b. the mean levels decreased significantly at 4, 6, and 8 weeks when compared with mean values at 2 week. however, when values at 8 week were compared with baseline (at 0 week) values, the difference was still significant which suggests that drugs were able to decrease serum uric acid level but could not attain the baseline level. it was observed that at 8 week, 63.33% of patients were having serum uric acid level > 6 mg / dl and 53.33% of patients were having > 6.76 mg / dl in group a, and in group b, 70% of patients were having serum uric acid level > 6.0 mg / dl and 53.33% of patients were having > 6.8 mg / dl at 8 week. the mean serum uric acid levels at all the weeks, i.e. 0, 2, 4, 6, and 8 week were compared by applying student 's t - test between group a and b, group a and c, and group b and c and were found to be nonsignificant at all the weeks. when the anova was performed for all the weeks within the three groups, comparison of serum uric acid level during weeks in various groups one patient responded well to febuxostat but developed hypersensitivity (developed itching after starting the drug which subsided after stopping it) to it and was switched to allopurinol but did not respond to allopurinol. one patient who was treated with febuxostat, uric acid lowered only slightly but responded well to allopurinol. serum uric acid level increased in spite of taking febuxostat in one patient but responded to allopurinol. one patient developed hypersensitivity to allopurinol (developed itching and rashes after taking the drug which subsided after stopping it). in one patient, allopurinol was not able to reduce serum uric acid level to a great extent and hence switched over to febuxostat and responded well. one patient responded well to febuxostat whose serum uric acid level increased in spite of taking allopurinol. expenditure of the patient on including either of the two drugs was compared, and the difference was calculated. 2.33/tablet, and it has to be taken thrice daily, i.e. cost / day was found to be rs. 7.70/tablet, and it has to be taken once daily, i.e. the cost / day was found to be rs. difference between the two drugs was found to be 71 paise / day or rs. tb was virtually wiped out with the help of antibiotics which were developed in 1950s, but the disease resurfaced in potent new and dangerous forms such as multidrug - resistant tb and extensive drug - resistant tb. pza is an important constituent of att but at times forcing its withdrawal due to hyperuricemia. in this study, an attempt was made to continue att without withdrawing pza with the help of adding drugs which decrease synthesis of uric acid by inhibiting xanthine oxidase enzyme. a number of previous studies including three randomized controlled trials have been conducted to compare the two drugs in patients of gout and hyperuricemia, and results were in favor of febuxostat. results of our study suggested that it was possible to continue pza in the patients with the help of these drugs. both drugs were found to be equally efficacious in lowering the serum uric acid levels possibly the reason for same efficacy might be the same mechanism of action of inhibiting the synthesis of uric acid by inhibiting xanthine oxidase. according to singal. study, febuxostat is a great addition to the armamentarium for gout management particularly in patients in whom allopurinol has failed because of either lack of efficacy or due to adverse events because of intolerance to allopurinol. moreover, a committee of the british national institute for health and clinical excellence concluded that although febuxostat is found to be more effective than fixed - dose (300 mg) allopurinol, it had not been found to be clinically more efficacious or cost - effective than allopurinol. numbers of adverse events encountered across both the treatment groups were same with both the drugs. allopurinol is known to cause severe hypersensitivity reactions, but after reviewing literatures, it was discovered that such reactions are usually seen after long - term administration of the drug. the phase iii trials and long - term follow - up also showed the incidence of adverse events with febuxostat to be similar with that of allopurinol. the reasons for no or little response with febuxostat could be explained on the basis of that the dose was not increased further. no response with either of the two drugs can also be explained because of variation in response due to individual variation in metabolizing the drugs. favorable point for using febuxostat could be that it requires only once - daily administration resulting in better patient compliance. moreover, overall incidence of adverse effects is also few with febuxostat according to previous studies as compare to allopurinol. as stated earlier that a committee of the british national institute for health and clinical excellence also concluded that it had not been shown to be clinically more efficacious or cost effective than allopurinol and recommended febuxostat for people who are intolerant of allopurinol. this study concludes that both allopurinol and febuxostat were equally efficacious in lowering pza induced raised serum uric acid level in tubercular patients, and it was possible to continue att without withdrawing pza in patients who developed hyperuricemia due to it. similar studies are required in future because ethambutol, constituent of att, also known to increase serum uric acid levels in about 50% of patients was not considered in the present study. other factors such as genetic predisposition and genetic polymorphism could also affect, and alter serum uric acid levels have not been taken into consideration in our study. | objectives : to compare the efficacy and safety of febuxostat and allopurinol in pyrazinamide (pza)-induced hyperuricemia in patients taking antitubercular therapy (att).methods : this randomized controlled study was conducted at a tertiary care teaching institute of rajasthan in all the sputum - positive tubercular patients aged between 18 and 65 years of either sex. serum uric acid level was monitored at 0th, 2nd, 4th, 6th, and 8th week of att. patients whose uric acid level was found to be increased at 2nd week were finally recruited in the study. ninety patients who developed hyperuricemia due to att were divided randomly into three groups (group a - febuxostat, group b - allopurinol, and group c - control) of thirty patients each. mean serum uric acid levels were calculated at all the weeks in all the groups, and serum uric acid levels were compared by applying student 's t - test and anova.results:mean serum uric acid level decreased from 10.698 mg / dl (at 2nd week) to 7.846 mg / dl (at 8th week) in group a and from 11.34 mg / dl (at 2nd week) to 7.280 mg / dl (at 8th week) in group b. numbers of adverse events encountered across both the treatment groups were same with both the drugs.conclusion:allopurinol and febuxostat were equally efficacious in lowering pza induced raised serum uric acid level in tubercular patients, and it was possible to continue att without withdrawing pza. |
the adverse effects of asthma in pregnancy are related to asthma severity and the intensity of treatment [13 ]. acute asthma exacerbations have been identified as the most significant event to affect fetal morbidity and mortality in pregnancies complicated by asthma [4, 5 ]. a number of studies have examined the effects of maternal asthma during pregnancy, in the presence and absence of inhaled corticosteroid (ics) treatment, on placental function and fetal development. these studies have demonstrated that use of ics for the treatment of asthma did not affect fetal growth, and that maternal asthma without treatment had a greater impact on the fetus and placenta. overall, the evidence clearly demonstrates no increased risk of adverse maternal or fetal complications in situations where asthma is well controlled throughout pregnancy [8, 9 ]. in contrast, poor asthma control, as indicated by the need for more intensive treatment, is a greater risk factor for an adverse outcome during pregnancy than ics use. further, regular use of oral corticosteroids (ocs), used in the management of acute asthma exacerbations and poorly controlled asthma during pregnancy, has been associated with an increased incidence of low birth weight babies and preterm birth. previous studies have defined asthma exacerbations as any asthma - related event that involved one or more of the following : a hospital admission, an unscheduled visit to a doctor, a course of oral steroids, an increase in medication use or decreased peak expiratory flow rate. among those with mild, moderate and severe asthma the exacerbation rates have been reported as 13%, 26% and 52%, respectively. relying solely on asthma severity, however, can be misleading as asthma can worsen during pregnancy regardless of maternal pre - pregnancy severity [4, 5 ]. exacerbations can occur at any time during gestation, but are most likely in the second and third trimesters between weeks 17 and 34, with peak incidence at around 25 weeks gestation. many interventions for pregnant asthmatic women are focused on reducing asthma exacerbations as it is likely to have the greatest impact on improving perinatal outcomes. a major comorbidity of pregnant women with asthma is cigarette use [13, 14 ]. we previously found that 25% of asthmatic women smoked during pregnancy relative to 17% of non - asthmatic women. asthmatic women who were current smokers during pregnancy had more severe asthma exacerbations during pregnancy relative to women who were nonsmokers. the combination of asthma and cigarette use also significantly increased the incidence of low birth weight neonates and preterm deliveries. it is recognised that social disadvantage encompasses a range of contributing factors including health literacy, income, occupation, housing, health behaviours (i.e. smoking and poor diet) and comorbidities (i.e. mental health) as well as race and/or ethnicity. the prevalence of asthma is greatest among those living in lower socioeconomic status areas, with low socioeconomic status also associated with almost double the rate of smoking, increased rates of hospitalisations for asthma and an increased risk of asthma - related mortality. children from low socioeconomic status communities are more likely to have poorly controlled asthma relative to children of high socioeconomic status and it has been proposed that socioeconomic status index is a strong predictor of poor asthma outcomes. the bioair study demonstrated that low socioeconomic status is a risk factor for exacerbations. however, poor asthma control and exacerbations in low socioeconomic status populations may be due to inadequacies in health literacy. apter. reported that reduced literacy and numeracy skills in association with ethnicity, age, income and educational attainment were all associated with level of asthma control, with those individuals with reduced literacy being more likely to have uncontrolled asthma. in the current study, we have examined asthma control and exacerbations in a group of pregnant women who live in a socially disadvantaged community in south australia. for example, 36% of pregnant women from this population have reported they were abused as children, 35% have suffered major life stressors and 30% of these pregnant women were diagnosed with depression during antenatal care. population - based data report that 40% of the inhabitants in this community do not finish year 10 of high school, > 22% of the population are unemployed, 27% are housed by the government and 22% of families have a sole female parent. our objective was to examine patterns and predictors of asthma control and exacerbations during pregnancy in a population of women from a socially disadvantaged community, and to determine the association of this data with perinatal outcomes. approval for the study was provided by the queen elizabeth hospital, lyell mcewin hospital and modbury hospital human research ethics committee and the university of adelaide human research ethics committee. pregnant women with a doctor diagnosis of asthma (n=189) were recruited through the lyell mcewin hospital antenatal clinics (south australia, australia) between may 2009 and may 2012 to take part in a non - interventional prospective cohort study evaluating the impact of maternal asthma on perinatal outcomes. women were assessed by a midwife, with additional respiratory training, at 12, 20, 28, and 36 weeks gestation. the midwife utilised a standardised data collection tool to collect demographic data including maternal weight, height, previous obstetric history and medical history. maternal smoking was assessed by maternal self - report, with women classified according to smoking status during the first antenatal visit. socioeconomic status for each woman was determined using her residential postcode at the time of delivery. women were then ranked according to their level of advantage, or relative disadvantage, based on data from the socio - economic indexes for areas, calculated from the australian bureau of statistics ' 5-yearly census of population and housing. in addition to demographic variables, data were collected to determine current asthma therapy and control, current asthma triggers, comorbidities and past history including frequency of ocs use and hospital admissions for asthma. the acq is a seven - item validated questionnaire which covers asthma symptoms and lung function, with scores > 1.5 indicative of uncontrolled asthma. forced expiratory volume in 1 s (fev1) and forced vital capacity (fvc) were measured using an easy one spirometer (ndd medical technologies, inc., fev1 % predicted was calculated based on the patient 's age, ethnicity and height using the equations of gore.. data were prospectively collected on asthma medications (dose, frequency and duration across trimesters). patients assessed as unstable and requiring medical review were referred to their primary care physician or to a respiratory physician. details of the exacerbations were obtained from the study visit notes and confirmed by consulting the medical record if necessary. an exacerbation event was defined as moderate / severe if it required a hospital admission, emergency department presentation, an unscheduled visit to a doctor or a course of oral steroids due to asthma. a loss of control was defined as an increase in asthma symptoms or an increase in medication use, which was managed by the subject and did not require medical intervention, or an acq score of > 1.5 during an asthma study visit. most women were identified as having a loss of control using the acq score at a study visit. an additional asthma outcome included recurrent uncontrolled asthma, defined as an acq score of > 1.5 during two or more study visits. data on perinatal outcomes were collected at delivery from the medical records, including infant sex, gestational age, birthweight, birth length, head circumference, method of delivery and apgar scores. gestational age was determined by the date of the last menstrual period and confirmed by ultrasound at 18 weeks. preterm birth was defined as 1.5 indicative of uncontrolled asthma. forced expiratory volume in 1 s (fev1) and forced vital capacity (fvc) were measured using an easy one spirometer (ndd medical technologies, inc., fev1 % predicted was calculated based on the patient 's age, ethnicity and height using the equations of gore.. data were prospectively collected on asthma medications (dose, frequency and duration across trimesters). patients assessed as unstable and requiring medical review were referred to their primary care physician or to a respiratory physician. details of the exacerbations were obtained from the study visit notes and confirmed by consulting the medical record if necessary. an exacerbation event was defined as moderate / severe if it required a hospital admission, emergency department presentation, an unscheduled visit to a doctor or a course of oral steroids due to asthma. a loss of control was defined as an increase in asthma symptoms or an increase in medication use, which was managed by the subject and did not require medical intervention, or an acq score of > 1.5 during an asthma study visit. most women were identified as having a loss of control using the acq score at a study visit. an additional asthma outcome included recurrent uncontrolled asthma, defined as an acq score of > 1.5 during two or more study visits. data were prospectively collected at each study visit. data on perinatal outcomes were collected at delivery from the medical records, including infant sex, gestational age, birthweight, birth length, head circumference, method of delivery and apgar scores. gestational age was determined by the date of the last menstrual period and confirmed by ultrasound at 18 weeks. preterm birth was defined as 1.5 (table 1). table 1maternal characteristics for the entire cohortsubjects n189maternal age years26.15.5bmi kgm28.47.2bmi category normal weight76 (40.2) overweight46 (24.3) obese67 (35.4)weight gain during pregnancy kg10.76.5socioeconomic status 1 (lowest)100 (52.9) 261 (32.3) 36 (3.2) 412 (6.3) 5 (highest)10 (5.3)ethnicity caucasian176 (93.1) other13 (6.9)parity 1111 (58.7)smoking status never / former smoker128 (67.7) quit smoking during pregnancy23 (12.2) current smoker38 (20.1)baseline ics use yes61 (32.3) no128 (67.7)baseline ics type ics13 (21.3) ics + laba48 (78.7)spirometry fev1 l3.020.43 fev1 % pred9211 fvc l3.700.56 fev1/fvc0.820.06new ics in pregnancy no102 (54) yes26 (13.8) using ics pre - pregnancy61 (32.3)any severe exacerbation41 (21.7) ocs use14 (7.4) general practitioner visit31 (16.4) emergency department visit10 (5.3) admitted to hospital2 (1.1)persistent uncontrolled asthma30 (15.9)data are presented as meansd or n (%), unless otherwise stated. bmi : body mass index ; ics : inhaled corticosteroid ; laba : long - acting 2-agonist ; fev1 : forced expiratory volume in 1 s ; fvc : forced vital capacity ; ocs : oral corticosteroid. maternal characteristics for the entire cohort data are presented as meansd or n (%), unless otherwise stated. bmi : body mass index ; ics : inhaled corticosteroid ; laba : long - acting 2-agonist ; fev1 : forced expiratory volume in 1 s ; fvc : forced vital capacity ; ocs : oral corticosteroid. the percentage of women who experienced an exacerbation or loss of control (figure 1a) increased linearly as gestation progressed. similarly, the percentage of exacerbations and loss of control also increased as gestation progressed (figure 1b), which was comparable to the number of exacerbations or loss of control throughout gestation for the entire population (figure 1c). overall there were more asthmatic women identified with a loss of asthma control (50%) as gestation progressed than with an exacerbation (22%). figure 1examination of the prevalence of asthma exacerbations and loss of asthma control throughout pregnancy in women with asthma. a loss of control was defined as an increase in asthma symptoms or an increase in medication use, which was managed by the subject and did not require medical intervention, or an asthma control questionnaire score of > 1.5 during an asthma study visit. an exacerbation event was defined as moderate / severe if it required a hospital admission, emergency department presentation, an unscheduled visit to a doctor or a course of oral steroids due to asthma. a) the percentage of women who experienced an exacerbation or loss of control as gestation progressed. b) the percentage of all asthma exacerbations or losses of control according to the number of weeks of gestation. c) the cumulative number of exacerbations or losses of control as gestation progressed within the entire population. examination of the prevalence of asthma exacerbations and loss of asthma control throughout pregnancy in women with asthma. a loss of control was defined as an increase in asthma symptoms or an increase in medication use, which was managed by the subject and did not require medical intervention, or an asthma control questionnaire score of > 1.5 during an asthma study visit. an exacerbation event was defined as moderate / severe if it required a hospital admission, emergency department presentation, an unscheduled visit to a doctor or a course of oral steroids due to asthma. a) the percentage of women who experienced an exacerbation or loss of control as gestation progressed. b) the percentage of all asthma exacerbations or losses of control according to the number of weeks of gestation. c) the cumulative number of exacerbations or losses of control as gestation progressed within the entire population. when examining the relative risk of a severe exacerbation there were no significant maternal characteristics that contributed to an exacerbation during pregnancy (table 2). however, maternal age, normal body mass index (bmi) and quitting smoking appeared protective against an exacerbation during pregnancy. the presence of an asthma exacerbation during pregnancy was not associated with any statistically significant increase in adverse perinatal outcomes (table 3). table 2maternal characteristics associated with moderate or severe asthma exacerbations during pregnancyexacerbationno exacerbationp - valuerelative risk (95% ci)subjects n41148maternal age years25.95.826.25.60.7950.99 (0.951.04)bmi kgm29.17.528.37.20.5361.01 (0.981.05)bmi category0.701 normal weight17 (41.5)59 (39.9)reference overweight8 (19.5)38 (25.7)0.78 (0.371.66) obese16 (39.0)51 (34.5)1.07 (0.591.94)weight gain during pregnancy kg9.87.211.06.50.2730.98 (0.931.02)parity0.699 018 (43.9)60 (40.5)0.90 (0.521.55) 123 (56.1)88 (59.5)smoking status0.557 never / former smoker29 (70.7)99 (66.9)reference quit smoking during pregnancy3 (7.3)20 (13.5)0.58 (0.191.73) current smoker9 (22.0)29 (19.6)1.05 (0.542.01)baseline ics use0.155 yes17 (41.5)44 (29.7)1.49 (0.872.55) no24 (58.5)104 (70.3)data are presented as meansd or n (%), unless otherwise stated. table 3perinatal characteristics of pregnancies complicated by a moderate or severe exacerbationmalefemalesevere exacerbationno exacerbationp - valuesevere exacerbationno exacerbationp - valuesubjects n24731775gestational age days27712270200.10827712273120.288birthweight g349646133237580.294346146032915910.271birthweight centile47.932.448.331.20.95457.426.245.630.50.143delivery method0.5600.756 vaginal14 (58.3)50 (68.5)11 (64.7)55 (73.3) elective lscs3 (12.5)9 (12.3)3 (17.6)10 (13.3) emergency lscs7 (29.2)14 (19.2)3 (17.6)10 (13.3)labour onset0.7721.000 sol14 (58.3)37 (50.7)9 (52.9)40 (53.3) iol6 (25.0)24 (32.9)6 (35.3)23 (30.7) none4 (16.7)12 (16.4)2 (11.8)12 (16.0)birth length cm50.22.149.73.90.55750.11.949.02.20.069head circumference cm35.41.134.62.30.11434.61.734.31.70.455preterm birth0.1761.000 no23 (95.8)61 (83.6)16 (94.1)67 (89.3) yes1 (4.2)12 (16.4)1 (5.9)8 (10.7)sga0.7261.000 no22 (91.7)64 (87.7)15 (88.2)64 (85.3) yes2 (8.3)9 (12.3)2 (11.8)11 (14.7)apgar score 1 min82810.96781820.191 5 min91910.44991910.700data are presented as meansd or n (%), unless otherwise stated. lscs : lower segment caesarean section ; sol : spontaneous onset labour ; iol : induction of labour ; sga : small for gestational age. maternal characteristics associated with moderate or severe asthma exacerbations during pregnancy data perinatal characteristics of pregnancies complicated by a moderate or severe exacerbation data are presented as meansd or n (%), unless otherwise stated. lscs : lower segment caesarean section ; sol : spontaneous onset labour ; iol : induction of labour ; sga : small for gestational age. interestingly, there were significant associations between maternal characteristics and the presence of recurrent uncontrolled asthma in this cohort (table 4). women with uncontrolled asthma were older (relative risk 1.06, 95% ci 1.011.11), overweight (relative risk 1.89, 95% ci 0.894.04), current smokers (relative risk 2.92, 95% ci 1.535.58) and prescribed an ics (possibly a reflection of asthma severity) (relative risk 2.40, 95% ci 1.254.60) (table 4). following stratification by fetal sex, the presence of recurrent uncontrolled asthma at two or more study visits was associated with an increased risk of sga (33.3% versus 9.5% ; p=0.018), but not preterm birth (11.1% versus 9.5% ; p=1.000) among women pregnant with females (table 5). by contrast, a nonstatistically significant increased risk of preterm birth was observed for males (25.0% versus 11.8% ; p=0.201), but not sga (8.3 versus 11.8% ; p=1.000) (table 5). table 4maternal characteristics associated with recurrent uncontrolled asthmarecurrent uncontrolled asthmacontrolled asthmap - valuerelative risk (95% ci)subjects n30159maternal age years28.05.625.75.40.0391.06 (1.011.11)bmi kgm30.47.828.17.00.11.03 (0.991.07)bmi category0.189 normal weight9 (30.0)67 (42.1)reference overweight6 (20.0)40 (25.2)1.10 (0.422.89) obese15 (50.0)52 (32.7)1.89 (0.894.04)weight gain during pregnancy kg7.97.211.36.20.0090.92 (0.840.99)parity 18 weeks gestation) [7, 12 ] and miss an important early time point for intervention and maintaining asthma control. our previous work in a mixed socioeconomic status population of pregnant asthmatic women observed a decrease in the frequency of exacerbations after 34 weeks gestation, but this may be due to study design rather than a real decrease over gestation. the current data suggest that the risk of poor asthma control and exacerbations continues until the end of pregnancy. this may be an effect specific to this particular population or it could be due to a major strength of this particular study in collecting data on asthma outcomes up until delivery. loss of control and in particular recurrent loss of control as measured at each visit appears to have a greater impact on perinatal outcome in this population of women. aside from all the problems associated with social disadvantage, the risks for a loss of control were age, bmi, cigarette use and ics use, which are similar to previous studies examining risk factors associated with exacerbations. a population - based study of 4434 asthmatic women examined ics treatment pre - pregnancy and during pregnancy. it was found that 50% of all women stopped or reduced their ics intake with pregnancy and 8.2% exacerbated with cessation, while 20% of all other women exacerbated. women using ics were more likely to exacerbate, which is due to the confounder of asthma severity. severe asthma increases the requirement for ics treatment and asthma severity increases the risk of an exacerbation. in our cohort, women with recurrent loss of control are probably similar to findings from previous studies in that ics use is a reflection of asthma severity, and therefore, they are more susceptible to worsening disease in pregnancy especially if there is a lack of compliance with medication. previous work has demonstrated that cigarette use in pregnancy is associated with worsening asthma and an increased incidence of exacerbations, which may also apply to an increased risk of loss of control as demonstrated in this study. age being a risk factor is interesting, but may be an interaction with asthma and smoking as the combination of these factors are known to contribute to more airway obstructions with age. there was a high prevalence of overweight and obese women in the current study population, which is in line with state wide prevalence. there is some data to suggest overweight and obesity is associated with worsening asthma in nonpregnant adults. overweight or obesity may both impact on the airway via similar mechanisms that include systemic inflammation, airway hyperresponsiveness and the mechanical impact of weight itself on thoracic cavity function. the data from this study suggests obesity itself has no impact on the risk of recurrent loss of control, but overweight women are at risk. this may be due to a difference in other risk factors such as smoking between obese and overweight women. furthermore it may also be related to dietary intake in this population as we have reported recently that asthmatic women who consumed a diet high in fat, sugar and refined grains were twice as likely to have uncontrolled asthma during pregnancy regardless of bmi. poor perinatal outcomes associated with maternal asthma appeared to vary with fetal sex. in the current study we observed a greater incidence of sga female babies in the presence of recurrent uncontrolled asthma. previous studies have reported a higher incidence of sga females with the combination of maternal smoking and asthma. several mechanistic studies have shown that there are sex - specific differences in the fetal response to maternal asthma that may be conferred by a difference in glucocorticoid sensitivity of the male and female placenta [31, 32 ]. these findings suggest males and females respond differently to the presence of maternal asthma and institute different strategies in relation to growth, which may also contribute to difference in perinatal outcomes. asthma was not observed to worsen in the presence of a female fetus in the current study, but previous studies have reported a higher incidence of maternal hospitalisations for asthma in women pregnant with a female, and reduced maternal lung function and an increased requirement for ics in women pregnant with a female. however, this remains controversial as large epidemiological studies did not find any difference in maternal exacerbations or daily ics dose in asthmatic women pregnant with a male or female fetus. while this study was sufficiently powered to assess asthma exacerbations and control during pregnancy, it was underpowered to examine less common perinatal outcomes such as preterm birth. the strengths of this study are reflected in the detailed and prospective collection of data on asthma status and perinatal outcomes. a key challenge related to studies involving asthmatic pregnancies lies in the accurate identification of asthma control status and exacerbations. while validated tools such as the acq can be used in the evaluation of asthma control at any point in time, no such validated tools are available for identifying exacerbations [35, 36 ]. a common approach for defining exacerbations has been to use any event requiring a hospital admission, emergency department presentation, an unscheduled visit to a doctor or a course of oral steroids due to asthma. for example, women who have greater self - awareness of potentially deteriorating asthma may more readily visit their doctor or the emergency department to seek additional help, while those with poor self - awareness may avoid seeking necessary additional help. this could lead to a situation in which those with greater health - seeking behaviours may appear to experience more exacerbations during pregnancy, but could in turn have better pregnancy outcomes as a result of subsequent improved asthma self - management. in contrast, those with impaired health - seeking behaviours may appear to experience fewer exacerbations, but have worse pregnancy outcomes due to poor asthma self - management. the results of this study in a socially disadvantaged population lend support to these criticisms of the common definition of asthma exacerbations and provide an argument for the preferred use of validated tools such as the acq, which does not rely on self - awareness or health - seeking behaviours for assessing asthma - related outcomes during pregnancy. many previous studies required retrospective collection of exacerbation data from medical records [10, 37, 38 ] and we speculate, based our current data, that while exacerbations identified from medical records may be a surrogate marker for persistently poorly controlled asthma during pregnancy, their predictive value in identifying persistently poorly controlled asthma during pregnancy could be biased by the characteristics of the population being studied. therefore, a major strength of this study is that it provides new evidence that chronically or persistently uncontrolled asthma may be a greater driver for poor perinatal outcomes than an acute exacerbation. the current study provides some new and novel insights into the course of asthma during pregnancy, especially the identification that 50% of all exacerbations and losses of control occur before 20 weeks of gestation. this highlights the need for clinicians to focus on maintaining asthma control during pregnancy through the introduction of asthma education and self - management skills pre - pregnancy to avoid early gestation events that may detrimentally affect placentation and fetal growth. interestingly, recurrent uncontrolled asthma had the greatest impact on perinatal outcomes, rather than exacerbations, suggesting there is a need for asthma management support during pregnancy to ensure women can maintain asthma control. we speculate that in combination with the identified risk factors for recurrent uncontrolled asthma reported in this paper, which include body weight, cigarette use, ics use and maternal age, there may also be financial stressors, different purchasing priorities and poor health literacy that could influence ics use and adherence in this socially disadvantaged population. the key to resolving these issues is to have a more structured approach to asthma education and self - management pre - pregnancy and then a clinically integrated service that encompasses respiratory care with antenatal care. the current study provides some new and novel insights into the course of asthma during pregnancy, especially the identification that 50% of all exacerbations and losses of control occur before 20 weeks of gestation. this highlights the need for clinicians to focus on maintaining asthma control during pregnancy through the introduction of asthma education and self - management skills pre - pregnancy to avoid early gestation events that may detrimentally affect placentation and fetal growth. interestingly, recurrent uncontrolled asthma had the greatest impact on perinatal outcomes, rather than exacerbations, suggesting there is a need for asthma management support during pregnancy to ensure women can maintain asthma control. we speculate that in combination with the identified risk factors for recurrent uncontrolled asthma reported in this paper, which include body weight, cigarette use, ics use and maternal age, there may also be financial stressors, different purchasing priorities and poor health literacy that could influence ics use and adherence in this socially disadvantaged population. the key to resolving these issues is to have a more structured approach to asthma education and self - management pre - pregnancy and then a clinically integrated service that encompasses respiratory care with antenatal care. | there exists a paucity of data for socially disadvantaged populations describing patterns and predictors of asthma control status and exacerbations during pregnancy, and their relationship to adverse perinatal outcomes.asthmatic women (n=189) were followed prospectively during pregnancy, with visits at 12, 20, 28 and 36 weeks gestation. data on loss of control, recurrent uncontrolled asthma and moderate / severe exacerbations were collected at each visit and their relationship to perinatal outcomes examined following stratification for fetal sex.50% of asthmatic women experienced a loss of control or moderate / severe exacerbation during pregnancy, with 22% of women experiencing a moderate / severe exacerbation. factors associated with an increased risk of women experiencing recurrent uncontrolled asthma during pregnancy included smoking (relative risk 2.92, 95% ci 1.535.58), inhaled corticosteroid use at the beginning of pregnancy (relative risk 2.40, 95% ci 1.254.60) and increasing maternal age (relative risk 1.06, 95% ci 1.011.11). no factors were associated with moderate / severe exacerbations. asthma control rather than exacerbations during pregnancy appeared to be most strongly correlated with perinatal outcomes. following stratification by fetal sex, the presence of recurrent uncontrolled asthma was associated with an increased risk of being small for gestational age in women pregnant with females (33.3% versus 9.5% ; p=0.018). in contrast, there was a nonsignificant increased risk of preterm birth in women with recurrent uncontrolled asthma that were pregnant with males (25.0% versus 11.8% ; p=0.201)these results suggest that the key to improving perinatal outcomes lies in improving asthma control as early as possible in pregnancy and monitoring throughout pregnancy, rather than focusing on preventing exacerbations alone. |
bipolar disorder (bd) associated seasonal pattern (mania during spring and summer together with depression during fall and winter) referred to as seasonal affective disorder (sad) is associated with disrupted circadian rhythms. aryl hydrocarbon receptor nuclear translocator - like (arntl) protein is a transcription factor and a core component of mammalian circadian rhythms regulatory network. convergent functional genomics approach, integrating the genetics with functional genomics in human as well as animal models identified the arntl gene as the top candidate associated with bd. several polymorphisms in arntl gene are reported to be associated with bd and sad in different ethnic groups. as the family members in the study were experiencing sad, this study was undertaken to delineate the role of arntl gene in sad. we attempted to decipher the five single nucleotide polymorphisms (snps) (rs2279287, rs1982350, rs7126303, rs969485, and rs2290035) in arntl gene [figure 1 ] and correlated it to differential seasonal behavior. however, the interaction between photoperiodic mechanisms (light - dark cycle) and the circadian system in the onset of sad is obscure. distribution of the five single nucleotide polymorphisms in aryl hydrocarbon receptor nuclear translocator - like gene the subjects comprised 30 members of close - knit family with sad (dsm-5 criteria was used in diagnosis) and age- and sex - matched 30 controls of the same caste with no prior history of psychiatric illness. in addition, 30 age- and sex - matched controls with no prior history of psychiatric illness belonging to 17 different castes formed as a negative control. the human ethical committee of bharathiar university, tamil nadu, india approved the study. the clinical investigations of all the study participants were carried out in kg hospital, coimbatore, tamil nadu, india. dna was isolated from the blood samples using hi - pura mini blood dna isolation kit (himedia, india). five snps across the arntl gene namely rs2279287 (a / g), rs1982350 (c / t), rs7126303 (c / t), rs969485 (a / g), and rs2290035 (a / t) were selected to test for their association as the risk factor in sad. briefly, polymerase chain reaction (pcr) reactions were run in a total volume of 5 l, containing 10 ng dna, 2.5 l 1 pcr buffer, and 0.125 l 40 allelic discrimination primer - probe mix. reactions were run in abi 7500 real - time system (applied biosystems, usa) with the following cycle parameters : 95c for 10 min ; followed by 40 cycles at 95c for 15 s and 60c for 30 s. the post - pcr run was done at 60c for 1 min. statistical significance was assessed by more powerful quasi - likelihood score test (mqls)-xm, a program written in c, that performs single - snp, case - control association testing on the autosomal chromosomes in samples with related individuals. the program is applicable to association studies with completely general combinations of related and unrelated individuals, where the relationship among the sampled individuals is assumed to be known. for each snp, for autosomal snps, the three test statistics computed are mqls, wqls and corrected. the link to the statistical software used is https://galton.uchicago.edu/~mcpeek/software/mqls_xm/index.html. the subjects comprised 30 members of close - knit family with sad (dsm-5 criteria was used in diagnosis) and age- and sex - matched 30 controls of the same caste with no prior history of psychiatric illness. in addition, 30 age- and sex - matched controls with no prior history of psychiatric illness belonging to 17 different castes formed as a negative control. the human ethical committee of bharathiar university, tamil nadu, india approved the study. the clinical investigations of all the study participants were carried out in kg hospital, coimbatore, tamil nadu, india. dna was isolated from the blood samples using hi - pura mini blood dna isolation kit (himedia, india). five snps across the arntl gene namely rs2279287 (a / g), rs1982350 (c / t), rs7126303 (c / t), rs969485 (a / g), and rs2290035 (a / t) were selected to test for their association as the risk factor in sad. briefly, polymerase chain reaction (pcr) reactions were run in a total volume of 5 l, containing 10 ng dna, 2.5 l 1 pcr buffer, and 0.125 l 40 allelic discrimination primer - probe mix. reactions were run in abi 7500 real - time system (applied biosystems, usa) with the following cycle parameters : 95c for 10 min ; followed by 40 cycles at 95c for 15 s and 60c for 30 s. the post - pcr run was done at 60c for 1 min. statistical significance was assessed by more powerful quasi - likelihood score test (mqls)-xm, a program written in c, that performs single - snp, case - control association testing on the autosomal chromosomes in samples with related individuals. the program is applicable to association studies with completely general combinations of related and unrelated individuals, where the relationship among the sampled individuals is assumed to be known. for each snp, the three test statistics computed are mqls, wqls and corrected. the link to the statistical software used is https://galton.uchicago.edu/~mcpeek/software/mqls_xm/index.html. the polymorphic count and allele frequency distribution of different snps in arntl gene in cases and controls of the same caste are given in table 1. the polymorphic count and allele frequency distribution of different snps in arntl gene in cases and controls of different castes are given in table 2. among the five snps of arntl gene, there was a significant variation in snp rs2279287 between the cases and controls of same caste [table 1 ]. the allelic frequency of the mutant allele (g) was found to be 0.75 in cases. genotyping of the cases for this snp showed that 50% of the cases are homozygous, and the remaining individuals are heterozygous for the mutant allele. none of the cases were found to be wild. in the control group belonging to the same caste, 20% were homozygous, 47% were heterozygous, and 33% were wild type. in the control group of different castes, the other 4 snps (rs1982350, rs7126303, rs969485, and rs2290035) were almost equally distributed both in the cases and controls [tables 1 and 2 ]. among the 90 subjects (30 cases and 30 controls of the same caste and 30 controls of different castes), only one individual is wild for all the five snps studied. polymorphic count and allele frequency distribution of different db - single nucleotide polymorphisms in arntl gene in cases and controls of same caste polymorphic count and allele frequency distribution of different db - single nucleotide polymorphisms in arntl gene in cases and controls of different castes snp rs2279287, which is a significant marker in the family under study is located in the promoter region of arntl gene and hence the polymorphism in this region could affect the clock regulated processes. the controls in this study carried the mutant alleles, and the same finding was reported by other studies. the significant marker rs2279287 in the present study is a part of the most significant haplotype of arntl gene of caucasians similar to this study, association of arntl gene variation with a seasonal pattern in bd has been reported in different populations recently. people with wild - type genotype of snp rs2290035 in arntl gene are associated with less seasonal variation in energy level. about 96% of patients with the mutant genotype of arntl gene have a routine seasonal variation of energy level. the functionality of the arntl protein in terms of homozygous, heterozygous, and wild polymorphic nature is not elucidated still. a fully functional arntl protein is hypothesized to inhibit mania by inactivating dopamine through monoamine oxidase a and thus inhibiting the promanic effects of dopamine and hence any defect in arntl gene (homozygous / heterozygous) is expected to promote mania. arntl protein has period - arnt - single - minded domains which regulate biological responses to light and hence mutations in the arntl gene could alter the sensitivity to light which is observed in sad subjects. thus, one can not rule out the essential nature of fully functional arntl gene. similar to previous reports, the present study also identified a potentially functional polymorphism, rs2279287 in arntl gene in an indian family diagnosed with sad. conclusively, we propose that polymorphisms in arntl gene disrupt the circadian rhythms causing sad, and genetic predisposition becomes more deleterious in the presence of adverse environment. this is the first report on arntl gene mutations aggravated by environment associated with sad in indian population. | background and aim : polymorphisms in aryl hydrocarbon receptor nuclear translocator - like (arntl) gene, the key component of circadian clock manifests circadian rhythm abnormalities. as seasonal affective disorder (sad) is associated with disrupted circadian rhythms, the main objective of this study was to screen an indian family with sad for arntl gene polymorphisms.materials and methods : in this study, 30 members of close - knit family with sad, 30 age- and sex - matched controls of the same caste with no prior history of psychiatric illness and 30 age- and sex - matched controls belonging to 17 different castes with no prior history of psychiatric illness were genotyped for five different single nucleotide polymorphisms (snps) in arntl gene by taqman allele - specific genotyping assay.statistical analysis : statistical significance was assessed by more powerful quasi - likelihood score test-xm.results:most of the family members carried the risk alleles and we observed a highly significant snp rs2279287 (a / g) in arntl gene with an allelic frequency of 0.75.conclusions:polymorphisms in arntl gene disrupt circadian rhythms causing sad and genetic predisposition becomes more deleterious in the presence of adverse environment. |
it is the combination of feelings and perceptions which can affect and be affected by the culture of a society. moreover, individuals appearance plays a substantial role in their social relationships. for centuries, humans have been concerned about their beauty as well as grooming and adorning themselves ; therefore, they have always sought to improve their physical attractiveness using makeup and cosmetics surgeries. currently, the increasing trend toward cosmetic and aesthetic surgeries has placed iran among the top 10 countries in the world in this regard. some genetic factors such as the shape of their face, especially nose, as well as ethnic and racial factors are considered as the major reasons of iranians tendency towards such surgeries. since iranians, typically, have large noses with a bony and cartilaginous hump, rhinoplasty causes such a significant change in the appearance of the surgery applicants which is not comparable with other types of cosmetic surgeries. besides, human body s limitations and potentials have always been under the influence of socio - cultural values of the society.. a closer look at different communities shows that dissatisfaction with one s appearance is now considered normative. such concern, which can be intensified by individual aesthetic standards promoted by advertisement industry, may affect the people s body image negatively and increase their willingness to undergo cosmetic surgeries. besides, research shows that the demands for such surgeries decrease by a decline in the rate of cognitive impairment. personality is defined as the realization of the innate idiosyncrasy and perceptions of an individual which determines the specific patterns of thought, emotion and behaviors in interaction with physical and social environment. cloninger also analyzes the underlying biogenic structures of personality by assessing the two dimensions of temperament and character. according to cloninger., temperament refers to genetically individual differences in behavioral reactions which are constant throughout the life ; however, character refers to the learned psychosocial and sociocultural influences on personality which matures throughout one s life. temperament domain includes four dimensions of novelty seeking (ns), harm avoidance (ha), reward dependence (rd) and persistence (p). ns is defined as behavioral reactions in response to novel stimulation which are reinforced by pursuit of rewards and escape from punishment. ha is manifested as inhibition of behavior in response to non - reward, null and punishing stimuli. rd is the tendency to respond to signals of reward and maintain previously rewarded behaviors. the three dimensions of character are as follows : self - directedness (sd), cooperativeness (co) and self - transcendence (st). st is the ability to accept and understand the rules and the environment as an integrated whole. biopsychosocial model of cloninger consists of psychobiological aspects of temperament and character which are developed to evaluate an individual s personality specifications through inheritance (temperament) or the environment (character). previous studies revealed that cosmetic surgeries can change several psychological constructs such as the sense of competence and self - esteem. in a study on temperament and character traits of women who underwent mammoplasty, most of the previous studies have focused on the variables of self - esteem, character and satisfaction changes after rhinoplasty. accordingly, it seems necessary to investigate temperament and character, as biological components of personality, in the applicants of rhinoplasty. in the present study, we aimed to evaluate these two traits in the patients referred to rhinoplasty surgeons in shiraz, southern iran. convenience sampling was used to recruit the participants in both groups among people who gave their consent to participate and had the inclusion criteria. we recruited 250 rhinoplasty applicants referred to shahriar and dastgheib hospitals in shiraz, southern iran during february to april 2015. the 250 participants of control group were selected from the students of universities and other educational institutes and clerks who were resident of shiraz during the study and had no history of cosmetic surgeries. inclusion criteria for enrollment in the case group were age between 18 - 60 years and intention to undergo cosmetic surgery only for aesthetic improvement. exclusion criteria were history of anxiety, depression, delirium, body dysmorphic disorder and psychotropic medication consumption according to the applicant s declaration. the sample size was calculated 250 in each group based on the data of similar studies and the following formula (power : 80% ; : 0.05 ; =0.2 ; z=1.96). written informed consent was obtained from all the participants after explaining the aim and method of the study. data were collected using a demographic questionnaire and temperament and character inventory (tci-125). demographic questionnaire contained questions on age, sex, marital status, education, occupation, place of residence, employment status, income, purpose and people who encourage the patients for undergoing cosmetic surgery as well as history of depression and anxiety. (1994) to assess the 7 dimensions of personality with a total of 29 subscales. it is self - rating and paper - and - pencil test with the true - false format. questions were about the respondent s interests, desires, attitudes, emotional reactions, goals and values. the questions are designed in a way that the right or the wrong answers increase the score of the same scale depending on the content of the expression. the validity and reliability of the questionnaire were confirmed by kaviani and poornaseh (2005) in a study on 1212 residents of tehran, iran. reliability coefficient was obtained for each dimension using test - retest (ns= o.86 ; ha= 0.88 ; rd=0.73 ; p=0.79 ; co=0. 86 ; sd=0.90 ; st=0. statistical qualitative tests including chi - square test and t - tests were used as appropriate. women (70.8%) had a higher prevalence rate than men (29.2%) in the study. the case and control groups were matched with respect to the variables of age, sex and educational level ; however, there was a significant difference between the two groups in terms of marital status (p0.05). the effect size was medium (0.30.05) ; however, a significant difference was reported regarding co dimension and its other subscales including social acceptance (co1), compassion (co4) and integrated conscience (co5) (p<0.05). the effect size was medium for co and co5 (0.3<|ef|<0.5) but weak for the subscales of co1, co2, co3 and co4 (|ef|<0.3). a statistically significant difference was found between the case and control groups in terms of st dimension and its subscales including self - forgetfulness (st1) and spiritual acceptance (st3) (p<0.05) except for the subscale of transpersonal identification (st2) (p=0.032). the effect size was medium for st and st3 but weak for st1 and st4. our results showed that the number of women seeking rhinoplasty is more than men. accordingly, our findings are consistent with other researchers who investigated the personality traits of the patients seeking cosmetic rhinoplasty. however, our results were in contrast with those of another study that examined the relationship between psychological symptoms and satisfaction after rhinoplasty. another study in iran revealed that appearance and beauty have been more often an issue for women than men so that they receive more recommendations from friends to undergo cosmetic surgeries. moreover, major appearance changes have been more acceptable in women than men as they usually pay more attention to their physical attractiveness. in other words, a woman values herself when she can continue her perfect life by undergoing cosmetic surgeries. rhinoplasty applicants have different age ranges ; the age group most likely applying for rhinoplasty is the late teens to young adulthood. new concerns about physical appearance begin almost from the age of 20 years and one of the most important concerns for both men and women about their appearance is the size and shape of their nose. other researchers reported that internet has always been the main source of obtaining information on rhinoplasty as most internet users are young people. similar to the findings of another study, the majority of our participants expressed that their purpose of undergoing cosmetic surgeries is their desire to become beautiful. accordingly, it can be concluded that a successful rhinoplasty makes the applicants satisfied with their new nose appearance and, consequently, enhances their satisfaction with their whole body. furthermore, people are encouraged to undergo cosmetic surgeries after seeing the results of successful surgeries performed on other people. our results are, also, in the same line with those of previous studies, indicating that the majority of the women seeking cosmetic surgeries have been single ; that might be due to the instrumental view which exists about women in the society and forces them to act and behave based on the men s desire. furthermore, bachelorhood and marriage can also play an important role in people s interest in undergoing cosmetic surgeries. the results of this study revealed higher ns scores in the case group compared to the control group. furthermore a significant difference was seen between the participants who have had cosmetic surgeries and their peers in the case group with respect to ns scores in its all subscales. our findings were consistent with those of haktanir. and shahi. we can conclude that the people who are interested in cosmetic surgeries commonly have personality traits such as feeling bored of monotony, persistence in making changes and willingness to take risks. hence, several factors such as variety seeking, possibility of making changes in the face, decreasing dissatisfaction with physical appearance, durability of changes, recent advances in cosmetic surgery techniques, television advertisement, promises to improve the appearance and increasing self - confidence play important roles in encouraging people to experience cosmetic surgeries. the results of the present study revealed a significant difference between the case and control groups in terms of ha scale scores (p<0.05) which is similar to the findings of other studies. our results showed higher ha scores reported by the participants of the case group compared with their counterparts in the control group. accordingly, higher ha score signifies the personality traits such as sensitivity to criticism and punishment, not being comfortable when meeting strangers, shyness, pessimism and dependence on others help in normal situations. hence, such people would like to undergo cosmetic surgeries due to their low self - esteem, friends recommendation, the importance of community s attitudes toward beauty and appearance, enhancing the shape and appearance of their nose, looking more attractive and improving their appearance, achieving success in marriage, gaining more confidence in social interactions and satisfying their family and friends. furthermore, we observed a significant difference between the case and control group in terms of rd scale scores in (p<0.05), which is similar to other researches. accordingly, people with lower rd scores are practical, though minded, cold, objective and socially insensitive with logical serious and beliefs as well as self - centered attitudes. all these factors could encourage the youth and even the adults to seek a greater share of beauty and beauty enhancements based on their orientations and age requirements without considering its heavy cost and even with accepting all its physical and psychological consequences. the results of the present study showed a significant difference between the case and control groups in terms of p scores in (p<0.05) which is similar to the findings of another study. therefore, the participants in the case group with lower p scores are action - oriented and easily get discouraged when faced with challenges, failures, and criticism. such people may have their own high standards and criteria, so they feel disappointed and dissatisfied when facing with problems. they are vulnerable to criticism and everything should be ideal for them ; otherwise, they feel dissatisfied. the mean of self - directedness (sd) scores reported by the participants of the case group was significantly different from those reported by their peers in the control group in the subscale of self - acceptance and intelligence. the individuals with higher sd scores are mature, purposeful, self - sufficient and fantasize about beauty and eternal youth. they try to increase their quality of life, self - confidence and positive mood by undergoing cosmetic surgeries. it seems that such surgeries increase the quality of life in the individuals with low self - directedness. individual s perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns. there was a significant difference between the case and control group with respect to the mean scores of co in all subscales except for empathy and helpfulness. the participants of the case group reported lower c scores ; therefore, they are opportunistic, self - centered, self - indulgent, impatient and critical and behave based on their prejudices and personal interests. moreover, those with lower co score are more likely to have cosmetic surgeries as they judge their appearance more negatively. they may find the surgeries as a means to improve their appearance and overcome their weaknesses. we found a significant difference between the case and control groups with respect to the mean scores of st in all subscales except for transpersonal identification. the participants of the case group reported lower st scores as they were impatient, proud, materialistic, self - conscious, and dissatisfied with a strong imagination. it can be concluded that, nowadays, life is a personal choice as people can do whatever they like and act based on their personal interests and benefits which indicate the trend of individualization. accordingly, cosmetic surgeries help people to achieve a good self - image and self - ideal sense through which they cure the feeling of not being desirable and dissatisfied with their appearance. the effect size estimated by the comparison of the mean scores was < 0.5 in the subscale of adventurer, attachment and compatible habits and < 0.3 in the other subscale indicating a statistically significant difference between the case and control groups. besides, the effect of such difference is in the medium and weak ranges which could happen due to our large sample size. considering the significant difference of the scores between the case and control groups, it can be concluded that both temperament and character traits, which are influenced by genetics and environment respectively, can affect the tendency toward rhinoplasty. the strengths of this study are the large sample size and also assessing the temperament and character of rhinolplasty applicants for the first time in the world based on our researches. firstly, the case and control groups were not matched in terms of their marital and employment status variables, and secondly the family income was not considered as an effective factor. considering the increasing number of rhinoplasty applicants and the importance of temperaments and characters in predicting biological - genetic traits of individual s personality, evaluating such traits in rhinoplasty applicants will be so helpful in identifying and predicting ideal candidates for such surgery. selecting the good patients can not only reduce the costs resulting from rhinoplasty imposed on families and society but also enhance the satisfaction of the applicants and the surgeons. | background : rhinoplasty is the most common cosmetic surgery which has been dramatically increasing in iran. currently, iran is ranked the first in the world in rhinoplasty. in the present study, we aimed to assess the character and temperament traits of the applicants referred to rhinoplasty surgeons in shiraz, southwest iran in 2015.methods:in this cross - sectional study, we recruited 500 participants among rhinoplasty applicants for case and among students and clerks residing in shiraz by convenience sampling method in 2015. the two groups were matched regarding the gender, age and educational level. data were collected using a demographic questionnaire and temperament and character inventory to assess the four dimensions of temperament (including novelty seeking ; harm avoidance ; reward dependence ; persistence) and the three dimensions of character (including self - directedness ; cooperativeness ; self - transcendence). data were analyzed using spss software, version 20. chi- square and t - test were used as appropriated.results:the meansd age of the participants was 27.436.6. the results showed a significant difference between the case and control groups with respect to the temperaments of novelty (9.472.80), harm avoidance (9.123.3), persistence (2.691.04), the characters of cooperativeness (15.384.02), and self - transcendence (9.483.41).conclusion : evaluating character and temperament traits in rhinoplasty applicants will be so helpful in identifying and predicting good candidates for such cosmetic surgery. selecting the ideal patients can not only reduce the costs resulting from rhinoplasty imposed on families and society but also enhance the satisfaction of the patients and the surgeons. |
the chinese atrial fibrillation registry (cafr) prospectively enrolled consecutive af patients from multiple centers, confirmed by objective tests such as 12-lead electrocardiograms (ecgs) or 24-hour holter monitorings before registration. the protocol was approved by the institutional ethics committee first in beijing anzhen hospital and then in each participating hospital. baseline characteristics of patients before and after matching values are given as meansd or numbers (percentages). chads2 scores, congestive heart failure : 1 score, hypertension : 1 score, age 75 years : 1 score, diabetes mellitus : 1 score, previous stroke or transient ischemic attack : 2 score. af indicates atrial fibrillation ; cad, coronary artery diseases ; copd, chronic obstructive pulmonary diseases ; dm, diabetes mellitus ; ehra, european heart rhythm association ; htn, hypertension ; lad, left atrium diameter ; lvdd, left ventricular diastolic diameter ; lvef, left ventricular ejection fraction ; mi, myocardial infarction ; non - rfa, non radiofrequency ablation ; nyha, new york heart association ; rfa, radiofrequency ablation. history of hypertrophic cardiomyopathy, dilated cardiomyopathy and thyroid disease were not listed because no cases matched based on propensity score matching. because only tertiary hospitals provided catheter ablation facilities, data of cafr from 4 tertiary hospitals in beijing, china, between april 2011 and february 2013 were selected to reduce confounders caused by different centers (beijing anzhen hospital, beijing tongren hospital, beijing chaoyang hospital, and peking university third hospital). patients with baseline chads2 scores (congestive heart failure [chf ] : 1 score, hypertension [htn ] : 1 score, age 75 years : 1 score, diabetes mellitus [dm ] : 1 score, previous stroke or transient ischemic attack [tia ] : 2 scores) 1 were considered eligible for the study regardless of their af types. afeqt questionnaires with at least 50% of responses finished for each domain were used for final analyses. those who received rfa other than circumferential pulmonary vein isolation, which was described precisely before, were excluded.12 to minimize the variation of the length of the follow - up, the date of baseline was defined as the index date. patients who underwent rfa more than 5 days before or after the index date were excluded. the follow - up started from the index date to 6 months5 days through outpatient, telephone, visiting at home every month, or admission into hospital whenever necessary. patients were advised to record any possible symptomatic episodes of af by doing 12-lead ecgs in the nearest clinics. furthermore they were routinely scheduled to do 12-lead ecgs or 24-hour holter monitorings at every follow - up regardless of af - related symptoms. patients in the following cases were excluded : patients with af of rheumatoid valvular diseases ; age younger than 18 years ; previous ablation for af ; life expectancy of less than 6 months ; patients who switched to ablation therapy when they refused the procedure at inclusion ; and patients who received a second ablation during the 6-month follow - up. the full cohort was then divided into those who received a single rfa (rfa group) and those who did not (non - rfa group). to reduce the impact of selection bias on the estimation of treatment effects, baseline differences in af patients with low chads2 score were adjusted by propensity score matching, which was based on 1:2 matching within a prespecified caliper width and without replacement. we estimated the propensity score by regressing treatment status based on the covariates using a multinomial logistic regression model. the matched samples were obtained by matching subjects on the logit of the propensity score by nearest neighbor matching, with caliper of 0.6 of the pooled standard deviations of the logit of the propensity score.13 covariates for propensity score matching were baseline characteristics of getting af and adjustment confounders and biases according to previous literature14 and earlier knowledge. they included age, sex, type of af, admission type, educational status, insurance plan type, european heart rhythm association (ehra) classification, new york heart association (nyha) stratification, clinical history (history of htn, dm, myocardial infarction, coronary heart diseases, hypertrophic cardiomyopathy, dilated cardiomyopathy, thyroid disease, and lung diseases), echocardiographic parameters (left atrial diameter, left ventricular [lv ] end - diastolic diameter, and lvef), and successive medications for rate or rhythm control, as well as anticoagulant drugs. the primary outcome was the comparison of each domain and the global score of afeqt questionnaire at 6 months between groups based on the propensity score matched cohorts. secondary outcomes were : (1) changes from baseline to 6 months within groups and the between - group differences of changes in each domain and the global score of afeqt questionnaire based on the propensity score matched cohorts. (2) af recurrence rate in rfa group and rate of treatment failure in the non - rfa group. af recurrence was defined as documented af / atrial flutter / atrial tachycardia (at) 30 seconds by 24-hour holter monitorings or 12-lead ecgs beyond 3-month blanking period and within 6-month follow - up postablation.15 documented af of at least twice in the non - rfa group was considered as treatment failure during the 6-month period.16 (3) complications or adverse effects related to the procedure or medicines used during the 6-month follow - up. the afeqt questionnaire (see appendix s1) is an af - specific qol evaluation according to its authors descriptions,11 which adequately correlates with other commonly used, well - established questionnaires. the impact of af on their qol during the previous 4 weeks are indicated with 20 items from 4 individual domains, including symptoms (4 items), daily activities (8 items), treatment concern (6 items), and treatment satisfaction (2 items). the afeqt questionnaire can be divided into 2 parts : evaluation of health status and treatment satisfaction. global health status is determined based on the sum of scores of the first 3 domains. raw scores within each domain are transformed to a 0 to 100 scale, where a score of 0 indicates the most severe symptoms or disability and a score of 100 indicates no limitation or disability. each domain contributes to an insight into a different aspect of patients qol, with higher scores representing a better life and less mental burden. both in unadjusted and adjusted cohorts, descriptive variables were expressed as meansd or median (range), and categorical variables were given as numbers (percentages). baseline characteristics, including baseline afeqt domains and global score, were compared between the 2 groups using the student t test or chi - squared test or fisher s exact analysis, as appropriate. a series of paired t tests were conducted to compare scores between baseline and 6 months and thus changes from baseline to 6 months of each domain and the global score within each group. scores of all afeqt domains and the global score at 6 months and their changes during the 6-month follow - up between the 2 propensity - matched groups were compared using an independent t test. the cohen approach of defining effect sizes of 0.2, 0.5, and 0.8 were used as indication of small, moderate, and large clinical changes for interpretation.17 the study provided at least 90% power at a 2-sided alpha level of 0.05 for a difference of a 20% score increase for the health status domains and treatment satisfaction domains of the afeqt at 6 months. exploratory subgroup analyses stratified by af type and age strata of 65 years were conducted in fear that qol may be affected by these factors and to minimize the modification. to further confirm our findings, baseline and 6-month score of each domain and the global score of afeqt questionnaire in af patients with low cha2ds2-vasc score (chf : 1 score, htn : 1 score, age 75 years : 2 scores, dm : 1 score, previous stroke or tia : 2 scores, vascular disease : 1 score, age 65 to 74 years : 1 score, sex category of female : 1 score) we performed all analyses using the sas statistical package (version 9.1 ; sas institute inc., cary, nc) and spss 18.0 for windows (spss, inc., chicago, il). because only tertiary hospitals provided catheter ablation facilities, data of cafr from 4 tertiary hospitals in beijing, china, between april 2011 and february 2013 were selected to reduce confounders caused by different centers (beijing anzhen hospital, beijing tongren hospital, beijing chaoyang hospital, and peking university third hospital). patients with baseline chads2 scores (congestive heart failure [chf ] : 1 score, hypertension [htn ] : 1 score, age 75 years : 1 score, diabetes mellitus [dm ] : 1 score, previous stroke or transient ischemic attack [tia ] : 2 scores) 1 were considered eligible for the study regardless of their af types. afeqt questionnaires with at least 50% of responses finished for each domain were used for final analyses. those who received rfa other than circumferential pulmonary vein isolation, which was described precisely before, were excluded.12 to minimize the variation of the length of the follow - up, the date of baseline was defined as the index date. patients who underwent rfa more than 5 days before or after the index date were excluded. the follow - up started from the index date to 6 months5 days through outpatient, telephone, visiting at home every month, or admission into hospital whenever necessary. patients were advised to record any possible symptomatic episodes of af by doing 12-lead ecgs in the nearest clinics. furthermore they were routinely scheduled to do 12-lead ecgs or 24-hour holter monitorings at every follow - up regardless of af - related symptoms. patients in the following cases were excluded : patients with af of rheumatoid valvular diseases ; age younger than 18 years ; previous ablation for af ; life expectancy of less than 6 months ; patients who switched to ablation therapy when they refused the procedure at inclusion ; and patients who received a second ablation during the 6-month follow - up. the full cohort was then divided into those who received a single rfa (rfa group) and those who did not (non - rfa group). to reduce the impact of selection bias on the estimation of treatment effects, baseline differences in af patients with low chads2 score were adjusted by propensity score matching, which was based on 1:2 matching within a prespecified caliper width and without replacement. we estimated the propensity score by regressing treatment status based on the covariates using a multinomial logistic regression model. the matched samples were obtained by matching subjects on the logit of the propensity score by nearest neighbor matching, with caliper of 0.6 of the pooled standard deviations of the logit of the propensity score.13 covariates for propensity score matching were baseline characteristics of getting af and adjustment confounders and biases according to previous literature14 and earlier knowledge. they included age, sex, type of af, admission type, educational status, insurance plan type, european heart rhythm association (ehra) classification, new york heart association (nyha) stratification, clinical history (history of htn, dm, myocardial infarction, coronary heart diseases, hypertrophic cardiomyopathy, dilated cardiomyopathy, thyroid disease, and lung diseases), echocardiographic parameters (left atrial diameter, left ventricular [lv ] end - diastolic diameter, and lvef), and successive medications for rate or rhythm control, as well as anticoagulant drugs. the primary outcome was the comparison of each domain and the global score of afeqt questionnaire at 6 months between groups based on the propensity score matched cohorts. secondary outcomes were : (1) changes from baseline to 6 months within groups and the between - group differences of changes in each domain and the global score of afeqt questionnaire based on the propensity score matched cohorts. (2) af recurrence rate in rfa group and rate of treatment failure in the non - rfa group. af recurrence was defined as documented af / atrial flutter / atrial tachycardia (at) 30 seconds by 24-hour holter monitorings or 12-lead ecgs beyond 3-month blanking period and within 6-month follow - up postablation.15 documented af of at least twice in the non - rfa group was considered as treatment failure during the 6-month period.16 (3) complications or adverse effects related to the procedure or medicines used during the 6-month follow - up. the afeqt questionnaire (see appendix s1) is an af - specific qol evaluation according to its authors descriptions,11 which adequately correlates with other commonly used, well - established questionnaires. the impact of af on their qol during the previous 4 weeks are indicated with 20 items from 4 individual domains, including symptoms (4 items), daily activities (8 items), treatment concern (6 items), and treatment satisfaction (2 items). the afeqt questionnaire can be divided into 2 parts : evaluation of health status and treatment satisfaction. global health status is determined based on the sum of scores of the first 3 domains. raw scores within each domain are transformed to a 0 to 100 scale, where a score of 0 indicates the most severe symptoms or disability and a score of 100 indicates no limitation or disability. each domain contributes to an insight into a different aspect of patients qol, with higher scores representing a better life and less mental burden. both in unadjusted and adjusted cohorts, descriptive variables were expressed as meansd or median (range), and categorical variables were given as numbers (percentages). baseline characteristics, including baseline afeqt domains and global score, were compared between the 2 groups using the student t test or chi - squared test or fisher s exact analysis, as appropriate. a series of paired t tests were conducted to compare scores between baseline and 6 months and thus changes from baseline to 6 months of each domain and the global score within each group. scores of all afeqt domains and the global score at 6 months and their changes during the 6-month follow - up between the 2 propensity - matched groups were compared using an independent t test. the cohen approach of defining effect sizes of 0.2, 0.5, and 0.8 were used as indication of small, moderate, and large clinical changes for interpretation.17 the study provided at least 90% power at a 2-sided alpha level of 0.05 for a difference of a 20% score increase for the health status domains and treatment satisfaction domains of the afeqt at 6 months. exploratory subgroup analyses stratified by af type and age strata of 65 years were conducted in fear that qol may be affected by these factors and to minimize the modification. to further confirm our findings, baseline and 6-month score of each domain and the global score of afeqt questionnaire in af patients with low cha2ds2-vasc score (chf : 1 score, htn : 1 score, age 75 years : 2 scores, dm : 1 score, previous stroke or tia : 2 scores, vascular disease : 1 score, age 65 to 74 years : 1 score, sex category of female : 1 score) we performed all analyses using the sas statistical package (version 9.1 ; sas institute inc., cary, nc) and spss 18.0 for windows (spss, inc., chicago, il). during 2011 to 2013, 2178 patients with af were enrolled in the cafr from 4 tertiary hospitals. a total of 1196 (54.9%) of them were excluded for their chads2 score 2. another 39 (1.8%) were also excluded because their afeqt questionnaires were not filled either at baseline (n=11) or 6 months (n=28 : 2 died, 12 were lost, and 14 failed to complete the afeqt questionnaire at 6 months). of the remaining patients, 43 (2.0%) were excluded because of other exclusion criteria (4 second rfa during 6-month follow - up, 21 previous ablation, and 18 switched from non - rfa to rfa during 6-month follow - up). of 900 eligible patients, 34 (1.6%) were excluded because they did not meet the requirement that at least 50% of responses of each domain should be completed. no significant difference was noted in baseline characteristics between eligible and ineligible patients, except that eligible patients were older (table s1). to report this observational study transparently, a selection flow diagram consistent with the best - practice guidelines (strobe statement) for cohort studies is listed in figure1. for current analysis, 866 subjects with chads2 scores of 0 and 1 were included with 144 (mean age : 64.469.79 years ; 59.14% male) in the rfa group and 722 (mean age : 61.2012.63 years ; 63.19% male) in the non - rfa group. the baseline characteristics of the 2 groups were summarized and many of them were different before adjustment (table1, left column). from this sample, 74 patients in the rfa group and 148 in the non - rfa group were selected based on a 1:2 matching within a prespecified caliper width. characteristics of the final propensity - matched cohorts (rfa vs. non - rfa) are also listed in table1, as shown in the right column. after matching, it seemed that the characteristics were well balanced, with the only significant difference in the proportions of af type (paroxysmal af : 70.3% in the rfa group vs. 55.4% in the non - rfa group ; p=0.03). afeqt indicates atrial fibrillation effect on quality of life ; non - rfa, nonradiofrequency ablation ; rfa, radiofrequency ablation. the first 3 domains and the global score of the baseline afeqt questionnaire between groups were different before matching (table2, left), whereas differences disappeared after matching (table2, right and figure2a). when domains and the global score at 6 months were compared between the 2 groups (figure2a), the statistically significant differences were noted in the symptoms domain (83.0712.37 units in the rfa group vs. 77.6817.14 units in the non - rfa group ; p=0.008) and the treatment satisfaction domain (76.3414.92 units in the rfa group vs. 70.3816.81 units in the non - rfa group ; p=0.01). differences of qol at 6 months were small (daily activities : 0.2, treatment concern : 0.2) or moderate (symptoms : 0.4, global score : 0.35 and treatment satisfaction : 0.4) in between - group analyses using cohen effect sizes. afeqt indicates atrial fibrillation effect on quality of life ; da, daily activities ; gs, global score ; non - rfa, nonradiofrequency ablation ; rfa, radiofrequency ablation ; tc, treatment concern ; ts, treatment satisfaction. a, domains and the global score of afeqt questionnaire at baseline and 6 months in rfa and non - rfa groups. p<0.001 vs baseline ; p<0.05 rfa versus non - rfa group at 6 months. b, changes of domains and the global score of afeqt questionnaire from baseline to 6 months. afeqt indicates atrial fibrillation effect on quality of life ; da, daily activities ; gs, global score ; non - rfa, nonradiofrequency ablation ; rfa, radiofrequency ablation ; tc, treatment concern ; ts, treatment satisfaction. when scores between baseline and 6 months of each domain and the global score were compared, statistically significant within - group changes were observed in each domain and the global score in both groups (all p<0.001), except for the treatment satisfaction domain in the non - rfa group (p=0.10 ; figure2a). using the cohen effect size to evaluate clinical importance, mean changes of treatment satisfaction in the non - rfa group was 0.3 (moderate) whereas the other domains and the global score were more than 0.8 (large), which were considered to be clinically meaningful. the differences between the rfa and non - rfa groups in score changes from baseline to 6 months are presented in figure2b. however, no significant difference was noted, except for the treatment satisfaction domain (13.1728.33 units in the rfa group vs. 2.9221.81 units in the non - rfa group ; p=0.007). the cohen effect sizes of differences in symptoms change and treatment satisfaction change were 0.3 and 0.4, respectively, which can be considered as moderate effect size. qol changes in 644 patients (rfa : 70 and non - rfa : 574) excluded after propensity matching are presented in figure s1. results of the health status of the primary endpoint for subgroup analysis are shown in table3. in subgroup analysis classified by af type, the global score at 6 months was higher in the rfa group compared to the non - rfa group in paroxysmal af (79.3110.89 units in the rfa group vs. 72.4015.74 units in the non - rfa group ; p=0.003) whereas no significant difference was found in nonparoxysmal af from the matched cohort. in patients stratified by age strata of 65 years, no significant differences were noted in the global score of the afeqt questionnaire between the rfa and non - rfa groups among the 127 af patients with age < 65 years. nonetheless, the global scores of the afeqt questionnaire achieved a significantly higher improvement after rfa in 95 patients with age 65 years (79.2911.16 units in the rfa group vs. 67.9616.04 units in the non - rfa group ; p<0.001). detailed subgroup analyses of the primary outcome in the domains of afeqt can be seen in table s2. health status of primary outcome in subgroup analysis af indicates atrial fibrillation ; non - paf, nonparoxysmal atrial fibrillation ; non - rfa, nonradiofrequency ablation ; paf, paroxysmal atrial fibrillation ; rfa, radiofrequency ablation. cohorts of patients with low baseline cha2ds2-vasc score (ie, cha2ds2-vasc 1) were also constructed based on 1:2 propensity score matching. of the 105 af patients with low cha2ds2-vasc score, 44 were from the rfa group and 61 were from the non - rfa group. no significant differences were observed in any domain and the global score of afeqt questionnaire at 6 months between the 2 groups (see table s3). during 6-month follow - up, af recurrence rate was 28.3% (21 of 74), with 9 paroxysmal af and 12 nonparoxysmal af. treatment failure in the non - rfa group was 48.6% (72 of 148), with occurrence of af more than twice in 31 paroxysmal af and failing to restore sinus rhythm in 41 nonparoxysmal af. detailed changes in domains and the global score of the afeqt questionnaire of patients with and without af recurrence in both groups are listed in figure s2. stroke occurred in 1 (0.96%) patient in the rfa group, 3 in the rfa group, and 2 in the non - rfa group were reported to have minor bleedings, respectively. no complications of clinical importance associated with rfa and medicines used were reported (see changes of medicine use in the non - rfa group in figure s3). during 2011 to 2013, 2178 patients with af were enrolled in the cafr from 4 tertiary hospitals. a total of 1196 (54.9%) of them were excluded for their chads2 score 2. another 39 (1.8%) were also excluded because their afeqt questionnaires were not filled either at baseline (n=11) or 6 months (n=28 : 2 died, 12 were lost, and 14 failed to complete the afeqt questionnaire at 6 months). of the remaining patients, 43 (2.0%) were excluded because of other exclusion criteria (4 second rfa during 6-month follow - up, 21 previous ablation, and 18 switched from non - rfa to rfa during 6-month follow - up). of 900 eligible patients, 34 (1.6%) were excluded because they did not meet the requirement that at least 50% of responses of each domain should be completed. no significant difference was noted in baseline characteristics between eligible and ineligible patients, except that eligible patients were older (table s1). to report this observational study transparently, a selection flow diagram consistent with the best - practice guidelines (strobe statement) for cohort studies is listed in figure1. for current analysis, 866 subjects with chads2 scores of 0 and 1 were included with 144 (mean age : 64.469.79 years ; 59.14% male) in the rfa group and 722 (mean age : 61.2012.63 years ; 63.19% male) in the non - rfa group. the baseline characteristics of the 2 groups were summarized and many of them were different before adjustment (table1, left column). from this sample, 74 patients in the rfa group and 148 in the non - rfa group were selected based on a 1:2 matching within a prespecified caliper width. characteristics of the final propensity - matched cohorts (rfa vs. non - rfa) are also listed in table1, as shown in the right column. after matching, it seemed that the characteristics were well balanced, with the only significant difference in the proportions of af type (paroxysmal af : 70.3% in the rfa group vs. 55.4% in the non - rfa group ; p=0.03). afeqt indicates atrial fibrillation effect on quality of life ; non - rfa, nonradiofrequency ablation ; rfa, radiofrequency ablation. the first 3 domains and the global score of the baseline afeqt questionnaire between groups were different before matching (table2, left), whereas differences disappeared after matching (table2, right and figure2a). when domains and the global score at 6 months were compared between the 2 groups (figure2a), the statistically significant differences were noted in the symptoms domain (83.0712.37 units in the rfa group vs. 77.6817.14 units in the non - rfa group ; p=0.008) and the treatment satisfaction domain (76.3414.92 units in the rfa group vs. 70.3816.81 units in the non - rfa group ; p=0.01). differences of qol at 6 months were small (daily activities : 0.2, treatment concern : 0.2) or moderate (symptoms : 0.4, global score : 0.35 and treatment satisfaction : 0.4) in between - group analyses using cohen effect sizes. afeqt indicates atrial fibrillation effect on quality of life ; da, daily activities ; gs, global score ; non - rfa, nonradiofrequency ablation ; rfa, radiofrequency ablation ; tc, treatment concern ; ts, treatment satisfaction. a, domains and the global score of afeqt questionnaire at baseline and 6 months in rfa and non - rfa groups. p<0.001 vs baseline ; p<0.05 rfa versus non - rfa group at 6 months. b, changes of domains and the global score of afeqt questionnaire from baseline to 6 months. afeqt indicates atrial fibrillation effect on quality of life ; da, daily activities ; gs, global score ; non - rfa, nonradiofrequency ablation ; rfa, radiofrequency ablation ; tc, treatment concern ; ts, treatment satisfaction. when scores between baseline and 6 months of each domain and the global score were compared, statistically significant within - group changes were observed in each domain and the global score in both groups (all p<0.001), except for the treatment satisfaction domain in the non - rfa group (p=0.10 ; figure2a). using the cohen effect size to evaluate clinical importance, mean changes of treatment satisfaction in the non - rfa group was 0.3 (moderate) whereas the other domains and the global score were more than 0.8 (large), which were considered to be clinically meaningful. the differences between the rfa and non - rfa groups in score changes from baseline to 6 months are presented in figure2b. however, no significant difference was noted, except for the treatment satisfaction domain (13.1728.33 units in the rfa group vs. 2.9221.81 units in the non - rfa group ; p=0.007). the cohen effect sizes of differences in symptoms change and treatment satisfaction change were 0.3 and 0.4, respectively, which can be considered as moderate effect size. qol changes in 644 patients (rfa : 70 and non - rfa : 574) excluded after propensity matching are presented in figure s1. results of the health status of the primary endpoint for subgroup analysis are shown in table3. in subgroup analysis classified by af type, the global score at 6 months was higher in the rfa group compared to the non - rfa group in paroxysmal af (79.3110.89 units in the rfa group vs. 72.4015.74 units in the non - rfa group ; p=0.003) whereas no significant difference was found in nonparoxysmal af from the matched cohort. in patients stratified by age strata of 65 years, no significant differences were noted in the global score of the afeqt questionnaire between the rfa and non - rfa groups among the 127 af patients with age < 65 years. nonetheless, the global scores of the afeqt questionnaire achieved a significantly higher improvement after rfa in 95 patients with age 65 years (79.2911.16 units in the rfa group vs. 67.9616.04 units in the non - rfa group ; p<0.001). detailed subgroup analyses of the primary outcome in the domains of afeqt can be seen in table s2. health status of primary outcome in subgroup analysis af indicates atrial fibrillation ; non - paf, nonparoxysmal atrial fibrillation ; non - rfa, nonradiofrequency ablation ; paf, paroxysmal atrial fibrillation ; rfa, radiofrequency ablation. cohorts of patients with low baseline cha2ds2-vasc score (ie, cha2ds2-vasc 1) were also constructed based on 1:2 propensity score matching. of the 105 af patients with low cha2ds2-vasc score, 44 were from the rfa group and 61 were from the non - rfa group. no significant differences were observed in any domain and the global score of afeqt questionnaire at 6 months between the 2 groups (see table s3). during 6-month follow - up, af recurrence rate was 28.3% (21 of 74), with 9 paroxysmal af and 12 nonparoxysmal af. treatment failure in the non - rfa group was 48.6% (72 of 148), with occurrence of af more than twice in 31 paroxysmal af and failing to restore sinus rhythm in 41 nonparoxysmal af. detailed changes in domains and the global score of the afeqt questionnaire of patients with and without af recurrence in both groups are listed in figure s2. stroke occurred in 1 (0.96%) patient in the rfa group, 3 in the rfa group, and 2 in the non - rfa group were reported to have minor bleedings, respectively. no complications of clinical importance associated with rfa and medicines used were reported (see changes of medicine use in the non - rfa group in figure s3). in the present study, 6-month qol in af patients with low stroke risk after rfa versus non - rfa were retrospectively evaluated using the afeqt questionnaire based on data from a multicenter registry. surprisingly, no significant superiorities of qol were noted when rfa was compared to non - rfa treatment at 6 months from the propensity - matched cohorts, except for domains of symptoms and treatment satisfaction. changes of health status in the rfa group were more than 19 units, which were considered to be clinically meaningful.18 however, their score changes in the non - rfa group were near the cut - off point of 19 units. although qol improvement was observed in patients post - rfa, a similar degree of improvement could also be observed in patients from the non - rfa group. first, significant qol increase within groups may be the results of increasing frequency of regular visits to the physicians and receiving more clinical care from cardiologists after recruitment. given that medical resources might be relatively limited in china, a country with a huge population, patients were often restricted from seeking for professional cardiologists directions before they were enrolled in the registry. in our study, patients both in the rfa and non - rfa groups had similar experiences with respect to convenient access to af clinics. the second possible reason was that complications and adverse effects associated with the procedure and medicines were uncommon owing to a relatively short follow - up period. rfa treatment is perceived to be effective in improving qol in the study population, but it seems to have only small or moderate superiorities to non - rfa treatment according to the ratings of differences in 6-month qol assessed by cohen effect size. a recently published randomized study19 also suggested a negative impact of ablation using the euroqol - five dimensions (eq-5d), with similar results with our study. no significant difference in 1-year qol improvement was noted between patients receiving ablation treatment and antiarrhythmic drugs, although a lower rate of recurrent atrial tachyarrhythmias was achieved in the ablation group. however, in previous studies using short form 36 score or eq-5d comparing symptomatic20 or paroxysmal10,21 af patients receiving rfa versus antiarrhythmic drugs, a higher rate of successful rhythm control was achieved and qol improved and was maintained much better after ablation during follow - up. the varying range of qol improvements may reflect differences in study design, patient selection, baseline qol impairment, questionnaires used for qol measures, procedural techniques, and period of follow - up. the results from our study mainly reflect qol outcome regardless of af type after a single rfa, different from some,10,19 but not all,20,21 previous trials. another possible reason, at least as a partial explanation for the similar qol at 6 months, was that most previous studies comparing qol in af patients treated with rfa were not conducted based on analysis stratified by low and high stroke risk.10,1922 chads2 and cha2ds2-vasc scores are widely used for evaluating risk stratification of stroke in nonvalvular af patients1 and the former is more popular in china.23 concerns of stroke in low - risk patients are much weaker before rfa, but may increase owing to an at least 3-month anticoagulation period after the procedure,5 which can lead to psychological pressure that offsets the minimal benefits of rfa from rhythm control.14 though differences of health status at 6 months were small, a significant difference in 6-month symptoms was demonstrated to be in favor of patients treated with rfa. the influence of symptoms relief and treatment effect on qol were investigated in several ablation studies.19,24 studies conducted by savelieva.25,26 found that symptoms and qol in af patients were not necessarily correlated because they were partially overlapped. symptomatic af may become asymptomatic af after rfa treatment, but the risk of stroke was not reduced accordingly. additionally, positive results concerning significant differences in symptoms domain should be interpreted with caution owing to risk of false - positive results originating from multiple comparisons. patients who moved from the non - rfa group to the rfa group had to be excluded in order to maintain the consistency of the length of 6-month follow - up, though these patients might have high symptoms load. although propensity matching was performed to minimize or avoid confoundings and ensure the balance of the baseline characteristics between the 2 groups, the existence of residual bias could not be neglected because of other unmeasured characteristics. subgroup analyses stratified by af type were further performed to adjust the imbalance of af type, which still existed after propensity score matching. rfa treatment was found to be significantly superior to non - rfa with respect to health status in paroxysmal af patients. the results that higher qol improvement was observed in older patients after rfa, compared to non - rfa, based on subgroup analyses stratified by age provided evidence for the assumption that patients eligible in the study would more likely benefit from rfa treatment than ineligible patients because they are relatively older. nonetheless, subgroups in this study were formed after propensity matching and baseline measures made it very difficult to determine whether a statistically significant improvement was the result of af type, age category, or other factors. no significant improvement of qol in the rfa group was noted when based on the more efficient cha2ds2-vasc score stroke stratification. the result of higher treatment satisfaction at 6 months in the rfa group was speculated to be partially attributable to a placebo effect of rfa, which was assumed to be a defect of the study similar to previous studies.22 af patients were instructed to fill in the afeqt questionnaires by trained nurses under conditions that patients completely understood the questions. the main differences of between - group settings in this study were whether patients were treated with rfa. there were still no significant superiorities in qol improvement compared to the non - rfa group, though several factors in favor of af patients with rfa treatment were adopted. first, a short - term follow - up of 6 months was considered to be beyond the duration of the anticoagulation period5 and with an acceptable af recurrence rate (28.38%).27 second, exclusion of af recurrent patients receiving a second rfa during the 6-month follow - up were probably in favor of af patients in the rfa group19 because they usually had symptoms leading to impairment of well - being after a single rfa. third, the fact that a higher proportion of patients in paroxysmal af after propensity matching is favorable for the rfa group according to the results of the subgroup analysis in paroxysmal af, in keeping with previous studies,19,20,28 given that patients with paroxysmal af often have larger qol improvement. nonetheless, it is hard to draw conclusions because no sufficient evidence was obtained for lacking an efficiently constructed placebo model for control of rfa. in spite of 6-month improvement of qol post - rfa, the absence of a statistically significant difference between rfa and non - rfa treatment in health status were to be taken seriously before making a clinical decision whether af patients with low stroke risk should be treated with rfa. we suggested that our study provided a basis for prospective, randomized design evaluating outcomes on qol in low - stroke - risk patients by rfa versus non - rfa. in this retrospective, observational study of a propensity - matched cohort, improvement of qol was observed from baseline to 6 months both in rfa and non - rfa groups using the afeqt questionnaire. health status at 6 months in patients treated with rfa was small - to - moderately superior to those without. it is discreetly supposed that rfa has not lived up to the high expectations for improvement of qol in af patients with low stroke risk during a 6-month follow - up. this study was supported by grants (2013bai09b02 and 2013dfb30310) from the ministry of science and technology of the people s republic of china and grants (d111100003011004 and d131100002313001) from beijing municipal commission of science and technology and beijing municipal administration of hospitals clinical medicine development of special funding support (code : zylx201302). the construction of cafr was also granted by bristol - myers squibb (bms), pfizer, johnson & johnson, boehringer ingelheim (bi), and bayer. ma has received honoraria from bristol - myers squibb (bms), pfizer, johnson & johnson, boehringer ingelheim (bi), and bayer for giving lectures. appendix s1. atrial fibrillation effect on quality - of - life (afeqt) questionnaire. table s1. domains and the global score changes in rfa and non - rfa group with and without af recurrence during the 6-month follow - up. | backgroundimpacts of a single radiofrequency ablation (rfa) on quality of life (qol) were not well investigated in atrial fibrillation (af) patients with low stroke risk.methods and resultsnine hundred af patients with low chads2 score (ie, chads2 1) who completed both a baseline and 6-month atrial fibrillation effect on quality - of - life (afeqt) questionnaire were selected from the chinese atrial fibrillation registry between 2011 and 2013. a final cohort of 222 patients was constructed after a propensity score matching with 74 in the rfa group and 148 in the non - rfa group. domains of afeqt were balanced at baseline between the 2 groups. no statistically significant differences were noted in qol (all p>0.05) when afeqt at 6 months was compared between groups, except for the symptoms domain (83.0712.37 units in the rfa group vs. 77.6817.14 units in the non - rfa group ; p=0.008) and treatment satisfaction domain (76.3414.92 units in the rfa group vs. 70.3816.81 units in the non - rfa group ; p=0.01). within - group changes in all domains and the global score of the questionnaire were moderate to large, whereas between - group comparisons in baseline to 6-month changes and qol at 6 months were small to moderate according to cohen effect sizes.conclusionsqol was balanced at baseline and improved at 6 months in both groups from this observational propensity - matched cohort based on the afeqt questionnaire. however, rfa treatment was only associated with small - to - moderate superiorities over non - rfa treatment. the role of rfa in qol improvement among af patients with low stroke risk requires further research. |
takeuchi.1 explained that the transverse ligament becomes degenerated and hypertrophic because of chronic mechanical stress by atlantoaxial subluxation. then, a part of the ligament develops reactive granulation tissue with a small vessel formation. finally, rupture of these small vessels cause repeated episodes of microbleeding, resulting in formation of a cyst. direct excision of the cysts and fixation has commonly been performed for the surgical treatment of retro - odontoid cystic mass associated with chronic atlantoaxial subluxation.12345 however, when the cyst is located in the ventral side of spinal cord, direct resection is technically dangerous. here, we present a case of a retro - odontoid cyst associated with chronic atlantoaxial subluxation and located in the ventral side of spinal cord. the cystic mass disappeared after posterior c1 and partial c2 laminectomy and c1-c2 pedicle screws fixation without resection of the retro - odontoid cyst. a 64 year old woman reported experiencing a sudden onset of neck pain, hand and foot paresthesia when she woke early in the morning. she was sent to a local hospital, where she was treated conservatively with external stabilization of the neck and bed rest. at 2 weeks later x - ray showed that the atlantoaxial instability [figure 1a - c ]. computed tomography revealed an abnormality of the odontoid process [figure 1d and e ]. magnetic resonance imaging detected an oval retro - odontoid cystic mass, which compressed the spinal cord. the mass showed uniform low intensity on t1-weighted image [figure 1f ] and uniform high signal intensity on t2-weighted image [figure 1 g ]. (a - c) preoperative open - mouth anteroposterior radiograph and dynamic flexion and extension radiograph showing the atlantoaxial instability. (d and e) preoperative computed tomography revealed an abnormality of the odontoid process. (g) the mass showed uniform high signal intensity on t2-weighted image the surgery was carried out with the patient positioned prone with the head slightly flexed and a midline incision was made. subsequently, paraspinal muscles were peeled to expose the bilateral laminae of c1 and c2. the laminectomy of c1 and partial c2 were performed, and then the neck was slightly extended to reduce the atlanto - axial joint. finally, an autogenous iliac crest graft was implanted in to c1 and c2 bilateral joints to facilitate fusion. x - ray [figure 2a and b ], computed tomography [figure 2c ] and magnetic resonance imaging [figure 2d ] were taken on the day of surgery. at 24 months after surgery, the retro - odontoid cystic mass was no longer present [figure 2e ], and the patient remained symptom free and returned to independent daily living. magnetic resonance imaging scans at 24 months after surgery showed that atlantodental interval was wider, compared to the day of surgery. (a and b) postoperative open mouth anteroposterior radiograph and lateral radiograph showing the rigid fixation between c1-c2 and atlantoaxial joint had been reset. (d) postoperative sagittal t2-weighted magnetic resonance image showed that the mass still compressed the spinal cord. (e) sagittal t2-weighted magnetic resonance image taken 24 months after surgery showed that the mass was no longer present an atlantoaxial subluxation associated with a non tumorous lesion in the posterior region of the odontoid process had been described as a retro - odontoid cyst or retro - odontoid pseudotumor in the initial report by sze.6 it usually occurs in the elderly, without clear association to any antecedent trauma. most patients are between 60- and 80-years - old, with both sexes being equally affected. all of these patients are seronegative for rheumatoid arthritis or other inflammatory arthropathies.7 however, the etiology of the cysts remains controversial. in the postulated view on its pathomechanism, preexisting atlantoaxial instability was presumed to cause repeated tear and subsequent hypertrophy of the transverse ligament, thus leading to the formation of the pseudotumor.8 takeuchi.1 explained that the transverse ligament of axis became degenerated and hypertrophic because of chronic mechanical stress by atlantoaxial subluxation. then, a part of the ligament developed reactive granulation tissue with small vessel formation. finally, rupture of these small vessels caused repeated episodes of microbleeding, resulting in formation of a cyst. optimal treatment for a retro - odontoid cystic mass associated with chronic atlantoaxial subluxation has not been established.. transoral or anterolateral decompression is theoretically the optimal option.2345 takeuchi.1 reported a case that they incised the dura to reach the mass and resect through the posterior approach. however, the anterior and posterior surgical excision involve a lot of complex technical problems and requires more operating time and blood loses, higher risks and complications and should be performed only by an experienced surgical team. it is also reported that posterior fixation of such degenerative tissue would reduce repetitive mechanical stimulation, thereby inhibiting stress on the spinal cord and reducing the inflammation which had produced the degenerative lesions.910 similar to degenerative cysts, retro - odontoid hypertrophy of soft tissue mass in a non rheumatoid patient with atlantoaxial subluxation has been reported to decrease after posterior fixation.1112131415 nevertheless, the surgical procedure is appropriate for no neurologic symptoms and the small cystic mass. for patients with large cystic mass leading to severe canal stenosis, the atlanto - axial joint reduction is prone to trauma to the spinal cord, which might cause spinal damage or even paraplegia. we selected posterior c1 and partial c2 laminectomy and c1-c2 pedicle screw fixation without resection of the retro - odontoid cyst in the patient because the cyst was located in the ventral side of spinal cord and the neurological deficit was less obvious. the posterior arch of the atlas and partial laminae of c2 were resected, providing the available space for the spinal cord drift. as far as possible, avoid damaging the nervous system. c1-c2 pedicle screws fixation might provide immediate and long term stability of the cervical spine, and is helpful in preventing an increase in the size and also to shrink the cyst. during the 24 months followup period, the cyst disappeared completely, and the patient remained symptom free and returned to independent daily living. these findings suggest that posterior laminectomy and fixation without resection of the retro - odontoid cyst is relatively simple and safe, and the results are satisfactory when the large cyst was located in the ventral side of spinal cord and the neurological deficit was less obvious. however, if the neurological deficit is progressive and severe, then resection of the retro - odontoid cysts should be selected because it could take time for the pseudotumor to disappear. | retro - odontoid cysts associated with chronic atlantoaxial subluxation are extremely rare. this article describes a case of retro - odontoid cystic mass associated with chronic atlantoaxial subluxation and its management with posterior c1 and partial c2 laminectomy and c1-c2 pedicle screw fixation without resection of the retro - odontoid cyst. a 64-year - old woman experienced a sudden onset of neck pain, hand and foot paresthesia. atlantoaxial instability associated with a retro - odontoid cystic mass was found in the imaging. the patient underwent posterior c1 and partial c2 laminectomy and c1-c2 pedicle screws fixation without resection of the retro - odontoid cyst. during the 24 months followup period, the cyst disappeared completely and the patient remained symptom free and returned to independent daily living. these findings suggest that posterior laminectomy and fixation without resection of the retro - odontoid cyst is relatively simple and safe and the results are satisfactory. |
the residual or resistant clubfoot represents one of the most difficult treatment challenges for the pediatric orthopaedist. often residual deformity is not obvious at the time of initial correction. with continued growth of the child, the persistent or undercorrected deformity becomes more apparent (refer to case seen in fig. d). the resistant clubfoot must be carefully assessed, taking each deformity into account to plan appropriate treatment. dorsal rotatory subluxation of the navicular leading to residual cavovarus foot deformity is a common finding in a resistant postoperative clubfoot. previous series have reported incidences varying from 7.1% (12 of 168 feet), to 34.9% (22 of 63 feet), to 54.2% (13 of 24 feet) of previously operated feet [2, 4, 6 ]. barnett described the presentation of this deformity as a bimalleolar axis of less than 80, internal foot progression angle, curved lateral border of the foot, shallow sinus tarsi, equinus, and rotation of the medial column of the forefoot. this residual cavovarus can lead to several clinical problems including painful forefoot deformity, difficulty with shoe wear due to painful dorsal prominence, abnormal gait, and poor cosmesis. as a result, dorsal rotatory subluxation of the navicular is associated with a high rate of revision surgery. surgical correction of symptomatic dorsal rotatory subluxation of the navicular is challenging. both our own experience and that reported by others has shown repeat conventional soft tissue release results in persistence of the subluxation. this retrospective review was performed for the following purposes : (1) to describe the outcomes of talonavicular arthrodesis with respect to symptomatic relief of dorsal rotatory subluxation of the navicular, (2) to assess correction of radiographic deformity, and (3) to identify associated complications. we retrospectively reviewed the medical records of all 13 patients (13 feet) who underwent talonavicular arthrodesis to treat symptomatic dorsal rotatory subluxation of the navicular in resistant clubfoot between 1999 and 2007. symptoms necessitating surgical treatment included pain, difficulty with shoe wear, gait abnormality, and unacceptable cosmesis. the average age at the time of surgery was 11 6 years (range, 6 6 years to 17 6 years). the remaining patient had underlying spastic quadriplegia and developed a clubfoot deformity after an iatrogenic nerve injury prior to presentation at our institution. all children were treated with previous complete posteromedial clubfoot release, and one patient had a previous attempt at talonavicular fusion performed by another surgeon. the minimum followup after surgery was 6 months (average, 35.5 months ; range, 6 to 93 months). the surgical technique began with an incision made on the medial aspect of the foot beginning approximately 1 cm below the medial malleolus and extending past the tuberosity of the navicular toward the first metatarsal. the posterior tibial tendon was identified in the proximal aspect of the wound and traced distally to its insertion on the beak of the navicular. the talonavicular joint capsule was opened widely to expose the articular surfaces of the talar head and proximal navicular. the soft tissues and joint capsule were elevated from the navicular in order to allow reduction onto the talus. any scar tissue limiting this reduction dorsally occasionally the naviculocuneiform joint was opened as well to facilitate accurate reduction of the navicular. next all cartilage was denuded from the talar head and from the articular surface of the navicular using a rongeur and curettes. one must preserve the subchondral bone to prevent excessive shortening of the medial column but also thoroughly remove all articular cartilage in order to obtain the desired fusion. after realigning the navicular in a plantar direction onto the talus, it was held in a reduced position using kirschner wires placed in a distal to proximal fashion into the body of the talus. adequacy of reduction was verified using intraoperative imaging to confirm restoration of a normal lateral talo - first metatarsal angle. compression and fixation were achieved using one or two 4-mm or 4.5-mm partly threaded cannulated cancellous screws. osteotomies of the cuboid or calcaneus were occasionally necessary to facilitate the reduction and improve the position of the foot. the arthrodesis was performed with one screw in seven cases, two screws in four cases, and two kirschner wires in two cases. in most cases utilizing one screw, additional procedures performed in the same setting included sliding calcaneal osteotomy (five cases), closing - wedge cuboid osteotomy (three cases), cuneiform osteotomy (one case), first metatarsal osteotomy (two cases), vulpius - type tendo - achilles lengthening (three cases), phalangeal osteotomy (two cases), individual toe flexor tenotomies (five cases), anterior tibial tendon transfer (two cases), distal tibia / fibula varus osteotomy (one case), calcaneocuboid fusion (three cases), and naviculocuneiform fusion (one case). the wound was closed in layers and a well - molded short leg cast was placed, which was split and spread for swelling. after discharge patients were seen for followup at 1 week for cast overwrap. they were maintained non - weight bearing in a short - leg cast for 4 weeks. at that point if sufficient healing was observed, patients were then placed back into a short - leg walking cast and allowed to progressively weight bear as tolerated in the cast for an additional 4 weeks. from the medical records we recorded previous treatment for clubfoot, age at time of talonavicular arthrodesis, details of surgical procedure including any simultaneously performed procedures, complications, and need for further treatment. one of us (vts) not involved in the treatment assessed the percent subluxation of the navicular on the lateral radiograph, calculated as the vertical dorsal displacement of the superior pole of the navicular above the line connecting the dorsal aspect of the head of the talus and the medial cuneiform divided by the total vertical length of the navicular. the lateral talo - first metatarsal angle and ap talo - first metatarsal angle were also assessed. postoperative radiographs were also assessed for adequacy of bony fusion mass as demonstrated by lack of a visible joint space between the talus and navicular with presence of spanning bone as well as preservation of postoperative correction. one patient, a college baseball player, complained of lateral foot pain we attributed to calcaneofibular impingement. three patients had returned to sports ; the records contained no notes of other limitations from the foot and no difficulty with shoe wear. the average preoperative percent subluxation of the navicular was 42% (range, 5%76%). the lateral talo - first metatarsal angle improved from an average of 18 (range, 540) preoperatively to 8 (range, 016). the ap talo - first metatarsal angle changed only minimally, from a preoperative average of 16 (range, 346) to postoperative average of 14 (range, 039) (fig. b(a) preoperative standing lateral demonstrating dorsal rotatory subluxation of the navicular on the head of the talus. (b) postoperative standing lateral radiograph demonstrating talonavicular arthrodesis with restoration of calcaneal pitch. (a) preoperative standing lateral demonstrating dorsal rotatory subluxation of the navicular on the head of the talus. (b) postoperative standing lateral radiograph demonstrating talonavicular arthrodesis with restoration of calcaneal pitch. one patient initially treated by another surgeon with attempted talonavicular arthrodesis developed nonunion of the talonavicular joint. the patient developed a pseudarthrosis and then underwent a third fusion using autogenous iliac crest bone graft. all of the other operated feet went on to achieve bony fusion uneventfully, and no other patients developed complications. patients develop a cavovarus foot deformity and have pain, abnormal gait, and difficulty with shoe wear. we therefore reviewed our patients with persistent dorsal rotatory subluxation of the navicular treated with talonavicular arthrodesis. we described the results of arthrodesis in terms of symptomatic relief, radiographic correction, and associated complications. this study is limited by a small number of patients and relatively short followup period. however, we present the series in order to highlight the difficulties associated with treatment of persistent navicular subluxation and propose a treatment method which has been successful in the short - term. longer - term followup will be necessary to fully assess the results of talonavicular fusion. dorsal rotatory subluxation of the navicular is a common cause of progressive cavovarus foot deformity after posteromedial release for clubfoot (fig. the clinical examination of a patient with dorsal rotatory subluxation demonstrates cavovarus of the forefoot with plantarflexion of the metatarsal heads. this represents breakdown through chopart s joint, which may be related to a stiffened subtalar joint resulting from the initial operative intervention. when examining these patients, it is important to be aware that the heel may be in either varus or valgus depending on whether the foot has been previously over- or undercorrected. as a result, a sliding calcaneal osteotomy may be necessary in addition to treatment for the subluxated navicular in order to fully correct the foot position.fig. 2a dserial radiographs of a patient included in this series demonstrating progressive nature of cavovarus deformity and subluxation of the navicular. (a) standing lateral radiograph of the patient at 3 years of age. (b) standing lateral radiograph of the patient at 6 years of age. (c) standing lateral radiograph of the patient at 10 years of age. again noted is increased subluxation of the navicular, increase in talo - first metatarsal angle, and progressive loss of calcaneal pitch. (d) standing lateral radiograph of the patient 1 year after talonavicular arthrodesis. serial radiographs of a patient included in this series demonstrating progressive nature of cavovarus deformity and subluxation of the navicular. (a) standing lateral radiograph of the patient at 3 years of age. (b) standing lateral radiograph of the patient at 6 years of age. (c) standing lateral radiograph of the patient at 10 years of age. again noted is increased subluxation of the navicular, increase in talo - first metatarsal angle, and progressive loss of calcaneal pitch. (d) standing lateral radiograph of the patient 1 year after talonavicular arthrodesis. results from excessive dorsally directed forces at the talonavicular joint in patients with no dorsiflexion at the ankle secondary to a flattop talus. state that release of the spring ligament with inaccurate pinning may be a contributing factor and stress the importance of direct visualization during the pinning process. in agreement, davidson stresses that since the navicular is not yet ossified at the time of initial clubfoot release, the surgeon can not rely on intraoperative radiographs and should instead directly visualize the talonavicular joint during pinning. he suggests holding the foot in mild equinus during pinning to prevent unrecognized dorsal subluxation of the navicular. kuo and jansen performed an anatomic study in order to better delineate the nature of the deformity. they noted superior rotation of the medial end of the navicular together with an intact naviculocuneiform joint caused an increased cavus due to increased height of the medial arch. in addition, they proposed the increased varus and supination of the forefoot was caused by shortening and rotation of the medial arch while the normal lateral arch was maintained. they also suggested the unreleased lateral structure of the navicular was a tethering factor after extensive medial release and the ball - and - socket shape of the talonavicular joint facilitated a rotatory displacement. although they observed no association between type of treatment of the posterior tibial tendon and development of dorsal rotatory subluxation, the authors suggested an unopposed anterior tibial tendon may pull the forefoot into supination and force rotation of the navicular. kuo and jansen believed correction required a complete midtarsal joint release with derotation of the navicular and pin fixation, however they did not report on any case examples or provide any followup data. we have also attempted to treat dorsal rotatory subluxation of the navicular with comprehensive soft tissue releases without success. similarly, barnett described poor results with attempts at correction by repeat soft tissue release and reduction resulting in persistence of subluxation. schlafly. described a failed attempt to perform open reduction of a dorsally subluxated navicular in a previously treated clubfoot. they could not achieve reduction and subsequently state, corrective surgery for postoperative dorsal navicular subluxation is not recommended. third street operation in conjunction with a complete peritalar release as a proposed treatment for dorsal subluxation of the navicular. he suggested release was required to free the medial column of the foot from the lateral column in order to reduce the navicular. this procedure involves complete capsulotomy between the cuboid and navicular, cuboid and third cuneiform, and between the base of the third and fourth metatarsals. however this was described simply as a potential new technique and no report on results was given. wei. previously reported 15 patients with either dorsolateral subluxation of the talonavicular joint, triangular navicular, and/or degenerative changes of the talonavicular joint who underwent 17 talonavicular arthrodeses. one patient was partially satisfied due to development of naviculocuneiform arthritis 3 years after surgery. fusion was achieved using either kirschner wires (13 cases), screws (three cases), or staples (three cases). they stated that talonavicular fusion led to correction of the residual midfoot deformity citing improvement in talo - first metatarsal angle and the ap talo - first metatarsal angle. the operation we describe provided a reliable method for achieving a symptom - free result with a single surgery. similar to wei., we noted an improvement in talo - first metatarsal angle as a result of the arthrodesis. 1a b). also, the percent subluxation of the navicular decreased from 41.8% to 6% after fusion. this decrease in subluxation corrects the position of the foot, removes first ray pressure, and allows ease of shoe wear and improved cosmesis. at final followup, one of the patients initially treated by another surgeon developed nonunion ; however, in the 12 patients initially operated on by the senior authors, there were no incidences of nonunion. the authors recommend placing two screws as was performed towards the end of the study period in order to achieve better compression, prevent rotation, and decrease the risk for nonunion. what is the effect of motion now occurring through the navicular - cuneiform joints rather than talonavicular ? we have encountered two patients with congenital talonavicular fusion who have excellent foot position, good motion, and no symptoms. whether this will hold true for dorsal rotatory subluxation of the navicular as sequelae of previously treated clubfoot can lead to symptomatic cavovarus deformity of the foot. talonavicular arthrodesis is an option for correcting the underlying cavovarus deformity associated with dorsal rotatory subluxation of the navicular and alleviating the accompanying symptoms of the plantar flexed first ray in a single surgical setting. | dorsal rotatory subluxation of the navicular, a common sequela of resistant surgically treated clubfeet, presents a challenging treatment problem. this subluxation typically progresses after posteromedial release. patients develop a cavovarus foot deformity and complain of pain, gait problems, and difficulty with shoe wear. previous attempts at soft tissue release and reduction have been largely unsuccessful. we reviewed 13 patients with dorsal rotatory subluxation of the navicular treated with talonavicular arthrodesis. the minimum followup after surgery was 6 months (average, 36 months ; range, 6 to 93 months). at last followup 12 of 13 patients were symptom - free. the mean preoperative subluxation of 42% was reduced to a mean of 6% at last followup. we noted improvement in the talo - first metatarsal angle from an average of 18 preoperatively to 8 postoperatively. one patient treated by another surgeon with attempted talonavicular arthrodesis developed a nonunion ; we observed no other complications. we believe talonavicular arthrodesis a reasonable option to correct the deformity and symptoms associated with dorsal rotatory subluxation of the navicular in a single surgical setting with a low complication rate.level of evidence : level iv, therapeutic study. see guidelines for authors for a complete description of levels of evidence. |
herpes zoster (hz) is an infectious disease caused by reactivation of latent varicella zoster infection. this common viral disease afflicts between 20% and 30% of the population at some point in their lifetime ; up to 50% of individuals over 80 years of age are affected.1,2 the dermatological symptoms of hz are typically preceded by prodromal symptoms that range from a tingling or burning sensation to a sharp stabbing pain. this is followed by a raised, reddish rash and vesicular lesions, typically over the region of skin affected by prodromal symptoms, 2 to 3 days later. this rash typically affects singular dermatomes resulting in a unilateral, stripe - like appearance that is diagnostic of hz. lesions continue to erupt for several days before the rash crusts over, generally within 7 to 10 days. symptoms then begin to subside and total resolution generally occurs within 3 to 4 weeks.3 while hz is generally self - limiting, longer lasting complications can occur. phn is defined as pain that persists after the resolution of the dermatological symptoms of hz. it is caused by damage to sensory neurons sustained during the initial active infection with pain persisting even after the virus has returned to its dormant state.4 there is no single standard definition of phn. pain persisting at 30 to 120 days measured from onset of hz or from healing of rash is used in the literature.57 pain persisting at 90 days from initial hz diagnosis is the most commonly used definition.57 the incidence of hz is estimated at 3.5 to 7 cases per 1,000 person - years (py), with the incidence in females slightly higher than in males.8,9 the incidence of hz increases steadily with age, rising to 67/1,000 py by 60 years, and continuing to increase thereafter.2,10,11 the rate of conversion to phn has been estimated at anywhere from 5% to 60% of hz cases.7,11,12 this wide range is partially explained by the lack of standard diagnostic criteria. studies that used pain persisting at 90 days to define phn have a more conservative conversion rate with a narrower range of between 5% and 15%.79,1113 phn is a challenging condition to treat, and the effectiveness of pharmacological therapy is limited. the canadian pain society consensus statement on the treatment of neuropathic pain recommends as first - line therapy for phn one of the tricyclic antidepressants (tcas), gabapentin, or pregabalin.14 opioids and topical lidocaine are listed as second - line alternatives. the european federation of neurological societies 2010 treatment guidelines for neuropathic pain largely echo these recommendations. opioids, while effective, are not recommended as first - line therapy due to side effects, development of tolerance, and risk of dependency.15 a variety of other drugs are also discussed in the literature, including those used for other forms of neuropathy, although their efficacy for phn is disputed. these include valproic acid / divalproex sodium, duloxetine, venlafaxine, carbamazepine, tramadol, nonsteroidal anti - inflammatory drugs (nsaids), and selective serotonin reuptake inhibitors.6,1418 while treatment of phn remains difficult, it is possible to reduce the risk of hz, and with it phn, with the hz vaccine (zostavax ii, merck & co., inc., kenilworth, nj, usa). this vaccine has been shown to reduce the relative risk of hz by 51% and of phn by almost 67%. vaccines may decrease the chance of developing hz, but hz and the associated phn are expected to continue to result in a significant burden to society in the near future. to determine the incidence of hz and rates of phn, a retrospective cohort study was conducted in manitoba, canada, over a period of 17 years. the objectives were to determine the burden of disease of zoster - related pain and to understand the major cost drivers in the changing cost of treating hz. a population - based cohort study was conducted in manitoba (canada) using administrative health care data from manitoba s universal public health care system gathered from april 1, 1995 to march 31, 2014 in the course of providing routine medical care. these data were accessed via the manitoba centre for health policy population health research data repository, a collection of databases containing records of contacts of manitoba residents with the health care system.19 databases utilized included the drug program information network, which processes all community pharmacy - based prescriptions for insurance coverage and drug utilization review ; the medical services database which contains records of all fee - for - service medical provider claims ; hospital discharge abstracts containing separations data for all hospitalizations ; and the manitoba health registry which contains basic demographic information on all persons registered with manitoba health, healthy living and seniors. all records are de - identified but contain a unique scrambled personal health number allowing for the cross - linkage of records over time and across data sets. individuals with one or more international classification of diseases-9 (icd-9) codes starting with 053 or icd-10-clinical modification (icd-10-cm) codes starting with b02 were considered as hz cases, the date of the first being the episode start date. the use of icd diagnostic codes to diagnose zoster was both highly selective (positive predictive value 93%) and sensitive (97.5%).20 multiple episodes per individual were allowed if 2 years had elapsed between episodes. to ensure only incident episodes were included in the analysis, a 2-year washout period was used ; thus, any episodes identified prior to 1997/98 were excluded. episodes where individuals were under 20 years at diagnosis were excluded to avoid misclassification of varicella zoster cases as hz. cases of phn were identified by hz pain drug treatment or medical claims with hz icd codes appearing past 90 days from diagnosis. prescriptions for opioids (anatomical therapeutic chemical codes starting with n02a), nsaids (m01), anticonvulsants (n03a), antidepressants (n06a), nabilone (a04ad), local anesthetics (d04a), glucocorticoids (h02ab), and acetylsalicylic acid and acetaminophen (n02b) associated with hz episodes identified in the previous step were collected. those dispensed between 90 days pre - hz diagnoses and 2 years postdiagnosis were evaluated against several criteria to assess if they were related to hz the use of each drug class was categorized as either incident or prevalent for that episode by evaluating prescriptions in the 90 days preceding diagnosis. a drug class was categorized as prevalent if a person received a total of 30 days supply or more within those 90 days, and all within - class prescriptions were excluded from the analysis for that episode. from the remaining incident class, all prescriptions from diagnosis to 1) 90 days postdiagnosis or 2) date of last hz medical claim were classified as hz phn treatment. a grace period of 100% of the prescription duration was added to its duration to create an end date. to be considered a continuous treatment, there should be no gaps between the end date of previous prescriptions and receipt of the next. discontinuity signaled the end of drug treatment and of that episode ; all later prescriptions were removed from further analysis of this episode. data were summarized by manitoba health fiscal years, which run from april 1 to march 31 for each year. incidence rates for hz were calculated for 1997/98 through to 2013/14, and age adjusted using the population of manitoba in 1997 as our standard. as 2 years follow - up was required to capture all data related to episodes of hz with phn, only episodes diagnosed from 1997/98 to 2011/12 contributed to the phn analysis. regression analysis was conducted on rates of conversion from hz to phn across this interval. drug costs were calculated directly from prescription records, included cost of both drug and dispensing fee, and were adjusted to 2013 canadian dollars using statistics canada s consumer price index for prescription drug costs in manitoba. annual total prescription costs were tabulated for all hz episodes, as well as within hz episodes that converted to phn (hz phn). prescription costs per episode were calculated, and t - tests were conducted to determine the significance of differences between mean costs in 1997/98 and 2011/12. the relationship between age at diagnosis and mean treatment cost was examined by regression analysis. prescription data were stratified by drug class for hz phn episodes, looking at anticonvulsants, antidepressants, nsaids, and opioids. regressions on the number of class - wise treated episodes were used to examine trends over time. approvals were granted by the university of manitoba health research ethics board and the manitoba health information privacy committee. individual written consent from patients was not required by the ethics board or the privacy committee for this retrospective analysis using de - identified data. between april 1, 1997 and march 31, 2014, there were 73,893 episodes of hz diagnosed in manitoba resulting in a mean of 4,347 (95% confidence interval [ci ] 4,067, 4,626) episodes per year. there were 5,749 episodes of hz diagnosed in 2013/14, a 50% increase from the 3,844 episodes diagnosed in 1997/98. the age - standardized incidence rate of hz increased by 21%, from 4.70 episodes/1,000 py in 1997/98 to 5.70 episodes/1,000 py in 2013/14. total prescription costs for treating pain resulting from hz and phn are shown in table 1. the annual cost of prescription drugs used to treat all hz - related pain rose by over 174%, from cad $ 121,438 in 1997/98 to $ 332,981 in 2011/12. phn accounted for a mean of 82.8% (95% ci 81.2%, 84.3%) of drug costs. this percentage did not change significantly over the course of the study period (p=0.57, r=0.026). from 1997/98 through to 2011/12, the period for which full phn results could be obtained, there were 6,038 episodes of hz that met the study criteria for phn, corresponding to a conversion rate of 9.6%. a linear regression analysis on conversion rates revealed no significant trend over this period of time (p=0.45, r=0.046). however, the increase in the total number of hz episodes resulted in a greater number of phn cases over time, rising from 331 in 1997/98 to 497 in 2011/12. the mean cost of treating hz - related pain increases linearly with age at diagnosis, each additional year increasing the mean episode cost by $ 1.04 (p<0.0001, r=0.65) (figure 1). the mean cost for persons aged 20 to 29 years was $ 24.15 (95% ci $ 19.95, $ 28.34), while for persons aged 60 to 69 years the cost was 143% higher at $ 58.72 (95% ci $ 52.92, $ 64.53). the average cost of treating an episode of hz, and separately those episodes with phn, increased over the study period (table 1). the mean cost per episodes to treat all hz - related pain rose significantly by 111% (t=5.369, p<0.0001) from 1997/98 to 2011/12, and by 94% for hz. changes in the treatment of hz phn over time were examined by linear regression (figure 2). only anticonvulsant use changed significantly with a 212% increase in the number of anticonvulsant - treated episodes from 1997/98 to 2011/12 (p<0.0001, r=0.92). the duration of treatment for hz phn varied widely between drug classes (figure 3). the median days supplied was highest for antidepressants (178 days) and anticonvulsants (134 days), with shorter durations for nsaids (50 days) and opioids (28 days). the median duration of anticonvulsant treatment increased from 55 to 141 days between 1997/98 and 2011/12. there was much less change observed in the median treatment duration of other drug classes, with antidepressants increasing from 161 to 190 days, opioids remaining relatively stable with an increase from 26.5 to 27 days, and nsaids decreasing from 38.5 to 30 days. the overall annual cost of opioid, nsaid, and antidepressant prescriptions underwent a moderate increase with antidepressants increasing by 91%, opioids by 67%, and nsaids by 35% (table 2). however, these changes were small compared to the increase in anticonvulsant use, which underwent a 755% rise in class cost, increasing from $ 15,520 to $ 132,685. this increase accounts for 56% of the total increase in prescription spending for the treatment of all hz - associated pain. the most frequently prescribed anticonvulsant drug for hz phn also changed over time. in 1997, carbamazepine prescriptions accounted for 62% (260 prescriptions) of all anticonvulsant prescriptions, while gabapentin accounted for only 21% (89 prescriptions). in contrast, in 2011, only 3.2% (80 prescriptions) of all anticonvulsant prescriptions were for carbamazepine. gabapentin accounted for 73% (1,798) of prescriptions, while pregabalin, which was not available in 1997, accounted for 9.2% (226). the economic burden of treating hz - associated pain, and phn in particular, increased significantly from 1997/98 to 2011/12 (table 1). this increase is the combined result of increases in both the incidence of diagnosed hz and in the mean episode cost of prescription drug treatment of pain. a dramatic increase in gabapentin was found to be a primary driver of mean episode cost. over the 17-year period between 1997/98 and 2013/14, there has been a sustained increase in the incidence of hz, with a resulting increase in the number of phn cases. changes in size and increasing age21 of the provincial population explain the majority of this increase. however, after taking both these factors into account, a significant increase of 21% in the age - adjusted incidence per thousand people was observed. as the rate of conversion from hz to phn did not change over the study period, the increased number of hz episodes resulted in an increase in the number of phn cases. this increase in phn cases is an important factor in determining the economic burden of hz, as these cases were responsible for over 80% of hz - related analgesic prescription costs. not only were there more hz and phn cases, there was also a significant increase in the mean cost per episode related to the treatment of pain. this higher episode cost was driven by the increased use of anticonvulsant drugs, primarily gabapentin. while the number of users of anticonvulsants rose, the use of other classes of drugs remained relatively unchanged (table 2). not only did the number of persons using anticonvulsant drugs increased, the median duration of treatment increased with them as well. from an evidence - based perspective, this change might seem surprising. there is limited evidence demonstrating superior efficacy and involves much greater cost compared to the other first - line therapies such as tcas.22 in fact, one systematic review estimated the number needed to treat (nnt) for a 50% reduction of pain for gabapentin to be 4.39 (95% ci 3.34, 6.07), while that for tcas was 2.64 (95% ci 2.1, 3.54).23 gabapentin was introduced to the canadian market in december 1994 when it was approved for the adjunctive treatment of partial seizures.24 it was aggressively marketed to physicians for a number of nonapproved indications, including phn, by parke davis. this promotion eventually led to a us federal lawsuit against the company for off - label marketing in 1996.25 it was not until 2004 that the us food and drug administration (fda) approved gabapentin for phn,25 although it has never received this indication in canada. some of this promotion suggested that tca use for phn was inappropriate in older patients from a safety perspective.22 however, gabapentin is not without its own adverse effects, and a systematic review suggests the number needed to harm (nnh) for major harms is actually similar for gabapentin (nnh=12.25, 95% ci 7.69, 30.2) and tcas (nnh=16.9, 95% ci 8.85, 178).23 concerns have been raised about the use of opioids in noncancer pain.26 these include the risk of dependency, the development of tolerance, and the potential for abuse, and are part of the rationale for listing opioids as second - line phn treatment options.15,27 however, opioids were used frequently to treat hz while the high rate of use may be concerning, the median of duration of opioid treatment (figure 3) reveals generally short - term use, possibly reflecting appropriate opioid stewardship. population level data from a universal single - payer health care system ensures virtually all contacts of residents with this health care system, and all prescription dispensations are captured in the study. these data sets also allow us to directly measure health system costs rather than inferring results from survey data or sampling, as it has been done in many previous studies.12,2830 there are also several limitations to this study. we were unable to measure the true incidence of hz in the population as only persons who sought medical treatment appeared in the administrative data. however, previous studies have reported that up to 95%99% of persons with hz will seek medical attention.8,28 second, hz diagnostic codes or prescription treatment past 90 days postdiagnosis were used as a proxy for a phn diagnosis as we only had access to the first three digits of icd codes in our data. thus, rates reported are of hz cases requiring ongoing medical management, sometimes referred to as clinically significant phn. this methodology is common in administrative data studies looking at phn.8 finally, the use of nonprescription medications, including low - dose ibuprofen and naproxen, acetaminophen, acetylsalicylic acid, and topical treatments, is not captured. the total costs of treating pain associated with hz and phn rose by 174% between 1997/98 and 2011/12. the first is the increasing number of hz cases. while some of this can be attributed to an aging population the second component is the increasing average cost of treating an episode of hz phn driven by the use of higher cost anticonvulsants for longer periods of time. management of the economic burden of hz phn analgesic treatment should include the use of evidence - based cost - effective therapies and measures such as vaccination that can help reduce the incidence of hz. | backgroundpain is a main symptom of herpes zoster (hz), and postherpetic neuralgia (phn) is a frequent complication occurring in 5% to 15% of cases, causing moderate to severe neuropathic pain. a population - based observational study was conducted to evaluate the treatment patterns and economic burden of prescription drug treatment of hz and phn pain in the province of manitoba (canada) over a period of 15 years.methodsadministrative health care data, including medical and hospital separation records, were examined to identify episodes of hz using international classification of diseases-9/10 codes between april 1, 1997 and march 31, 2014. episodes of phn were identified using medical and prescription claims. incident use of analgesic, antidepressant, or anticonvulsant drugs was used to determine prescription pain costs.resultsthe age - adjusted incidence of hz increased from 4.7 episodes/1,000 person - years in 1997/98 to 5.7/1,000 person - years in 2013/14. phn occurred in 9.2% of hz cases, a rate that did not change over the study period (p=0.57). the annual cost to treat hz pain rose by 174% from 1997/98, reaching cad $ 332,981 in 2011/12, 82.8% (95% confidence interval [ci ] 81.2%, 84.3%) of which was related to phn. the per episode cost of hz rose by 111% from $ 31.59 (95% ci $ 25.35, $ 37.84) to $ 66.81 (95% ci $ 56.84, $ 76.78) and by 94% for phn from $ 292 (95% ci $ 225, $ 358) to $ 566 (95% ci $ 478, $ 655). these increases were driven by increasing use of anticonvulsants, primarily gabapentin, which accounted for 57% of the increase in cost.conclusionthere has been an increase in the incidence of hz and phn and in the average cost associated with the prescription treatment of their resultant neuropathic pain. the primary driver of the increased episodic cost is the increased use of gabapentin. these changes have resulted in a substantial increase in the economic burden associated with hz and phn. |
an important goal of visual system studies in animals is to understand vision in humans. in past decades, emphasis has been placed on studying carnivores and nonhuman primates based on the belief that advanced species resemble humans in both behavior and brain organization more closely than less advanced species, such as rodents, whose visual system has been widely assumed to be much simpler by comparison. however, numerous studies in rats and mice have convincingly shown that the occipital cortex surrounding primary visual cortex (v1, striate cortex) is significantly more elaborate than previously thought, consisting of about a dozen of interconnected, topographically organized extrastriate visual areas [115 ]. this knowledge, together with the availability of genetic and molecular tools, especially in mice, has triggered a surge of studies using rodents as models for advancing our understanding of cortical visual processing in mammals. indeed, recent advances include evidence that visual areas in rats [12, 16 ] and mice [15, 1722 ] are functionally specialized and organized into processing streams that resemble the temporal and parietal parallel processing streams of primates and that progressive functional transformations along these pathways conform to general hierarchical principles. while these and other studies have also pointed out differences between rodents, carnivores, and primates [16, 22, 23 ], they nevertheless highlight the usefulness of rodents as models in mammalian visual research. however, the small size of the brains in rats and especially mice presents some challenges, such as greater difficulty in locating and recording from small cortical areas. experiments benefiting from, or requiring larger brains, such as studies using stimulating / recording microelectrode arrays to explore the potential for chronic recordings in visual cortex, or for restoring vision [25, 26 ], have often used rabbits, a lagomorph whose brain is lissencephalic as in rats and mice but about 6 and 30 times larger than the brain of rats and mice, respectively. rabbits have also been used in a variety of other investigations involving the visual cortex, including cross - modal and mri and fmri studies, but at present limited information about the organization of visual cortex beyond v1 is available in this species [2935 ]. studies of the distribution of extrastriate fields labeled following single anatomical tracer injections into rabbit v1 reported that the arrangement of extrastriate areas connected with v1 closely resembles the arrangement of visual areas in rats and mice [36, 37 ] and suggested that the rodent visual cortical plan may be more general, encompassing species within the lagomorphs and perhaps other orders [11, 36 ]. whereas these previous anatomical studies identified extrastriate areas connected with v1, they were unable to reveal their internal topography due to the use of only single v1 tracer injections. to address this issue, in a group of albino rabbits we injected multiple distinguishable tracers into different regions of v1 in the same animal and analyzed the topography of resulting extrastriate labeled fields with reference to the patterns of callosal connections and myeloarchitecture revealed in tangential sections of the flattened cortex, an approach that has been fruitful for delineating the location and topography of extrastriate visual areas in several species [6, 7, 38 ]. we also revealed the patterns of callosal connections in a group of dutch belted rabbits to investigate possible differences in the overall organization of this interhemispheric pathway between albino and pigmented strains of rabbits. surgery was performed in 5 albino and 5 adult dutch belted rabbits (weighing approximately 20002500 g) anesthetized with ketamine hydrochloride (33 mg / kg, i m) and xylazine hydrochloride (5 mg / kg, i m), supplemented with atropine sulfate (0.05 mg / kg sc). procedures followed protocols approved by the institutional animal care and use committees. to reveal the distribution of callosal connections in the right hemisphere, total volumes of about 9.010.0 l of a solution of horseradish peroxidase (hrp, sigma type vi, st. louis, mo ; 30% in saline) were injected into about 5060 sites across occipital cortex in the left hemisphere. to reveal striate - extrastriate connections, small volumes (0.020.1 l) of several additional tracers were injected at different loci in striate cortex of the right hemisphere (approx. these tracers included the anterograde tracer h - proline (25 ci/l solution in saline, l-[2,3-h ] proline, specific activity 40.0 ci / mmol, new england nuclear, usa) and up to three retrogradely transported fluorescent tracers (rhodamine beads, rb, green beads, gb, lumafluor, naples, fl, usa, concentrated stock solution, and fast blue, fb, sigma co, st louis. the approximate area of effective tracer uptake for restricted injections was estimated as described previously. all tracers were pressure - injected through glass micropipettes (50100 m tip diameter) using brief pressure pulses. after 35-day survival, animals were deeply anesthetized with pentobarbital sodium (100 mg / kg ip) and perfused through the heart with 0.9% saline followed by 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4). the right cortical mantle was resected, flattened between glass slides, and sectioned tangentially (60 m thick sections) as described previously. the rest of the brain received additional fixation and was cut into 60 m thick coronal sections. the transport of h - proline was revealed in sections processed for autoradiography, with an exposure time of 6 weeks at 4c, while hrp labeling was revealed using tetramethylbenzidine as the chromogen. digital images of the myelin- and hrp - labeling patterns were obtained by scanning photographs of the histological sections at 2400 dpi using an epson 4990 scanner. the distribution of cells labeled with fluorescent tracers was acquired with a custom, computer - assisted microscope system. tangential sections throughout the depth of the cortex were examined to ensure that injections analyzed were restricted to grey matter. the left hemispheres were extensively infiltrated with hrp and the ipsilateral visual thalamic nuclei were uniformly and densely labeled with reaction product (figure 1(a)), indicating that hrp was effectively transported from the injected cortex. in the right hemispheres, the locations of the injection sites into v1 [29, 35 ] were confirmed by analyzing the distribution of labeled fields within the ipsilateral dorsal lateral geniculate nucleus of the thalamus (dlgn) (figure 1(b)). using adobe photoshop cs2 (adobe systems), digitized images of anatomical tracers and myelin labeling patterns from the same animal were carefully aligned with each other using the border of v1, the edges of the sections, blood vessels, and other fiducial marks. cells labeled by the injections of rb, gb, and fb were represented by red, green, and blue dots, respectively, and overall labeling patterns were reconstructed from 3 - 4 sections. h - proline labeling was visualized under dark field microscopy and represented by outlines of densely labeled regions. callosal connections were illustrated either by photographic images, by outlines of the areas containing dense accumulations of hrp labeling, or by thresholded versions prepared after first applying a median filter to reduce noise, followed by a high - pass filter to remove gradual changes in staining density across the entire image. the results were carefully inspected to confirm that these versions accurately represented the labeling pattern observed in the sections. figures were prepared using adobe photoshop cs2, and all imaging processing used, including contrast enhancement and intensity level adjustments, was applied to the entire images. heavily myelinated areas are observed in occipital, temporal, and parietal regions (figure 2(a)). figure 2(b) striate cortex (area v1) appears as an oval region of dense myelination with sharp and smooth borders (figure 2(a)). subdivided v1 into a lateral region (oc2), which is binocular [30, 35 ], and a medial monocular region (oc1), but the border between these subdivisions is not as apparent in the myelin pattern (figure 2) as it is in other species (e.g., squirrel). area oc3.1, immediately lateral to v1, includes the physiologically defined area v2. area oc3.1 is less densely myelinated than v1 but more myelinated than neighboring temporal areas te2.1 and te2.2. in parietal cortex, heavily myelinated regions include poc1 (the barrel field), the rest of primary somatosensory cortex (s1), and poc3 (second somatosensory cortex, s2). in temporal cortex, densely myelinated regions include primary auditory cortex (te1) and a region within the most posterior and ventral portion of temporal cortex (te3) (figure 2(b)). in medial extrastriate cortex, the border between cytoarchitectonic areas oc3.2 and rsg was not obvious in myelin stained tangential section. the patterns of callosal connections were revealed in albino (figures 3(a) and 3(b)) and dutch belted rabbits (figures 3(c)3(e)). dense accumulations of retrogradely hrp - labeled cell bodies and anterogradely labeled terminations formed a band straddling the lateral border of v1. often, this band had a beaded appearance, consisting of a series of distinct patches of about 0.75 mm in diameter, separated by about 1.5 mm (arrows in figures 3(a)3(c)). in some cases, patches were present throughout most of the lateral border of v1 (figures 3(a) and 3(b)), while in other cases they were apparent only in posterior regions of v1 (figure 3(c)). at some places, pairs of patches (opposing arrows in figure 3(b)) were separated by a narrow region of low labeling density centered at the lateral border of striate cortex. the callosal band at the lateral border of v1 was consistently narrow posteriorly (0.71.0 mm in width), but it often tended to increase in width in more anterior regions of v1, reaching about 2.5 mm at its widest region in some cases (figure 3(c)). it is unlikely that the patchiness and anteroposterior difference in width are due to incomplete infiltration of hrp because hrp labeling in the ipsilateral dlgn was uniformly dense throughout the nucleus (figure 1(a)). in extrastriate cortex callosal connections formed patches and bands, and in some cases callosal bands partially or completely encircled areas devoid of callosal connections in lateral extrastriate cortex (figure 3). the appearance and location of several of these callosal features were consistent across rabbits and proved useful when comparing patterns of striate - extrastriate connections from different animals. no major differences between albino and pigmented rabbits were observed in the distribution of callosal connections (figure 3). striate - extrastriate connections were studied only in albino rabbits (n = 5). tracer injections into v1 were placed in regions representing upper and lower visual fields, as well as nasal and temporal fields (figure 4). labeled fields of different sizes were observed in lateral extrastriate cortex. the largest fields were typically arranged anteroposteriorly forming a first tier consisting of 4 - 5 fields located immediately lateral to v1, in area oc3.1, while 2 - 3 smaller fields formed a second tier distributed more laterally in oc3.1 and in neighboring temporal areas te2.1, te2.2, and te3. in medial extrastriate cortex, within area 29d, labeled cells usually formed an elongated field. we identified putative retinotopically - organized areas based on the analysis of local, systematic displacements of labeled fields in response to displacements of injection sites in v1. as in previous studies in the rabbit [36, 37 ], we have tentatively adopted the nomenclature established in the rat, in which visual areas are named according to their location relative to v1 [1, 6 ]. the case shown in figures 5(a) and 5(b) illustrates the distribution of extrastriate labeled fields resulting from injection sequences oriented along either the mediolateral or anteroposterior axes in v1. from these data we tentatively identified several visual areas in lateral extrastriate, which are summarized in figure 5(c). figure 5(a) illustrates that a mediolateral sequence injections across the width of v1 (from temporal to nasal representations in the lower visual field) result in a mirror image distribution of labeled fields in area oc3.1. the fb- and gb - labeled fields are elongated anteroposteriorly, occupying portions of areas that we identify as areas al (anterolateral) and lm (lateromedial) by their locations relative to v1 and the callosal pattern (figure 5(c)). the largest field of tritiated proline is restricted to area lm, while the two more lateral smaller fields of tritiated proline suggest the existence of additional areas lateral to lm. this animal also received an injection of rb more posteriorly in v1 (figure 5(b)). figure 5(b) shows a sparse field of rb - labeled cells in anterior portions of area al and a robust elongated field in posterior portions of putative area lm, whose position was more posterior than the fields produced by the other 3 anterior injections. further posteriorly, a callosal band extends across areas te2.2 and te3 in a lateroposterior direction separating two additional rb - labeled fields and partially overlapping with a fb - labeled field. the location and topography of the areas separated by this callosal band suggest that they correspond to areas pl (posterolateral) and p (posterior) described in the rat and mice [6, 7 ] (figures 5(b) and 5(c)). the field of fb - labeled cells overlapping the callosal band is assumed to straddle the border between pl and p (figures 5(b) and 5(c)). note that in area al, the fields of fb- and gb - labeled cells occupy more posterior portions than the field of rb - labeled cells, and the reverse is true in lm ; namely, fb- and gb - labeled cells occupy anterior regions of lm while rb - labeled cells occupy posterior regions of this area. as a result, while a region free of rb - labeled cells separates the fields of rb - labeled cells in al and lm, the fields of fb- and gb - labeled cells appear to form continuous fields extending from anterior lm to posterior al. in area pl, the distribution of rb- and fb - labeled cells reverses with respect to the distribution in area lm ; that is, fb - labeled cells are posterior, while rb - labeled cell are anterior. thus, as the injection site in v1 moves from anterior to posterior, the labeled fields move anteriorly in al and pl and posteriorly in lm, suggesting that, as in the rat, the anteroposterior axis in v1 (from lower to upper visual field representations) maps along the same direction in lm but in the reverse direction in areas al and pl. the anteroposterior map in pl appears to reverse again in area p. the rb - labeled field in lm extends further laterally at both its anterior and posterior ends forming two tongues. the posterior tongue is long, extending into areas te2.1 and te2.2 (field labeled li, laterointermediate, figures 5(b) and 5(c)), while the anterior tongue is short with only a few rb - labeled cells seen beyond the lateral border of area oc3.1 (field labeled lla, laterolateral anterior, figures 5(b) and 5(c)). note that both of these tongues of rb labeling are located between patches of callosal connections and overlap with the two small lateral fields of tritiated proline, providing support to the idea that these two regions may correspond to two separate visual areas. finally, labeling in medial area 29d suggests a mirror image representation of the mediolateral axis in v1, but rb labeling was not strong enough in this region to map the anteroposterior axis in v1. figure 6 provides further evidence that a mediolateral injection sequence (gb - fb - rb) produces a mirror image sequence of labeled fields (rb - fb - gb) in lm. moreover, this sequence reverses again (gb - fb - rb) as fb - labeled cells and rb - labeled cells are found immediately lateral to the gb - labeled field (indicated by an arrow). it appears to correspond to the area occupied by both the posterior isotope - labeled field and the posterior tongue - like rb - labeled field described in figure 5(b). together, these data provide further evidence that this region may correspond to area li in the rat, in which the mediolateral map reverses with respect to that in lm. thus, in figure 6, the gb - labeled field indicated by the arrow (temporal visual field representation) would mark both the lateral border of lm and the medial border of li. more posteriorly, figure 6 shows separate labeled fields that may correspond to areas pl and p described above. while distribution of rb - and fb - labeled cells in lp suggests a mirror image of the corresponding injection sites in v1, the topography in p is not clear. immediately lateral and posterior to lp, in area te3, we tentatively identify area tp (temporal posterior area, see below). labeling could not be assigned with certainty to area al. in medial extrastriate cortex, a narrow field of labeling was oriented anteroposteriorly, but its topography was not apparent. additional data on the mapping of the anteroposterior axis in v1 are presented in figures 7 and 8. following an injection of fb in a region of v1 representing lower visual fields, two dense, slightly separated fb - labeled projection fields were observed lateral to v1, in regions likely corresponding to areas al and lm (figure 7(a)). comparing these data with data from an injection of rb placed more posteriorly, in regions representing upper fields (figure 7(b)), shows that the distribution of rb - labeled cells is more extensive, occupying more anterior regions in al as well as more posterior regions in lm. these displacements in opposite direction are consistent with data in figure 5 suggesting that the anteroposterior map in v1 has the same orientation in lm but is inverted in al. a small field of fb- and rb - labeled cells is observed lateral to the larger rb- and fb - labeled fields associated with areas al and lm. this small field appears to be in a region corresponding to area lla, and the fact that fb - labeled cells are located more anterior than the rb - labeled cells suggests that the anteroposterior axis in v1 is mapped along the same orientation in area lla. further posteriorly, this case shows labeled fields resembling those seen in figures 5 and 6, in regions that appear to correspond to areas li, pl, p, and tp. anterior to al, there was sparse fb and rb labeling in a narrow region delimited laterally by a callosal band. this region may correspond to area rl (rostrolateral, figure 7(b)) described in the rat [12, 47 ] and mouse. labeling similar to that described in figures 5 and 6 was observed in medial extrastriate cortex. figure 8(a) correlates the pattern of callosal connections (outlined in yellow) and the labeled fields resulting from injections of tritiated proline (black outlines), rb, and fb with the underlying myeloarchitecture. the injection of tritiated proline in anterior v1 (lower visual fields) produced a circular labeled field in area oc3.1, which likely straddles the border between areas al and lm. a smaller labeled field is located immediately lateral, in an area we tentatively identify as area lla (figure 8(b)). another isotope - labeled field was observed anteriorly in area oc3.1, in a region we identify as area rl. the injection of rb was placed more posteriorly in v1 and, consistent with the topography of areas al and lm described above, the rb - labeled fields in al and lm appear to fuse at the site of the large isotope - labeled fields, presumably at the border between al and lm, where the representations of lower visual fields in al and lm meet. however, unlike this isotope - labeled field, rb - labeled fields extended more anteriorly in al and more posteriorly in lm, occupying regions that represent higher elevations. a tongue - like rb - labeled field extended laterally between callosally labeled regions. this field overlapped with the small, lateral isotope - labeled field, supporting the existence of a small area we call lla in this region (see figures 5(b) and 7(b)). in putative area rl, the rb labeling was located further lateral than the isotope labeling, suggesting that rl is topographically organized. in posterior regions, the rb injection produced labeled fields in areas te2.2 and te3 (figure 8(b)) that resemble the labeling localized in areas pl, p, and tp in figures 5, 6, and 7. the rb- and fb - labeled fields in area tp occupy a portion of a densely myelinated area observed in te3 (figure 8(a)), and the separation between these fields suggests that area tp is topographically organized. labeling in this region was observed following injections into different v1 sites, suggesting that area tp represents a large portion of the visual field. the injection of fb was placed very close to the posterior and medial border of v1, in a region representing peripheral portions of the upper visual field. in lateral extrastriate cortex, fb - labeled cells accumulated in lateral area oc3.1, in approximately the same location occupied by the gb - labeled field in figure 6 (black arrow), and by the two more posterior isotope - labeled fields in figure 5(b). these results support the interpretation drawn from figures 5(b) and 6 that peripheral visual fields are represented at the boundary between putative areas lm and li. a small fb - labeled field was located at the posterior end of lateral extrastriate cortex, likely in area p, and a few fb - labeled cells were located in area al. in medial extrastriate cortex, elongated fb- and rb - labeled fields overlapped extensively in the anteroposterior direction, while along the mediolateral axis they were segregated, suggesting a mirror - image representation of the mediolateral axis in v1. in a final case (87 - 1) we revealed the projections from an injection of tritiated proline placed at the medial border of posterior v1, in a region roughly similar to the fb injection in figure 8. we observed a labeling pattern resembling that produced by the fb injection in figure 8 (data not shown). in both cases the distribution of labeling in lateral extrastriate cortex was rather restricted, suggesting that extreme upper peripheral visual fields are not represented in all extrastriate areas. we confirmed studies showing that callosal connections form a band that straddles the lateral border of v1 [48, 49 ]. our results extend these previous observations by showing that in lateral extrastriate cortex of both albino and pigmented rabbits callosal connections form patches and several band - like regions oriented mediolaterally at different anteroposterior levels. in some cases, a band of callosal labeling was observed in anterior portions of medial extrastriate cortex. the location of several of these callosal features was constant across animals of both strains. however, we did not typically observe ring - like callosal configurations encircling separate extrastriate cortical regions, as described in rodents and some marsupials [50, 51 ]. the callosal band at the v1/oc3.1 border often had a beaded appearance in both albino and dutch belted rabbits. the presence of callosal patches along this band is in agreement with previous reports in rabbits and squirrels. a recent study in long evans rats correlated distinct periodicities in the pattern of callosal connections in v1 with ipsilateral ocular dominance columns, but a similar correlation may not exist in rabbits and squirrels because no evidence of ocular dominance columns has been found in these species [55, 56 ]. it remains possible that callosal projection patches at the v1 border in rabbits and squirrels relate to orientation selectivity or other forms of functional segregation. relative to the width of v1, the callosal zone in v1 is narrower in rabbits than in rats [30, 58 ]. a possible explanation comes from relating the width of the binocular regions in v1 and the projections from temporal retina in both species. the binocular region in rabbit v1 [30, 59 ] is narrower than in rats and other species, reflecting the fact that the rabbit temporal retina is relatively small due to the more lateral placement of the eyes. moreover, as in the rat, the entire temporal retina of rabbits projects both ipsilaterally and contralaterally. consistent with the hypothesis that the width of the callosal zone in v1 reflects the extent of temporal retina from which crossed projections originate [6264 ], the width of the callosal zone in v1 matches the width of the binocular region in v1 in both rats and rabbits [30, 59 ]. thus, relative to the width of v1, the difference in the width of the v1 callosal zone between rabbits and rats may simply reflect the difference in the width of the binocular zone between these species. it is worth adding that both the width of the binocular region in rabbit v1 [29, 30 ] and the strength of the ipsilateral eye input to this region tend to decrease posteriorly, which may explain our observation that the width of the callosal zone in rabbit v1 tends to decrease posteriorly. our results extend previous studies and provide further evidence that extrastriate areas identified anatomically in the rabbit [36, 37 ] resemble the pattern of visual areas in the rat not only in their general location with respect to v1 but also in their internal topography. our data are also consistent with the interpretation that, as in the rat, lateral extrastriate areas connected with v1 are arranged primarily in two tiers. figures 9(a) and 9(b) show a tentative diagram of the distribution and internal topography of extrastriate visual areas derived from this study. to facilitate comparison with studies in rodents, a diagram of visual areas in the rat is illustrated in figure 9(c). the anteroposterior and mediolateral oriented arrows (figure 9(b)) summarize the displacements of the injections sites in v1 and the resulting displacements of labeled fields in some of the identified extrastriate areas. most injections into rabbit v1 labeled fields widely distributed in the areas delineated in figures 9(a) and 9(b), with the exception of very medial and posterior injections (see figure 8). the latter injections produced more restricted labeling patterns, suggesting that only some extrastriate areas contain representations of extreme upper and temporal regions of the visual field. representations that are either incomplete or biased toward particular regions of visual space have also been described in mice. in rats (figure 9(c)) and mice, areas al, lm, pl, and p form a tier located adjacent to the lateral border of v1. in these areas, the representation of the mediolateral axis in v1 (from temporal to nasal visual fields) is inverted, such that tracer injections into medial or lateral regions of v1 produce labeled fields away or close to the lateral border of v1 in lateral extrastriate cortex, respectively [117, 47 ]. the similarity extended to the representation of the anteroposterior axis in v1 (from lower to upper visual fields). as in the rat and mice, we observed that the anteroposterior axis in v1 maps along the same direction in area lm but in the reverse direction in both al and pl. our data (figure 5) also suggest that the map reverses again in p. opposite orientations of the elevation maps in lm and al are illustrated by the fact that labeled fields in lm and al originating from progressively more anterior loci in v1 (representing progressively lower visual fields) moved closer and closer together, eventually merging. as such, the most anterior v1 injection results in a single field at the putative border between al and lm (see isotope labeled field in figure 8(b)). we also identified area rl whose location in anterior lateral extrastriate cortex resembles that of area rl in rats [12, 47 ] and mice. in these rodents, area rl is often associated with a small anterior callosal ring, and in some rabbits callosal connections form a ring - like configuration in this region (figure 3). in agreement with montero, we observed that area lm is the largest area in lateral extrastriate cortex and that it is elongated in the anteroposterior direction. on the basis of its location, size, and topographic organization, we concur with montero 's suggestion that lm corresponds to an area called v2 in previous electrophysiological studies in the rabbit [30, 31, 35 ] and that it is likely homologous to visual area v2 described in primates, carnivores, and other species. in addition to the first tier of areas located immediately adjacent to the lateral border of v1, we identified a second tier of smaller areas. in one of these, area li, the mediolateral topography was a mirror image of that in lm, as in rats [6, 9, 11, 16 ] and mice [7, 14 ]. a small area called ll has been identified further laterally in rats [6, 9, 11, 16 ] and mice, in which the mediolateral topography reverses again, resembling the map in lm. while next to li we may have failed to identify an area corresponding to area ll in rats and mice, we tentatively named lla a small area we identified further anteriorly (figure 9(a)). the second tier is somewhat variable in the rat, and in some studies area lla has been identified anterior to ll [6, 47 ]. thus, it is possible that area lla in the rabbit may correspond to area lla in the rat and mouse. the densely myelinated area we observed in te3 may correspond to a heavily myelinated area, called tp (temporal posterior), described in approximately this region in the squirrel [67, 68 ] and agouti. the portion connected with v1 may correspond to a visually responsive area in rabbit temporal cortex described in previous physiological and anatomical studies [33, 34 ]. in the rat, it may correspond to a region connected with v1 described in perirhinal cortex [6, 11, 12 ] (see area pr, pararhinal, in figure 9(c)) and to the caudal temporal area, while in the mouse it may correspond to area 36p. we did not observe projections anterior to v1 that could correspond to area a (anterior) in rats and mice. likewise, we did not observe projections to a site in somatosensory cortex called s in rats and mice. medially, in area 29d, we observed connections with an elongated region that we tentatively called area m (medial, figure 9(a)). labeling from different injections usually overlapped extensively, but in some animals the anteroposterior axis in v1 was represented as in v1, while the mediolateral axis in v1 was represented as a mirror image. this region may correspond in part to area am described in the rabbit and areas pm, am, and m described in rats [6, 9, 11 ] and mice [7, 14, 15, 18 ]. more detailed experiments will be necessary to correlate labeling in medial cortex with the architectonic subdivisions recognized by fleischhauer. in this region. in addition to areas v1 and v2, a previous physiological study described three small visual areas in regions corresponding approximately to areas al and lla in the present study. further posteriorly, the same study described an additional small area in regions that may correspond to area pl or tp in this study. these findings support the notion that areas identified anatomically in the rabbit likely correspond to separate representations of the visual field, as it has been demonstrated in the rat and mice. additional studies combining electrophysiological and anatomical methods will be required to further explore the topography and interconnectivity of the areas identified in this and previous anatomical studies [36, 37 ]. our study provides further information about the location and topography of extrastriate areas connected with v1 in the rabbit and relates these areas to the patterns of callosal connections and myeloarchitecture. our results should facilitate the interpretation of additional mapping and hodological data obtained in the rabbit with electrophysiological and other techniques and contribute toward comparative studies of the organization of visual cortex in mammals. in view of the similarity that appears to exist between rabbits, rats, and mice, the rabbit offers an alternative model for further studies of the rodent visual cortical plan, especially for projects benefiting from a larger brain. | previous studies in rabbits identified an array of extrastriate cortical areas anatomically connected with v1 but did not describe their internal topography. to address this issue, we injected multiple anatomical tracers into different regions in v1 of the same animal and analyzed the topography of resulting extrastriate labeled fields with reference to the patterns of callosal connections and myeloarchitecture revealed in tangential sections of the flattened cortex. our results extend previous studies and provide further evidence that rabbit extrastriate areas resemble the visual areas in rats and mice not only in their general location with respect to v1 but also in their internal topography. moreover, extrastriate areas in the rabbit maintain a constant relationship with myeloarchitectonic borders and features of the callosal pattern. these findings highlight the rabbit as an alternative model to rats and mice for advancing our understanding of cortical visual processing in mammals, especially for projects benefiting from a larger brain. |
the surgical extraction of an impacted mandibular third molar is one of the most common procedures performed by oral and maxillofacial surgeons. most mandibular third molars are impacted in the mandibular ramus area near the second molar, and the level of difficulty of extraction is classified according to the degree of impaction, position in the mandibular ramus, and angulation of the long axis of teeth. usually, a third molar impacted far away from its original site is affected by a cyst or a tumor. only a few cases of ectopic mandibular third molar in the region of pterygomandibular space without association of cystic lesion -- such as odontogenic keratocyst and dentigerous cyst -- have been reported1,2. we report a case of mandibular third molar located in the pterygomandibular space that seems to have been displaced by neither cyst nor tumor. a 46-year - old male patient visited the department of oral and maxillofacial surgery, seoul national university dental hospital, complaining of swelling and pain in the right preauricular region. he was previously told by a general dentist at a local clinic that he had a malposed tooth in the right mandible and was advised not to have the tooth extracted until symptoms appear. a panoramic radiograph showed a third molar located high in the ascending ramus of the right mandible.(fig. 1) to identify the exact location of the tooth, computed tomography (ct) was taken, showing the third molar in the pterygomandibular space associated with a radiolucent lesion.(fig. 2) the radiolucent lesion was evaluated as cystic lesion such as odontogenic keratocyst or dentigerous cyst or secondary inflammation accompanied by soft tissue involvement. an incision was made over the right external oblique ridge and extended from the second molar to the posterosuperior mandibular ascending ramus. subperiosteal dissection was done superiorly, exposing the anterior border of the ramus from the retromolar area almost to the tip of the coronoid process. medial subperiosteal dissection proceeded posteriorly, exposing the lingula and inferior alveolar neurovascular bundle up to the condyle neck. 3) the tooth was easily removed from the area between the lingula and the sigmoid notch. 4) sharp areas were smoothened, with the site curetted and cleaned with sterile saline solution. a small follicular space enveloping the crown of the tooth was also identified, suggesting inflamed granulation tissue.(fig. 5) a connection to the periodontal space of the mandibular second molar was detected. in view of the sclerotic change of the underlying mandible (fig. 3b), we assume that there had been prolonged communication with the oral cavity. several studies have reported ectopic mandibular third molar in the mandibular ramus3,4, mandibular condyle5 - 7, and coronoid process8. an aberrant eruption pattern has been suggested to occur when the tooth has been displaced by a lesion, usually an odontogenic cyst3,10,11. dentigerous cyst is the most common benign lesion related to impacted mandibular third molar12. over time, the pressure exerted by the intracystic fluid on the occlusal aspect of the third molar may cause its displacement, sometimes from its original location3,4,13. in the present case, the mandibular third molar was not displaced by a cystic lesion but by an uncertain cause. the development of the tooth germ in an aberrant position or aberrant tooth germ eruption pattern may be the most likely etiology. otherwise, primary and total dislocation of tooth base may be the cause8. in the process of mandibular skeletal growth, bone is typically added along the posterior ramus border and resorbed along the anterior border ; mandibular condyle develops as a result of bone apposition in the posterior ramus14. in this case, the presence of dental caries implies that tooth dislocation occurred after its exposure to the oral cavity. keros and susi8 reported the ectopic mandibular third molar in the coronoid process and assumed that the bone forming the mandibular base in childhood may shift to the region beneath the coronoid process in adulthood, with the ectopic mandibular third molar embedded. following the normal growth pattern, the third molar crown moved upward, eventually reaching the coronoid process of the mandible in non - inverted state. nonetheless, the etiology of ectopic impaction and migration of tooth is still unclear. peck15 reported that the intraosseous migration of impacted mandibular tooth is related to genetic determinants. according to marks and schroeder16, regional disturbance in the dental follicle might lead to local defective osteoclastic function, with an abnormal eruption pathway being formed. sutton17 believed that an abnormally strong eruption force or a change affecting the crypt of the tooth germ might lead to erroneous eruption. when teeth are located near the mandibular condyle, the preauricular approach can be used. the approach has the advantage of good exposure of the surgical site but may result in complications such as extraoral scar, damage to temporomandibular joint, and facial nerve injury11. the intraoral approach may avoid these problems, but access to and view of the severely displaced tooth may be limited ; thus making the surgery difficult. in this case, the impacted tooth was located on the lingual side of the pterygomandibular space, and the surgery was performed using the intraoral approach. during the surgery, the inferior alveolar nerve should be protected. moreover, excessive grinding of the coronoid process or mandibular condyle should not be done to prevent fracture. nowadays, most third molar extractions are performed when the patients are in their twenties, so the dislocation becomes rarer. moreover, there may be patients with ectopic tooth without clinical symptoms, not knowing that they have a dislocated tooth. therefore, annual panoramic radiograph taking from childhood is recommended for the early detection of ectopic third molar and its pathologic changes such as cyst formation and infection. impacted teeth diagnosed upon routine radiographic examination -- and which are not associated with any pathology -- usually do not require treatment, but they should be removed to prevent cyst formation, infection, and weakening of the bone predisposing to fracture7. the surgical extraction of the ectopic third molar should be carefully planned and performed to minimize complications induced by surgery. | impacted mandibular third molars are located between the second mandibular molar and mandibular ramus. however, ectopic mandibular third molars with heterotopic positions are reported in the subcondylar or pterygomandibular space. the usual cause of malposition is a cyst or tumor, and malposition without a pathology is rare. this case report described an impacted mandibular third molar in the pterygomandibular space without any associated pathology. |
cardiovascular disease (cvd) is leading to mortality among patients who were in developed countries. in the early of 20 century, < 10% of the world 's deaths were attributed to cvd. the half of deaths in developed countries and 25% of deaths in developing countries were due to coronary vascular disease. one way to improve the cardiac patient 's ability is cardiac rehabilitation program that was begun in 1960 for the first time. the aim of this program was the secondary prevention of coronary heart disease (chd), re - admissions to hospital and deaths from cardiovascular events. cardiac rehabilitation programs are generally including interventions for the management of disease and smoking cessation, correction of nutrition status and physical activity. diet has been recognize as a risk factor in preventing and reducing the risk of heart disease., reported that food guide for outpatients with heart failure are important in two aspects, awareness about nutrition and food and modification of diet quality. in recent years, the posterior and anterior dietary patterns are used to study of the relationship between food, nutrition, and chronic diseases. one of them is dietary diversity score (dds). to assessment of dds according to us department of agriculture 's food guide pyramid, five main groups, including grains, vegetables, fruits, meat, and meat substitutes and dairy products are used, and each of these five groups is divided into several subgroups. dds was used to assessment of diet quality in several disease for instant, a cross - sectional study that was conducted in 2006 among 581 tehranian adults with hypercholesterolemia, (hypertriglyceridemia and low - density lipoprotein cholesterol were diagnosed according to atp iii guidelines of the national cholesterol education program) showed that higher values of dds was associated with lower cvd risk factors. the role of oxidative stress and obesity in the etiology and prevalence of cvds is well known. assessed the association between dietary diversity and antioxidant markers and revealed that oxidative stress was inversely associated with dietary diversity. a study that was done in 2011 showed that overweight stunted children consumed food with low dietary diversity and high dietary energy density, also in another study that was conducted among 289 healthy female students, which was selected randomly from isfahan university of medical sciences revealed that low body mass index (bmi) and waist circumference was associated with high dds, so concluded that dds was reversely correlate with abdominal adiposity and obesity among female students. dds was assessed in a different disease, but no study has evaluated dds before and after nutrition counseling among patients undergoing cardiac rehabilitation, so this study was designed to examine this purpose. this was an experimental before - after study based on data that was collected from patients admitted to the cardiac rehabilitation research center, isfahan cardiovascular research institute between 2008 and 2013. the exclusion criteria include patients who cardiac rehabilitation was contraindications for them such as unstable angina, acute phase of myocardial infarction, and unstable arrhythmia, and inclusion criteria include all patients who complete 2-month cardiac rehabilitation program and two dietary records (before and after cardiac rehabilitation). selected the samples were conducted through all people counting method, so all patients who were admitted for rehabilitation program based on inclusion and exclusion criteria were enrolled. we admitted 250 patients to participate in a cardiac rehabilitation program but among them, 53 patients were not completed cardiac rehabilitation program and 3 days dietary records, so they excluded from the study. this study is registered by registration i d irct2015090923957n1 with iranian registry of clinical trials. demographic characteristics of patients (gender, age, education, income, marital status) having risk factors of heart disease (smoking, obesity, type 2 diabetes mellitus, hypertension, dyslipidemia), having a history of chd among family members, were complete through a questionnaire form. hip circumference, weight, height, and cause of recourse to cardiac rehabilitation and admission date of the people for cardiac rehabilitation were recorded. for measurement of weight, bmi was calculated through dividing weight (kg) by the square of height (m). elastic meter was used to measurement of waist and hip circumferences with an accuracy of about 0.5 cm. these measurements were conducted two times for those who completed cardiac rehabilitation program, once before and once after the period of rehabilitation. the rehabilitation program included 24 exercise sessions that was done three times per week, programmed over 8 weeks. each session lasted for 6090 min (1020 min for warm up the bodies, 2040 min for aerobic exercise according to the severity of disease and patient risk, 10 min to cool down and 20 min to relax the bodies after each session was ended). team of cardiac rehabilitation was consisted of general practitioner, a physiotherapist, a nutritionist, and a nurse in every exercise session. cardiac rehabilitation program was started 68 weeks after coronary artery bypass graft, 4 weeks after myocardial infarction and unstable angina, and immediately after percutaneous coronary interventions and chd diagnosed by angiography. during 8 weeks of rehabilitation program some educational session conducted for patients and their family, for example, educations about cardiovascular risk factors, diagnose and treatment cvd, drugs, methods for reduced their stress, cessation smoking, and activities to improve nutrition status. three days dietary records were completed by each patient at the beginning of the treatment period. the expert dietitian was trained participants about how they completed dietary records. ask to patients to complete the dietary record in 3 days, including 2 days a week and a holiday. after completing dietary records by subjects, dietary recommendation was done according to therapeutic lifestyle change (tlc) dietary pattern and individual status. tlc dietary pattern including : (fat : 25%35% of total calorie intake, saturated fatty acid : < 7% of total calories, trans fatty acid : 0 or as low as possible, poly - unsaturated fatty acid : up to 10% of total calories, monounsaturated fatty acid : up to 20% of total calories, carbohydrate : 50%60% of total calories especially from whole grains, fruit, and vegetables, fiber : 2530 g / day, plant sterols : 2 g / day, protein : approximately, 15% of total calories, cholesterol : < 200 mg / day). the patient could ask their question during 2 months that they referred to this center for exercise training. patients who were completed two dietary records (after and before cardiac rehabilitation) were enrolled to study. all patients had the same protocol treatment and nutritional counseling which was conducted by two experienced nutritionists. all food items that were collected from dietary records were converted to gram, then serving of food items were calculated. kant. method was used for scoring dietary diversity which was intended five food groups including bread and grains, vegetables, fruits, meat, and dairy products. bread and grains subgroups included whole grain, refine grain, pasta, rice, biscuit and flour, the subgroups of vegetable consisted of fresh vegetable, potato, tomato, legumes, starch vegetable, yellow vegetable, and green vegetable. for fruit groups, we considered two subgroups included fruit and fruit juice, berry and citrus fruit. the meat subgroups were fish, sausage, egg, poultry, soy, red meat. subgroups of dairy consisted of cheese, yogurt, kashk (iranian foods which made from yogurt), and milk. finally, the total score was calculated by the sum of these numbers, so the minimum dds was zero and the maximum was 10. if persons consumed half serving of each subgroup was considered as a consumer of that subgroup. data were analyzed using the spss software, version 20 (ibm spss, tokyo, japan). some variables including subgroups of food did not follow a normal distribution according to kolmogorov smirnov test so wilcoxon test were used to compare dds score for each subgroup but distribution of total dds after and before nutrition counseling was normal according to kolmogorov smirnov test so we used pair sample t - test to compare total dds before and after nutrition counseling. this was an experimental before - after study based on data that was collected from patients admitted to the cardiac rehabilitation research center, isfahan cardiovascular research institute between 2008 and 2013. the exclusion criteria include patients who cardiac rehabilitation was contraindications for them such as unstable angina, acute phase of myocardial infarction, and unstable arrhythmia, and inclusion criteria include all patients who complete 2-month cardiac rehabilitation program and two dietary records (before and after cardiac rehabilitation). selected the samples were conducted through all people counting method, so all patients who were admitted for rehabilitation program based on inclusion and exclusion criteria were enrolled. we admitted 250 patients to participate in a cardiac rehabilitation program but among them, 53 patients were not completed cardiac rehabilitation program and 3 days dietary records, so they excluded from the study. this study is registered by registration i d irct2015090923957n1 with iranian registry of clinical trials. demographic characteristics of patients (gender, age, education, income, marital status) having risk factors of heart disease (smoking, obesity, type 2 diabetes mellitus, hypertension, dyslipidemia), having a history of chd among family members, were complete through a questionnaire form. hip circumference, weight, height, and cause of recourse to cardiac rehabilitation and admission date of the people for cardiac rehabilitation were recorded. for measurement of weight, bmi was calculated through dividing weight (kg) by the square of height (m). elastic meter was used to measurement of waist and hip circumferences with an accuracy of about 0.5 cm. these measurements were conducted two times for those who completed cardiac rehabilitation program, once before and once after the period of rehabilitation. the rehabilitation program included 24 exercise sessions that was done three times per week, programmed over 8 weeks. each session lasted for 6090 min (1020 min for warm up the bodies, 2040 min for aerobic exercise according to the severity of disease and patient risk, 10 min to cool down and 20 min to relax the bodies after each session was ended). team of cardiac rehabilitation was consisted of general practitioner, a physiotherapist, a nutritionist, and a nurse in every exercise session. cardiac rehabilitation program was started 68 weeks after coronary artery bypass graft, 4 weeks after myocardial infarction and unstable angina, and immediately after percutaneous coronary interventions and chd diagnosed by angiography. during 8 weeks of rehabilitation program some educational session conducted for patients and their family, for example, educations about cardiovascular risk factors, diagnose and treatment cvd, drugs, methods for reduced their stress, cessation smoking, and activities to improve nutrition status. three days dietary records were completed by each patient at the beginning of the treatment period. the expert dietitian was trained participants about how they completed dietary records. ask to patients to complete the dietary record in 3 days, including 2 days a week and a holiday. after completing dietary records by subjects, dietary recommendation was done according to therapeutic lifestyle change (tlc) dietary pattern and individual status. tlc dietary pattern including : (fat : 25%35% of total calorie intake, saturated fatty acid : < 7% of total calories, trans fatty acid : 0 or as low as possible, poly - unsaturated fatty acid : up to 10% of total calories, monounsaturated fatty acid : up to 20% of total calories, carbohydrate : 50%60% of total calories especially from whole grains, fruit, and vegetables, fiber : 2530 g / day, plant sterols : 2 g / day, protein : approximately, 15% of total calories, cholesterol : < 200 mg / day). the patient could ask their question during 2 months that they referred to this center for exercise training. patients who were completed two dietary records (after and before cardiac rehabilitation) were enrolled to study. all patients had the same protocol treatment and nutritional counseling which was conducted by two experienced nutritionists. all food items that were collected from dietary records were converted to gram, then serving of food items were calculated. method was used for scoring dietary diversity which was intended five food groups including bread and grains, vegetables, fruits, meat, and dairy products. bread and grains subgroups included whole grain, refine grain, pasta, rice, biscuit and flour, the subgroups of vegetable consisted of fresh vegetable, potato, tomato, legumes, starch vegetable, yellow vegetable, and green vegetable. for fruit groups, we considered two subgroups included fruit and fruit juice, berry and citrus fruit. the meat subgroups were fish, sausage, egg, poultry, soy, red meat. subgroups of dairy consisted of cheese, yogurt, kashk (iranian foods which made from yogurt), and milk. finally, the total score was calculated by the sum of these numbers, so the minimum dds was zero and the maximum was 10. if persons consumed half serving of each subgroup was considered as a consumer of that subgroup. data were analyzed using the spss software, version 20 (ibm spss, tokyo, japan). some variables including subgroups of food did not follow a normal distribution according to kolmogorov smirnov test so wilcoxon test were used to compare dds score for each subgroup but distribution of total dds after and before nutrition counseling was normal according to kolmogorov smirnov test so we used pair sample t - test to compare total dds before and after nutrition counseling. the median (interquartile), mean (standard deviation [sd ]) and p value differences between dds score before and after nutrition counseling for each five subgroups and total dds was shown in table 2. the differences between dds of grain, fruit, vegetable, and meat before and after nutrition counseling were not significant (p = 0.635, p = 0.423, p = 0.826, p = 0.207, respectively), but differences of dds for dairy before and after nutrition counseling was significant (p = 0). demographic characteristics of participants the median (interquartile), meanstandard deviation and p value differences between dietary diversity score before and after nutrition counseling for each five subgroups and total dietary diversity score the mean sd of dds before and after nutrition counseling was 3.9 0.96 and 4.2 1.13 respectively and statistically significant differences were found between two periods (after and before nutrition counseling), (p = 0.001). figure 1 shows mean sd differences between dds score for each subgroup and total dds after and before nutrition counseling. the mean standard deviation differences between dietary diversity score for each subgroups and total dietary diversity score before and after nutrition counseling this experimental before - after study observed significant differences between dietary diversity before and after nutrition consultation and found that nutrition counseling in cardiac rehabilitation program could improve dietary diversity. many factors were known as a risk factor of cvd, for example, some nutrient deficiency, metabolic syndrome, and obesity. a study that was done by azadbakht. assessed usual dietary intake of 581 healthy controls which was selected randomly from participants of tehran lipid and glucose study by semi - quantitative food frequency questioner. they calculated dds and defined metabolic syndrome according to atp iii criteria ; finally, they concluded that dds improvement was associated with reduced risk of having metabolic syndrome. the other study showed that dds was associated with nutrient sufficiency proportion of calcium and vitamin c that they had an inverse association with cvd. they selected patient randomly from isfahan university of medical sciences, iran, then calculated bmi and dds and they found the possibility of obesity decreased with increased in dds. study which was carried out on sri lankan adults concluded that lower dds was associated with higher rate of obesity, the same result was shown in a study that was conducted among us women but in the us men, the association between dds and obesity was not clear. higher dietary diversity was correlated with lower risk of cardiovascular and cancer mortality and lower rate of morbidity. in this study, we revealed that dds increased after nutrition counseling so the risk of cvd may decrease but more assessment needs to explain this conclusion. in several study, the association between nutritional counseling and improvement of health outcome were assessed, for example, study that was done among type 2 diabetics patients and cvd patients showed a significant improvement in body mass index and treatment of cvd and type 2 diabetes in patients who received nutritional counseling, also health outcomes were improved among patients with premature coronary artery disease and dyslipidemia after nutritional counseling. study which was done by rhodes. found that nutritional knowledge and body mass index were improved in patients who received medical nutrition therapy by dietitian, so the nutrition counseling have a great role on health outcomes, that was consistent with finding of this study. first, as we could not morally apply intervention for a group of subjects while the other group did not receive any intervention so the study can not be designed as a randomized clinical trial, second, the dietary record that was used in this study affected the usual intake of patients, so under - reporting might be happened. however, design of this study was the strength of this study because dietary intake of each patient was compared with itself, so the effect of within variation was decreased. using of dietary records has some strength. this method is based on actual intake ; it is completely open - ended, and it does not rely on memory. nutrition counseling was successful in increasing dds which might be associated with improve diet quality of cardiac patients and it can reduce the risk of disease. studies with larger sample size and shorter time duration and assessment of dds in different kinds of cardiac disease were recommended. | background : dietary diversity score (dds) measurement was used to assessment of diet quality in different disease like cardiovascular disease. one way to improve the cardiovascular patient 's ability is cardiac rehabilitation program that include exercise training, nutrition consultation and psychological treatment. no study was designed to compare the dds before and after dietary consultation among cardiac rehabilitation patients, so this study was designed to examine this purpose.methods:subjects were participated in the 2-month cardiac rehabilitation program. all patients that completed the cardiac rehabilitation program and 2 dietary records (before and after nutritional counseling in cardiac rehabilitation program) enrolled in study. kant method was used for scoring dietary diversity. data were analyzed using the statistical package for social sciences (spss version 20). wilcoxon test were used to compare dds score for each subgroup and pair sample t test was used to compare total dds after and before nutrition counseling. p < 0.05 was considered as statistically significant.results:the differences between dds of grain, fruit, vegetable and meat before and after nutrition counseling were not significant (p = 0.635, p = 0.423, p = 0.826, p = 0.207 respectively), but differences of dds for dairy and total dds before and after nutrition counseling were significant (p = 0, p = 0.001).conclusions : dietary diversity was increased after nutrition counseling among patients with cardiac disease. |
congenital candidiasis (cc) is rare and usually caused by intrauterine candidial infection and manifests within first 6 days of life. it may be localized involving only skin or generalized resulting in respiratory distress, meningitis, sepsis, and death. a total of 10 - 35% of the women suffer from candidial vagintis during pregnancy, but less than 1% of them develop candidial chorioamnionitis that can affect the fetus. this is why cc is so rare and only 100 cases have been reported in the literature so far. a preterm (36 weeks), baby boy weighing 2.5 kg delivered by lower segment caesarean section (lscs) was brought to the dermatology department with pustular lesions all over the body on the second day of life. the attendant noticed erythema in both groins and over face within few hours after birth, followed by the appearance of pustular lesions on it in next 12 - 16 h. scalp, back of trunk, extremities, and palms were involved by the second day. mother was 31-year - old, gravida 6, para 3, live born 2, abortions 3, death 1[g6p3l2a3d1 ] with gestational hypertension. there was a history of cervical incompetence and cervical encirclage was done at 16 week of pregnancy. cutaneous examination revealed multiple, superficial, small pustules some coalescing to form lakes of pus over an erythematous back ground in groins, scalp, forehead, back, extremities, and palms. based on the history of vaginitis and cervical suture in the mother, whitish plaques on cord during delivery, pustular lesions on an erythematous background with in 24 h after birth, a clinical diagnosis of cc was considered. pustular lesions in groins pustules coalescing to form lakes of pus on the back hemogram revealed leucocytosis (24,900 cells / mm). koh mount from the pustular lesions revealed pseudohyphae suggestive of candidiasis [figure 3 ]. culture on sabourauds dextrose agar showed candida species, further confirming the diagnosis [figure 4 ]. the baby was given topical 2% ketaconazole cream twice daily along with fluconazole 6 mg / kg intravenously once daily for 3 days as there was leucocytosis. pustules began to dry by 3 day and desquamated completely by 5 day [figure 5 ]. oral fluconazole (6 mg / kg / week) and topical ketoconazole were continued for 1 week and there was no recurrence at 3 months of follow - up. cc is a very rare condition which presents at birth or with in first 6 - 7 days after birth and generally represents maternal chorioamnionitis occurring either from birth canal as an ascending infection or as transplacental infection. ascending infection may occur either from subclinical rupture of membranes or even through intact membranes resulting in whitish plaques on the membranes and umbilical cord along with skin lesions, described classically as various risk factors like < 27 weeks of gestation age, wt < 1000 g, intrauterine device, cervical sutures, invasive procedures, and extensive instrumentation have been reported. the role of maternal steroids or immunodeficiency in the infant is controversial. in the present case, cervical encirclage was done for cervical incompetence by mcdonold 's method at 16 weeks of pregnancy. cc manifest at birth or within a few hours of birth as extensive erythematous maculopapular eruption on head, trunk, and extremities that progress to vesicles and pustules on erythematous base in 1 - 3 days. palmar and plantar pustules are considered as hallmark of the disease, but mucosae and napkin area are spared. onychia and paronychia may occur and rarely cc may be limited to nails. scalded or burn - like appearance of skin lesions may herald systemic involvement. severe involvement of gastrointestinal and respiratory tract can occur due to aspiration of infected amniotic fluid that culminates in candidial septicemia manifesting as bronchopneumonia, meningitis, arthritis, endocarditis with microabcess in liver, brain, kidneys, or spleen. features like respiratory distress, leucocytosis with left shift, persistent hyperglycemia, glycosuria, positive cultures from blood, urine, cerebrospinal fluid (csf), and burn - like skin lesions suggest systemic involvement. neonatal candidiasis typically manifests after 6 days of life and differs clinically from cc [table 1 ]. cc should be differentiated from various other diseases presenting with pustules in the newborn [table 2 ]. comparision of congenital and neonatal candidiasis causes of neonatal pustular disorders diagnosis was established by koh mount of skin lesions showing budding yeasts and pseudohyphae and culture revealing candidial growth. blood, urine, and csf cultures should be obtained to rule out systemic involvement if there is clinical suspicion. amphotercin b is the first line agent given in doses of 0.5 - 1 mg / kg / day and liposomal amphotericin b is less toxic and preferred if there is preexisting renal insufficiency. fluconazole at 6 - 12 mg / kg / day dose is effective alternative if organism is susceptible. topical therapy is given till the resolution of skin lesions and systemic therapy continued for minimum of 21 - 28 days if systemic involvement is present. cc is very rare and needs to be differentiated from various diseases presenting with generalized maculopapular or pustular lesions at birth in order to avoid complications. | congenital candidiasis (cc) is a rare disease with less than 100 cases being reported in the literature. it presents within six days of life with manifestations ranging from localized skin disease to systemic involvement in the form of respiratory distress, sepsis, and death. we report a neonate who presented with diffuse pustular eruption on erythematous background involving face, trunk, and palms within 24 h after birth. candida albicans was identified in 10% potassium hydroxide (koh) smear and culture from the pustules. intravenous fluconazole and topical ketoconazole were given and the condition improved completely in two weeks. cc is rare and needs to be differentiated from other conditions presenting with pustular lesions at birth in order to avoid complications. early diagnosis and prompt treatment of this condition is important as untreated cases carry a mortality rate of 8 - 40%. |
epiretinal membrane (erm) formation reflects a number of pathological changes occurring in vitreoretinal junctions. retinal glial cells, fibrous astrocytes, and mller cells proliferate and migrate from neurosensory retina, through surface and breaks of the internal limiting membrane (ilm). in most cases, the disease is idiopathic but it can also be seen in eyes following retinal surgery, like vitrectomy or extracapsular lens extraction, in uveitic eyes or following vascular retinal diseases [14 ]. the epiretinal membrane itself is defined as a fine, semitranslucent, nonvascular, fibrocellular membrane on the inner retinal surface along the ilm [1, 2, 5 ]. affected patients may present with variable degrees of decrease in visual acuity (va) and disturbing metamorphopsia or micropsia. pars plana vitrectomy with membrane peeling is the current standard treatment for surgical removal of erm, with reported rates of visual improvement ranging between 67% and 82%. in addition, removing the ilm has been suggested as a measure to prevent cellular reproliferation. furthermore, a number of recent reports are dealing with an interesting correlation of macular function and morphology using sd - oct and microperimetry. disruptions of the photoreceptor inner and outer segment band seem to be a potential predictor for poor visual recovery in eyes having undergone macular surgery and some patients also seem to have paracentral microscotomas after membrane and ilm peeling. however, the induction of a potential mechanical trauma by using end - gripping forceps in areas of epiretinal membrane and ilm peeling resulting in potential functional or morphological damage has not yet been addressed. the aim of the present study was to analyze the correlation between morphological changes of the outer retina, such as ez (ellipsoid zone) and elm (external limiting membrane), and functional parameters, such as retinal sensitivity and visual acuity in the fovea and in the area of erm and ilm peeling, whether the manual peeling using forceps during surgery has an influence on postoperative functional outcome or not. in order to be able to identify these specific areas all operations were video - documented. in this prospective, observational nonrandomized study, 42 eyes of 42 patients who were diagnosed with epiretinal membranes, but no other retinal diseases, were included. in all subjects 23-gauge pars plana vitrectomy with peeling of the erm and ilm was performed at the department of ophthalmology, ludwig maximilians university of munich, germany, between july and december 2013. patients were informed about the use of their data for this study prior to surgery. all patients suffered from a decrease in best corrected visual acuity (bcva) below 20/30 snellen. patients with severe refractive medium opacity, proliferative diabetic retinopathy, age related maculopathy, advanced glaucoma, history of uveitis, previous retinal surgery, and intravitreal injections were excluded. standard 23-gauge pars plana vitrectomy was performed by two highly trained vitreoretinal surgeons (anselm kampik and christos haritoglou). in all cases heavy brilliant blue solution (fluoron gmbh, neu - ulm, germany) was applied for visualization of the ilm before or after erm removal. patients with relevant lens opacification (locs iii with grade > 3 of nuclear and/or cortical and/or posterior subcapsular opacification) underwent a combined surgery with cataract extraction and intraocular lens implantation. the surgery was documented on video in order to be able to postoperatively identify areas where peeling using end - gripping forceps was applied. a detailed eye examination including measurement of bcva, intraocular pressure, and slit lamp examination of the anterior segment, with documentation of lens opacities using the lens opacities classification system iii, thorough fundus examination by indirect binocular ophthalmoscopy, spectral - domain volume scan oct (heidelberg engineering sd - oct, heidelberg, germany) and central 2-degree and 18-degree microperimetry (maia, ellex medical lasers ltd., adelaide, australia) was performed at every visit. in addition, all video - documented areas of manipulation on the retinal surface using forceps were postoperatively evaluated using a focal 2 microperimetry at these areas (figure 1). morphological changes in the outer retina such as ez and elm were scored using a grading system (grades 02) already published in the literature [6, 11 ] and mean retinal thickness was analyzed with sd - oct pre- and postoperatively. briefly, in oct measurements grade 0 was defined as an intact ez / elm junction, as seen by a continuous hyperreflective line, grade 1 showed a focal disruption of the ez / elm junction 200 microns in length. these morphological changes were then correlated with functional results, such as bcva, expressed as a gain in lines, and mean retinal sensitivity measured by microperimetry. the macular area was divided into 5 sectors as published previously (modified edtrs grid) in order to allow a reliable standardized examination and correlation of findings. sector 1 was defined as the foveal area while the parafoveal area was divided into 4 quadrants labeled sectors 25. microperimetry was performed with the maia machine, which is a near - infrared, line scanning laser ophthalmoscope that incorporates a high frequency eye tracker and an automated macular perimeter to determine threshold sensitivity and fixation characteristics. the automated eye tracker locks onto the entire fundus image and captures fixation changes 25 times per second during testing. the system is using a 4 - 2 - 1-staircase strategy with a goldmann iii stimulus. preoperatively we performed two tests, a 25-stimulus test covering two degrees of the foveal area and a 68-stimulus test covering 18 degrees of the whole macular center field. these two regions were reassessed 3 and 6 months postoperatively. in brief, a fundus image is taken every time a new baseline test is performed with the maia machine. a follow - up exam then repeats the baseline expert test by accurately remeasuring the same points while comparing anatomical significant landmarks to the baseline test. in addition, we performed a 25-stimulus test covering two degrees of all video - documented peeling areas to observe any changes in functional sensitivity in these areas during follow - up. background luminance was set at 4 asb, the stimulus dynamic range was set up to 30 db, and maximum luminance was 1000 asb. for the sd - oct analyses, a volume scan was performed in each observational time step. five horizontal scans of the fovea and 22 horizontal scans of each parafoveal quadrant with a single scan distance of 11 microns were obtained and separately evaluated in the modified edtrs grid to cover the areas where peeling of membranes was performed during surgery. bcva was measured using a snellen chart and converted to the logarithm of minimum angle of resolution (logmar). the mann - whitney test was used to compare the statistical distribution of evaluated parameters. the mean retinal sensitivity of the fovea, overall and in areas of peeling, was correlated with mean sd - oct grading (02). chicago, il, usa). a p value 0.05). furthermore, investigated oct measurements in areas of peeling showed no worsening of preexisting outer retinal structure defects or new defects in a preexisting normal ez and elm band after surgical intervention. the current approach of correlating morphological alterations with functional ones in various macular diseases is to compare oct scans with microperimetry patterns, not only in the foveal but also in the parafoveal area. due to advances of newer oct machines (high resolution, eye tracker, and fast scan mode) it is possible to evaluate the vitreomacular interface and outer retinal structures in more detail. specifically, the ez, formerly is / os, and the elm have been thought to be of prognostic nature in case of pathological alterations or surgical intervention like pars plana vitrectomy with membrane peeling [6, 8, 9, 1113 ]. the formation of erm is a pathology of the vitreoretinal interface and the cellular proliferation is very likely related to an incomplete posterior vitreous detachment in idiopathic cases but can secondly occur after different retinal diseases or interventions such as retinal breaks, laser or cryotherapy, inflammatory diseases, or retinal detachment. as a first step tractional forces lead to morphological disorganization of the inner retina [1416 ] followed by changes in the outer retinal layers as the disease progresses. the extent and localization of outer retinal changes in eyes with erm formation may be very variable and correlate with the size of the membrane and the focus where tractional forces are most pronounced. these morphological alterations are often associated with a decrease of visual acuity and disturbing metamorphopsia, which are the main indications for surgical intervention [1720 ]. however, in some cases the functional result obtained postoperatively is not satisfying for both the surgeon and the patient despite clinically visible anatomic success. the reason for this discrepancy is correlated with alterations of the outer retinal layers, which can be depicted with high resolution imaging oct [7, 12, 2022 ]. furthermore, our study group recently showed that not only foveal but also parafoveal alterations in photoreceptor junction influence postoperative outcome. in addition to a positive correlation between outer segment restoration and functional results after macular surgery, itoh and associates observed that a recovery of the foveal cone microstructure may be seen as late as 12 months after anatomically successful surgery and that intact cone outer segment tips after erm surgery correlate with bcva [12, 21 ]. the influence of the used tamponade in the end of erm surgery (air or balanced salt solution) has also been discussed. however, so far published studies investigating the role of the outer retinal layers and their impact on functional recovery focused mainly on foveal sections obtained during oct examinations and did not include the area surrounding the fovea [7, 8, 10, 13, 17, 18, 2427 ]. the present investigation systematically analyzed both the foveal and the parafoveal region and established a correlation between morphological abnormalities detected in oct images and retinal function in these specific areas as measured by microperimetry and bcva after a surgical intervention. despite a successful surgical intervention some patients still complain about decreased visual acuity and/or microscotoma postoperatively. to exclude a mechanical injury to the retina by using end - gripping forceps we analyzed in detail the areas of peeling using oct and microperimetry to compare morphological alterations in the outer retina with functional ones. in our study we could find a significant correlation of decreased retinal sensitivity and ez or elm interruptions in oct measurements both overall and in areas of peeling. however, patients with more than two lines of visual acuity gain showed no significant improvement in retinal sensitivity compared to patients with a visual acuity gain below 2 lines. our results indicate that ez integrity on sd - oct is a statistically significant predictor of visual acuity in patients with erm formation, and statistical analysis illustrates that ez disruption, in contrast to elm disruption, increases the predictive power of oct measurements. furthermore, the present study indicates that the standard surgical approach of ilm and erm peeling using forceps has no significant negative influence on postoperative retinal sensitivity outcome, even if preexisting outer retinal alterations exist. a limitation of our study is related to the limited number of patients included and the evaluation of only outer retinal structure alterations. as we have already shown in our previous study, the present work confirms that morphological and functional tests (sd - oct and microperimetry) in patients with erm formation should not be focused on the foveal region alone but should also cover the parafoveal area. in addition, the surgical approach using manual forceps for membrane peeling in a highly trained surgeon setting does not influence functional outcome. therefore, standard 23-gauge vitrectomy with membrane peeling is a safe and efficient approach to treat erm formation. | purpose. to assess functional and morphological alterations following video - documented surgery for epiretinal membranes. methods. forty - two patients underwent video - documented 23-gauge vitrectomy with peeling of epiretinal (erm) and inner limiting membrane (ilm). patient assessment was performed before and 3 and 6 months including best corrected visual acuity (bcva), slit lamp biomicroscopy, sd - oct, and central 2 and 18 microperimetry. in addition, all video - documented areas of peeling on the retinal surface were evaluated postoperatively using an additional focal 2 microperimetry. retinal sensitivity and bcva were correlated with morphological changes (ez and elm) in the foveal region and in regions of membrane peeling. results. overall, bcva increased from 0.6 (0.2) to 0.2 (0.2) logmar after 6 months with an increase in retinal sensitivity (17.9 2.7 db to 26.8 3.1 db, p 0.05). in contrast, overall postoperative retinal sensitivity was significantly decreased in patients with a visual acuity gain lower than 2 lines (p < 0.05) correlating with ez defects seen in oct. conclusions. mechanical trauma of epiretinal membrane and ilm peeling due to the use of intraocular forceps may affect the outer retinal structure. nevertheless, these changes seem to have no significant impact on postoperative functional outcome. |
this is an institutional review board (irb) approved retrospective sequential review of case notes and x - rays of all children admitted to our institution between 2005 and 2009 with a displaced supracondylar humerus fracture. all children were followed at least 24 months. upon admission to the emergency department, all children were examined clinically and had a pulse oximeter probe placed over the ipsilateral hand. the inclusion criteria was a pulseless perfused hand following a supracondylar fracture of the distal humerus as defined by an objective finding of no waveform on the nellcor n395 pulse oximeter (coviden, boulder, co, usa). we reviewed the medical records to determine demographic information, mechanism of injury, perfusion and pulse oximeter readings on admission. we used the modified gartland 's classification system10) to ascertain the grade of the fracture. all patients had immediate postoperative evaluation of the hand in the area of color, perfusion and pulse oximeter waveform readings. as mentioned above, the pulse oximeter used in this study was a nellcor n395 pulse oximeter. a good waveform (fig. 1) and a poor or absent wave form (fig. we defined a pulseless supracondylar humerus fracture as having no palpable radial pulse despite full extension of the elbow and a well perfused hand with capillary refill of less than 2 seconds. all patients were followed for a minimum of 24 months and then referred back under the care of their regular physicians. patients with an absent radial pulse and who had an absent pulse oximeter waveform post crpp had open exploration of the brachial artery. open exploration of the brachial artery was undertaken by a consultant vascular surgeon using a standard anteromedial approach. a total of 3,182 patients presented to our department with a supracondylar humerus fracture from 2005 to 2009. 781 children sustained displaced supracondylar fractures of gartland grade iib or iii and were admitted for operative stabilization. in the emergency department, the above criteria for determining if a hand was pulseless were applied. we identified 37 (4.7%) patients presenting with a pulseless hand following a supracondylar humerus fracture. all 37 received closed reduction and percutaneous pinning emergently within 6 hours of presenting to the hospital. the remaining 26 patients had no palpable radial pulse but had a perfused hand with good capillary refill. each of these patients had a pulse oximeter applied intraoperatively and in retrospective analysis were divided into 2 groups. all patients had a good waveform and at the 1 hour postoperative review, had an intact radial pulse. the radial pulse remained present over the next 24 hours. at follow - up, all patients continued to have intact radial pulses. seven of the patients had a documented anterior interosseous nerve injury which recovered by the 3rd month postsurgery. no patient subsequently developed ischemic contractures or noted any forearm claudication in their school physical exercise classes. this group had four patients and all received emergent open exploration of the brachial artery. in three cases, anatomical pathology one case had entrapment of the brachial artery in the fracture site (table 1, subject 26). in two cases, the artery was tented by the supratrochlear branch of the brachial artery (table 1, subjects 12 and 21). 4), the supratrochlear branch of the brachial artery remained tethered by the unossified components of the fracture, the torn periosteum and the jagged edge of the fracture. the ligation of the binding supratrochlear branch and the freeing of the entrapped brachial artery in these 3 cases led to a prompt return of the radial pulse intraoperatively. the fourth case (table 1, subject 9), had a large hematoma in the anteromedial cubital fossa but no appreciable pathology over the brachial artery. following evacuation of the hematoma, radial pulse promptly returned. in the early postoperative period, the radial pulse remained present 24 hours postoperative and during all subsequent follow - up visits. all 4 patients remained well and at 24 months, had a palpable radial pulse with no intervening events of restenosis. of the 4 cases only 1 had concomitant nerve injury affecting the anterior interosseus nerve. no patients in group 1 or group 2 developed forearm compartment syndrome. at long term follow - up, no patients in our series had any long term sequelae of volkmann ischemic contracture. all patients achieved fracture union and were discharged from follow - up once they had reached at least 24 months of follow - up from the date of injury (table 1). of the remaining 744 supracondylar fractures requiring crpp, none developed ischemic contractures. table 2 summarizes the effectiveness of using this test in predicting the need for exploration based on the waveform obtained via pulse oximetry. all patients had a good waveform and at the 1 hour postoperative review, had an intact radial pulse. the radial pulse remained present over the next 24 hours. at follow - up, all patients continued to have intact radial pulses. seven of the patients had a documented anterior interosseous nerve injury which recovered by the 3rd month postsurgery. no patient subsequently developed ischemic contractures or noted any forearm claudication in their school physical exercise classes. this group had four patients and all received emergent open exploration of the brachial artery. in three cases, anatomical pathology one case had entrapment of the brachial artery in the fracture site (table 1, subject 26). in two cases, the artery was tented by the supratrochlear branch of the brachial artery (table 1, subjects 12 and 21). 4), the supratrochlear branch of the brachial artery remained tethered by the unossified components of the fracture, the torn periosteum and the jagged edge of the fracture. the ligation of the binding supratrochlear branch and the freeing of the entrapped brachial artery in these 3 cases led to a prompt return of the radial pulse intraoperatively. the fourth case (table 1, subject 9), had a large hematoma in the anteromedial cubital fossa but no appreciable pathology over the brachial artery. following evacuation of the hematoma, radial pulse promptly returned. in the early postoperative period, the radial pulse remained present 24 hours postoperative and during all subsequent follow - up visits. all 4 patients remained well and at 24 months, had a palpable radial pulse with no intervening events of restenosis. of the 4 cases only 1 had concomitant nerve injury affecting the anterior interosseus nerve. no patients in group 1 or group 2 developed forearm compartment syndrome. at long term follow - up, no patients in our series had any long term sequelae of volkmann ischemic contracture. all patients achieved fracture union and were discharged from follow - up once they had reached at least 24 months of follow - up from the date of injury (table 1). of the remaining 744 supracondylar fractures requiring crpp, none developed ischemic contractures. table 2 summarizes the effectiveness of using this test in predicting the need for exploration based on the waveform obtained via pulse oximetry. current literature points to many studies using color flow doppler ultrasound in the emergency department, perioperative and postoperative setting as a tool in determining the patency of the brachial artery in the setting of a pulseless supracondylar humerus fracture.4,11,12) to our knowledge, this is the first study in pulseless supracondylar humerus fractures that uses the pulse oximeter in diagnosis to conclusion of treatment with excellent results. none of our patients developed volkmann 's ischemic contracture and all of them had successful return of the radial pulse at 24 hours postprocedure and at 1 year follow - up. we recognize that a child who may have a diminished pulse at presentation may eventually have a pulseless hand as part of a continuum of fracture hematoma and compression of the brachial artery. a child with a brachial artery intimal damage which may progress to late brachial artery occlusion these subjects would not have been included in our study as a waveform would have been present at presentation. thus, we looked at the remaining 744 patients to detect if there was any delayed compartment syndrome, late ischemic contracture or forearm claudication but none was found in the postoperative examination at 24 hours or in subsequent outpatient follow - up. with that background, we propose a treatment algorithm using the pulse oximeter for children with supracondylar humerus fractures presenting with an absent pulse (fig. many authors have advocated observation as a treatment of choice.2,4,13) increasingly, many have advocated early intervention once there is recognition of vascular injury. noaman14) explored the brachial artery in 31 children in whom the radial pulse was absent after closed reduction and pinning in a series of 840 grade iii fractures. his indications were a pulseless forearm with a pink or cold hand, an absent radial pulse one hour after satisfactory closed reduction and percutaneous pinning and an absent radial pulse associated with an open fracture or signs of tethering of the brachialis muscle. there was arterial damage in 30 children and in one the artery was released from the site of the fracture. korompilias.,15) in his series of 5 children with a pink, pulseless hand also recommended surgical exploration to restore the patency of the brachial artery even in the presence of a viable and well - perfused hand after an attempt at closed reduction. most recently, white.7) systematically reviewed 19 papers in the english literature where pulseless and perfused supracondylar fractures were managed. of this group, 90% underwent surgical exploration and 82% was found to have brachial artery injury. this paper also went on to analyze the results of patency of the brachial artery following exploration and in 54 such explorations 91% remained patent at the 1 year follow - up. while the results of this review appear to indicate that brachial artery injury was much higher than our series, we believe that there is some selection bias using pooled retrospective data from 19 such studies. we experienced similar levels of patency of the brachial artery postsurgical exploration at the 1 year follow - up (100%). this systematic review further supports early intervention once there is recognition of brachial artery pathology and challenges early suggestions of ' watchful waiting '. in contrast, ramesh.16) reported a series of patients with well perfused hands but absent radial pulse following humerus supracondylar fracture fixation. the author cautioned that these patients did not have excruciating pain distal to the elbow that persisted beyond 12 hours after the injury as compared to the series by blakey.9) this demonstrates that the upper limb has an extremely good collateral blood supply. the abundant collateral supply of the elbow comes from the superior and inferior ulnar collateral artery medially and the profunda brachii artery laterally. these arteries branch from the brachial artery proximal to the olecranon fossa, where supracondylar humerus fractures frequently occur. the profunda brachii artery branches to give a radial recurrent branch and an interosseous recurrent branch. the superior ulnar collateral artery runs posterior to the medial epicondyle to form the posterior ulnar recurrent artery. the inferior ulnar collateral artery runs anterior to the medial condyle of the humerus to become the anterior ulnar recurrent artery. the radial recurrent and both the ulnar recurrent arteries then rejoin the radial and ulnar artery respectively at the level of the biceps insertion on the radial neck.17) this rich collateral network forms the basis for pulseless perfused hands (table 1, subject 26) despite brachial artery entrapment at the level of the supracondylar humerus. while we have no patient in our study with both an absent radial pulse and a well perfused limb on long term follow - up, we recognize that it may be possible for a waveform to be present despite brachial artery occlusion due to the rich network of collaterals at the elbow joint. our study however did not show any patients who did not have return of the radial pulse once intraoperative pulse oximetry showed a good waveform. this may be a limitation of our study due to its small sample size or may indicate that pulse oximetry is truly objective in determining the eventual return of the radial pulse. at a minimum of 24 months follow - up, all patients had well palpable radial pulses indicating that stenosis or delayed intimal damage resulting in occlusion unlikely occurred. we are of the opinion that given the readily available resource of the pulse oximeter in the operating room, the challenges of subjectivity in monitoring pain in the postoperative patient and the good results following surgical exploration of the brachial artery that surgical exploration should be undertaken once there is documented injury to the brachial artery. our study demonstrates that the pulse oximeter is an objective tool to support or reject the decision for surgical exploration. mangat.18) advocated that the likelihood of vascular injury was high with documented anterior interosseous nerve or median nerve injury in his series of 19 patients over a 14-year period. our study showed different results as only 1 patient who required surgical exploration with positive findings of brachial artery pathology had nerve injury at presentation. in the group which had a good waveform and which did not require exploration, 7 patients had nerve injury which recovered during follow - up. we surmise that nerve injury, while associated with the incidence of brachial artery pathology in a pulseless hand, is not an objective predictor of surgical exploration. the use of pulse oximetry allows easy access and is a relatively cheap alternative to doppler ultrasound. furthermore, it removes the subjectivity in examining the peripheral nerves in an already fretful and irritable child. previous authors have also suggested the use of angiography to assess the vascular status of the affected limb.3,19) shaw.11) expressed that this may potentially increase the time required before a vascular exploration. angiography is also invasive and has risks of iatrogenic injury to the artery due to its small size.20) in addition, there are also risks of contrast allergy. we recognize that the use of the pulse oximeter in colder environments may lead to false positives as a result of peripheral vasoconstriction. we acknowledge limitations to our study with respect to its limited sample size and the lack of imaging or exploration to confirm the absence of injury to the brachial artery. based on the eventual presence of a radial pulse, this study shows a positive predictive value of 0.75, and a negative predictive value of 1.0. we will need to continue evaluating this with increasing numbers that are added to our study population over time. we are optimistic that this test has a high specificity of 95.65%, and would want to embark on future studies that test the true effectiveness of the algorithm and to pit the results of the pulse oximeter with that of color flow doppler in a prospective context to assess the patency of the brachial artery. in conclusion, the pulse oximeter is an excellent and readily available tool in the emergency department and the operative room to assist diagnosis of the pulseless hand and subsequently, to discern arterial injury in well perfused, postfixation supracondylar humerus fractures. | backgroundthe management of the pulseless perfused hand in association with a supracondylar humerus fracture following operative stabilisation remains controversial. previous authors have suggested the use of color - flow duplex monitoring, magnetic resonance angiography and segmental pressure monitoring as objective steps to ascertain blood flow following adequate internal fixation. we examine the use of the waveform of the pulse oximeter in objectively determining a perfused limb and in predicting the need for surgical exploration in patients who present with a pulseless perfused hand after operative stabilisation for supracondylar fracture of the humerus.methodsa retrospective review of all supracondylar fractures over a 60 month duration (2005 - 2009) in our instituition was performed. each electronic record was reviewed and limbs which had absent radial pulse following admission were identified. x - ray films of each of the patients were reviewed. a search using the pubmed database was performed with the following keywords, supracondylar humerus fracture, pediatric, pulseless, vascular injury, arterial repair.resultsin this series of pulseless perfused hands following operative fixation of supracondylar fracture, a total of 26 patients were reviewed. all were gartland grade iii extension type fractures. postoperative pulse oximeter waveforms were present in all but 4 patients. these patients subsequently had exploration of the brachial artery with significant findings. in the remaining 22 patients, waveforms were present and the child had return of the radial pulse soon after operative fixation without any further need for surgical exploration. at 24 months follow - up, all children were well with no neurovascular compromise.conclusionsthe presence of a waveform on a pulse oximeter is a sensitive and easily available modality in determining vascular perfusion as compared to other more complex investigations. the high sensitivity of this test will allow surgeons to objectively determine the requirement for surgical exploration of the brachial artery. |
motor branch or recurrent branch of the median nerve, also called as the thenar branch, innervates the intrinsic muscles of the thumb (abductor pollicis brevis, opponens pollicis, and the superficial head of the flexor pollicis brevis). it has an important role in the movements of the thumb and thus in many activities of the hand. accordingly, hand surgeons pay attention not to injure the motor branch while performing carpal tunnel surgery. herein, a rare anatomic variation regarding the motor branching of the median nerve is being reported. during surgery for releasing the left carpal tunnel in a 62-year - old lady, the thenar muscular branch of the median nerve was observed to originate 2.5 cm above the proximal border of the transverse carpal ligament [figure 1 ]. it traveled along the medial side of the flexor carpi radialis tendon. by entering the carpal tunnel, the motor branch, along with the median nerve, the medial and lateral divisions of the median nerve were normal in appearance under the carpal tunnel and there were no pseudoganglion formation. intraoperative photograph demonstrating motor branching of the median nerve proximal to the transverse carpal ligament illustration showing the course of motor branching of the median nerve the motor branch of the median nerve was, in relation to the transverse carpal ligament, was found to be of extraligamentous, subligamentous, transligamentous types in 46%, 31%, 23%, respectively in 246 carpal tunnel release operations by lanz. while studying 72 hands of 36 cadavers, reported the classical distal (extraligamantous - subligamantous) branching in 86%, and transligamentous motor branching in the rest. while linburg and albright, rockwell reported multiple motor branching anomalies, graham referred to a case with motor branching in the ulnar region. lanz and gruber found upper level median and lateral division branching (in the distal forearm) in four cases. eiken. reported three similar cases and kessler reported one case with the same anomaly. however, all those reports were of medial and lateral divisions of the median nerve at the upper level. the upper level branching of only the motor branch is reported in the series of olave. in one case. likewise in our case, the sensory branch of the median nerve (medial and lateral divisions) was inside the carpal tunnel. while releasing carpal tunnels a proper understanding of the anatomic variations of the motor branch is paramount in order not to cause iatrogenic injuries that may compromise hand functions severely. | we present a 62-year - old female patient who had an anatomic variation in the median nerve of the left hand. during surgery for releasing the left carpal tunnel, an abnormally high level of origin of the thenar muscular branch of the median nerve was detected, at 2.5 cm above the proximal border of transverse carpal ligament. it traveled between the medial side of the flexor carpi radialis tendon and median nerve and entered the carpal tunnel. after exiting the carpal tunnel distally, the nerve, was noted to course towards the thenar area. such variations in the median nerve should be kept in mind while performing carpal tunnel release. |
catatonia was first described by kahlbaum in 1874, as a brain disorder, which has cyclic, alternating and progressive course. over the years, understanding about catatonia has increased and it is now well - known that besides the primary psychiatric disorders, catatonia is associated with many neurological and medical disorders. catatonia in adolescents has been reported to be associated with affective, psychotic, autistic, developmental, drug induced and medical conditions. further, as in adults, catatonia in children and adolescents also responds to benzodiazepines and electroconvulsive therapy (ect). however, the literature on the use of ect in adolescents with catatonia is limited. in this case report, we present a case of catatonic schizophrenia, treated with ect and review the literature on the use of ect in adolescent catatonia. a 16-year - old single girl presented with an insidious onset illness of 3 year duration. for the initial 1 year, the symptoms were characterized by fearfulness, anxiety, derealization and poor academic performance. during the 2 and 3 years of symptomatic phase, she developed additional symptoms of social withdrawal, poor initiative, irritability, muttering and gesturing in air, suspiciousness, delusions of reference and persecution and delusion of misidentification, poor self - care and stopped studying. about a month prior to presentation to our center, her speech output started reducing, she had perseveration and later became mute, had marked psychomotor retardation, ambitendency, active and passive negativism, posturing and refusal to eat. she was taken to a psychiatrist for her symptoms and was given intravenous lorazepam up to 8 mg / day along with risperidone up to 4 mg / day, but did not show any improvement. following this, there was no history suggestive of other delusions, hallucinations in any other modality, made phenomenon, somatic passivity, delusional perception, depressive symptoms, manic symptoms, head injury, fits, loss of consciousness, fever, substance use and thyroid dysfunction. patient was found to have immobility, posturing, withdrawal, gegenhalten, mutism, negativism, staring and drooling of saliva. her bush - francis catatonia rating scale (bfcrs) score was 20. in view of her symptomatology, she was admitted to the in - patient unit of the hospital. on investigation, hemogram, liver function test, renal function test, serum electrolytes, thyroid function test, ultrasound abdomen, computerized tomography of brain, x - ray chest (posterior anterior view) and electrocardiogram did not reveal any abnormality. on the basis of the history and mental status examination, a diagnosis of catatonic schizophrenia was considered. she was initially treated with injection lorazepam 4 mg every 6 h for initial 24 h along with tablet olanzapine 7.5 mg / day but did not show any improvement. following this, her parents were educated about the electroconvulsive therapy procedure and the indications for the same in patient. attempt was also made to take patient 's assent before initiating ects, but could not be done considering her condition. she was administered nine ects (details shown in table-1) over the period of 3 weeks and dose of olanzapine was increased to 15 mg / day. with this treatment, gradually she showed improvement in all her symptoms. as her clinical condition improved, her assent was sought for subsequent ects. during this period, assessment for memory impairment was also undertaken following each ect, but patient did not have any subjective complaints or objective signs of memory impairment. by her ninth ect, her bfcrs score came down from 20 to 2 and following this, no more ects were given in view of a plateau of clinical response. on mental status examination, she only had blunted affect with no other active psychopathology at the end of the course of ect. it is recommended that catatonia should be managed with high doses of benzodiazepines (i.e., lorazepam or other benzodiazepines) or ect, in addition to the treatment of underlying medical and psychiatric disorder with specific treatment. however, the literature with regard to the use of ect in adolescents is limited. in a review of the published literature from 1985 to 2009, the authors located 31 reports, which described 59 cases of catatonia in children and adolescents treated with ect. majority of the patients described in the literature who were treated with ect were males (57%). the age range of patients varied from 6 to 19 years with only three patients less than 11 years of age. most of the cases were diagnosed with mood disorders (47.5%) and this was followed by schizophrenia (27.1%), pervasive developmental disorder (13.5%), organic catatonia (5%), psychotic disorders other than schizophrenia (5%) and one case (1.7%) each of idiopathic catatonia and familial catatonia with seizures. in terms of response to ect, the data suggested that 45 patients (76.27%) had a favorable response, 3 (5%) cases had a partial response and in one case there was no response to ect. the authors cautioned that while interpreting the efficacy data it is important to remember that there may a potential bias for reporting only those cases, which show improvement with ect. in another review, authors noted that bilateral (bitemporal or bifrontal) ect has better efficacy than the unilateral ect. further, it is suggested that if a patient responds partially to lorazepam it should be used concurrently with ect for a better outcome in the acute management of catatonia. we carried out a search in pubmed and could locate 10 reports of use of ect in adolescents with catatonia published after the review of consoli. eight out of the 10 reports, described 11 cases in which ect was used in adolescents for treatment of catatonia in patients with varied clinical conditions [table 2 ]. one case series described the use of maintenance ect in three children with autism, in which ect had led to resolution of acute symptoms of catatonia. in the retrospective study from our center, which described the use of ect in adolescents, out of the 25 patients, 17 had catatonic symptoms and the response rate in patients with catatonia was 91.6%. the present case adds to the limited data, which is available and shows that use of ect can be life - saving in adolescents with severe mental disorders. case reports / case series of use of electroconvulsive therapy in adolescents published after consoli. review from the above review, it can be concluded that ect is an effective treatment for management of catatonia in adolescents. in view of the same, rather than banning the use of ect in adolescents, it would be better to leave the decision to use and not to use ect in adolescents in the hands of the clinicians. | there is lot of skepticism about the use of electroconvulsive therapy (ect) in children and adolescents. however, available literature suggests that use of ect can be at times life - saving in adolescents, especially those presenting with severe catatonia. we treated a 16-year - old female who presented to us with catatonia with a course of nine ects, with which she showed marked improvement. review of the literature suggests that ect should be considered as the second line treatment in the management of catatonia in adolescents. |
a 15-year - old female developed fever, stinging eyes and headache, followed one day later by sudden appearance of flaccid blisters on the face with subsequent dissemination to more than 70% of the whole integument and development of mucosal erosions. thirty days before, she had been operated on the nose and medicated with cefuroxime (for 8 days), paracetamol (for approx. 10 days) and metamizol (for approx. 10 days), the latter of which was later identified by lymphocyte transformation test as the most likely causative compound. four days before admission, she had taken acetylsalicylic acid and paracetamol for flu - like symptoms including conjunctival irritation and rhinitis. upon physical evaluation, disseminated flaccid blisters filled with serous liquid were present on 70% of her skin surface. her eye-, mouth-, pharyngeal- and genital mucosae were also affected by erosive lesions. she received intravenous immunoglobulins (ivigs), in total 3 g / kg body weight over three consecutive days, hydration and nutritional support as well as careful wound care. topical therapy included silver - coated dressings on erosions, and after reepithelialization, skin washes with water and wax emulsion were performed, followed by the application of hydrocortisone 0.5% in hydrophilic unguent. from day 1 after onset of ivig therapy onwards, no new development of blisters was observed. during the clinical course, desquamation of dusky areas of skin leaving an intact epidermis below was observed. we noted frequent spontaneous bleeding upon changes of the silver - coated dressings during reepithelialization period in the second week. the remaining detached skin fragments that overlied the progressively regrowing epidermis as of day 7 were progressively shed between day 7 and day 28. the patient was discharged from the intensive care unit (icu) at day 28 after admission, with almost complete skin reepithelialization. at the last follow - up control 6 months later, the patient showed full recovery, except for residual alopecia and dry eyes. to date, no specific treatment with a high evidence level of efficacy has been reported for ten. the standard of care consists of best supportive therapy in an icu setting, and includes hydroelectrolytic and nutritional support as well as regular wound care and the appropriate detection and treatment of infectious complications. whereas best supportive care is an accepted standard of care for ten patients, none of the specific treatment strategies described to date, including ivig (adequately dosed at 3 g / kg body weight ; there is an apparent dose dependence, as the reduction in mortality appears to be greatest for doses of 3 - 4 g / kg), corticosteroids, and infliximab, has shown solid evidence in support of a survival advantage in ten patients. to date, only one prospective, randomized - controlled clinical trial has been reported in ten, demonstrating no therapeutic benefit for thalidomide in the treatment of ten. ivig is a safe therapeutic modality that has been shown in several case series to potentially provide a benefit in ten. controversy as to the exact efficacy does, however, exist. in the recently established guideline for the use of high - dose immunoglobulins in dermatology, early administration of high - dose immunoglobulins (3 g / kg) indeed, several published studies reporting the use of ivig for the treatment of ten have shown this therapy to potentially provide a survival benefit. the studies published to date are unfortunately heterogeneous, and often of limited size, retrospective nature, and noncontrolled. on the other hand, ten is a rare disease, and randomized prospective controlled studies analyzing the effect of ivig are very difficult to perform, and do not exist to date. eight of the 11 studies published to date reporting the use of ivig in ten suggest a positive effect of ivig on mortality if compared to the mortality predicted by scorten where applied, or the 25 - 35% average mortality rate for ten reported in the literature (table 1). it must be kept in mind, however, that as for many other therapies, the total dose (in g / kg body weight) of ivig administered may have an effect on the therapeutic outcome. indeed, a recent publication by trent., analyzing in detail the studies published to date, showed that a dose - response relationship likely exists : per 1-g / kg increase in ivig dose, there was an observed 4.2-fold increase in patient survival. moreover, no mortality was observed amongst the 30 patients that had received 3 g / kg ivig or more. taking into account the lack of a proven specific therapy, the severity of ten, the safety of ivig, and the data to date concerning the efficacy of ivig in ten, we are of the opinion that in the absence of proven superior therapies, ivig therapy should be considered in patients with ten. | toxic epidermal necrolysis is a rare but clinically well - described dermatological pathology. however, clinical pictures of this disorder in text books do not reflect its dynamic evolution. usually, the desquamative post - bullous stage is represented, neglecting the initial bullous stage as well as the skin healing. with one clinical case, we provide a day - after - day illustration of the evolution of a patient suffering from toxic epidermal necrolysis. during one month, a skin area of a limb was regularly photo - documented. |
neuroepithelial cells, which constitute the wall of the neural tube, proliferate by repeating symmetric cell division, where each neuroepithelial cell divides into two neuroepithelial cells (progenitor expansion phase, figure 1 ; alvarez - buylla., 2001 ; fishell and kriegstein, 2003 ; fujita, 2003 ; gtz and huttner, 2005 ; miller and gauthier, 2007). as the wall of the neural tube becomes thicker, neuroepithelial cells elongate and become radial glial cells, which have cell bodies in the ventricular zone, and radial fibers reaching the pial surface (figure 1). radial glial cells were previously thought of as specialized glial cells that guide neuronal migration. later, it was found that radial glial cells undergo asymmetric cell division, where each radial glial cell divides into two distinct cell types, a radial glial cell and an immature neuron or a basal progenitor (neurogenic phase, figure 1 ; malatesta., 2000 immature neurons migrate outside of the ventricular zone along radial fibers into the cortical plate, where these cells become mature neurons, whereas basal progenitors migrate into the subventricular zone (svz), proliferate further and give rise to more neurons. radial glial cells give rise to many different types of neurons, initially deep layer neurons and then superficial layer neurons later, by repeating asymmetric cell division. radial glial cells also give rise to oligodendrocytes and ependymal cells and finally differentiate into astrocytes (gliogenic phase, figure 1). initially, neuroepithelial cells undergo repeated self - renewal by symmetric division (progenitor expansion phase). as development proceeds, neuroepithelial cells elongate to become radial glial cells, which have cell bodies on the inner side (called the ventricular zone) of the neural tube and radial fibers that reach the outer surface after the production of neurons, some radial glial cells give rise to oligodendrocytes and ependymal cells. both neuroepithelial cells and radial glial cells are considered embryonic neural stem / progenitor cells. if neural stem / progenitor cells are prematurely depleted, not only is the total number of cells reduced but also later - born cell types are lacking, indicating that the maintenance of neural stem / progenitor cells until the final stage of development is essential to produce the proper number of cells and the full diversity of cell types (hatakeyama., 2004). notch signaling has been shown to play an important role in the maintenance of neural stem / progenitor cells (fortini, 2009 ; kopan and ilagan, 2009 ; pierfelice., 2011). here we review the recent progress on the mechanism and role of notch signaling in neural development. proneural genes such as the basic helix - loop - helix (bhlh) transcriptional activators mash1 and neurogenin2 (ngn2) induce neuronal differentiation (bertrand., 2002 ; ross., 2003). these genes also induce the expression of notch ligands such as delta - like1 (dll1) and jagged1, which activate notch signaling in neighboring cells (figure 2 ; castro., 2006). the ubiquitin ligase mindbomb is required in dll1-expressing cells for the dll1-induced activation of notch signaling (ito., 2003 ; yoon., 2008). upon activation of the transmembrane protein notch, the notch intracellular domain (nicd) is released from the transmembrane portion and transferred to the nucleus, where nicd forms a complex with the dna - binding protein rbpj and the transcriptional co - activator maml (figure 2). maml complex induces the expression of bhlh transcriptional repressors such as hes1 and hes5 (honjo, 1996 ; artavanis - tsakonas., 1999 ; ohtsuka., 1999 ; hes1 and hes5 then repress the expression of proneural genes and dll1, thereby leading to the inhibition of neuronal differentiation and the maintenance of neural stem / progenitor cells (figure 2). thus, neurons inhibit their neighboring cells from differentiating into the same cell types, a process known as lateral inhibition. this regulation maintains asymmetric division into a neural stem / progenitor cell and a differentiating daughter neuron. however, this regulation also suggests that the maintenance of neural stem / progenitor cells depends on neighboring neurons expressing dll1, raising the question as to how neural stem / progenitor cells are maintained during the early stages before neurons are generated. proneural genes such as mash1 and ngn2 promote neuronal differentiation and induce the expression of dll1, which in turn activates notch in neighboring cells. upon activation of notch, the notch intracellular domain (nicd) is released from the transmembrane portion and transferred to the nucleus, where it forms a complex with the dna - binding protein rbpj and the transcriptional co - activator maml. hes1 and hes5 then repress the expression of proneural genes and dll1, thereby leading to the maintenance of neural stem / progenitor cells. proneural genes and dll1 are expressed in a salt - and - pepper pattern by neural stem / progenitor cells as early as e9.0 (progenitor expansion phase) in the developing mouse dorsal telencephalon (our unpublished observation), where neuronal formation starts around e10.5e11.0. in addition, hes1 is expressed at various levels by neural stem / progenitor cells (shimojo., 2008). it is likely that proneural genes induce the expression of dll1, which up - regulates hes1 expression in neighboring cells, suggesting that notch signaling is active in neural stem / progenitor cells before neurons are formed. this observation raises the question of why neurons are not formed during the progenitor expansion phase even though proneural genes are expressed. time - lapse imaging analysis revealed that hes1 expression oscillates in neural stem / progenitor cells (masamizu. this negative feedback leads to the disappearance of hes1 mrna and protein because they are extremely unstable, but the disappearance of hes1 protein allows the next round of its expression. in this way, hes1 expression oscillates with a period of 23 h in neural stem / progenitor cells (figure 3 ; hirata., 2002). protein expression exhibits an inverse correlation with ngn2 protein and dll1 mrna expression in neural stem / progenitor cells, suggesting that hes1 oscillation induces the oscillatory expression of ngn2 and dll1 by periodic repression (shimojo., 2008). indeed, time - lapse imaging analysis revealed that ngn2 and dll1 expression oscillates in neural stem / progenitor cells, where hes1 expression oscillates, whereas their expression is sustained in differentiating neurons, where hes1 expression is repressed (figure 4 ; shimojo., 2008). it is likely that ngn2 can not induce neuronal differentiation when its expression oscillates, probably because many downstream genes respond rather slowly to ngn2 expression. it seems that ngn2 induces neuronal differentiation only when its expression becomes sustained, and when its expression oscillates, only quickly responding genes such as dll1 are selectively induced. dll1 oscillations may lead to the mutual activation of notch signaling and the maintenance of neural stem / progenitor cells. thus, depending on its expression dynamics, ngn2 may lead to two opposite outcomes : when its expression oscillates, ngn2 induces the maintenance of neural stem / progenitor cells, but when its expression is sustained, ngn2 induces neuronal differentiation. hes1 expression oscillates with a period of 2 h in many cell types such as neural stem / progenitor cells and fibroblasts. this negative feedback leads to the disappearance of hes1 mrna and protein, because they are extremely unstable, allowing the next round of its expression. in this way hes1 expression oscillates with a period of 23 h in neural progenitor cells. in these cells, in contrast, the expression of ngn2 and dll1 is sustained in post - mitotic neurons, which lose hes1 expression. this reciprocal regulation is particularly important for the maintenance of neural stem / progenitor cells before the formation of neurons. the expression dynamics of ngn2 (oscillatory versus sustained) are regulated by hes1 (oscillatory versus no expression). one possible mechanism is numb - mediated inhibition of notch signaling (figure 2). numb is asymmetrically distributed into a prospective neuron during asymmetric cell division (shen. numb - expressing cells lose hes1 expression and differentiate into neurons, while numb - negative cells maintain hes1 expression (ohtsuka., 2006). the above observations raise the possibility that ngn2 and dll1 oscillations enable the maintenance of neural stem / progenitor cells without the aid of neurons. when hes1 expression is low in a subset of cells (cell 1 in the left panel of figure 5), ngn2 and dll1 expression becomes high, leading to the activation of notch signaling and the up - regulation of hes1 in neighboring cells (cell 2 in the left panel of figure 5). in the latter cells, high levels of hes1 repress ngn2 and dll1 expression, but due to oscillations, hes1 expression becomes low after 1 h, while ngn2 and dll1 expression becomes high (cell 2 in the right panel of figure 5), leading to the activation of notch signaling in the former cells (cell 1 in the right panel of figure 5). in this way, dll1 oscillations lead to the mutual activation of notch signaling between neural stem / progenitor cells (shimojo., 2008). these observations also suggest that notch signaling is not a one - way mechanism (neuron to neural stem / progenitor), but functions by reciprocal transmission (neural stem / progenitor to neural stem / progenitor). at later stages (neurogenic phase), many differentiating neurons express dll1 in a sustained manner, thereby activating notch signaling in neural stem / progenitor cells. at these stages, ngn2 and dll1 expression mostly occurs in neurons but not in neural stem / progenitor cells, and therefore their expression is sustained, although hes1 expression in neural stem / progenitor cells oscillates even at these later stages. ngn2 and dll1 oscillations are regulated by hes1 oscillation in neural stem / progenitor cells. ngn2 oscillation may be advantageous for the maintenance / proliferation of neural stem / progenitor cells during the early stages of development, because it induces dll1 expression and activates notch signaling without promoting neuronal differentiation. a salt - and - pepper pattern induced by lateral inhibition is just a snapshot of oscillatory expression. it has been thought that lateral inhibition amplifies stochastic variation, creates a salt - and - pepper pattern in gene expression and selects subsets of cells for neuronal differentiation : ngn2-positive or dll1-positive neural stem / progenitor cells are initially selected to become neurons when the neurogenic phase starts, while negative cells remain neural stem / progenitor cells (artavanis - tsakonas., 1999). however, time - lapse imaging analysis indicated that a salt - and - pepper pattern is just a snapshot of oscillatory expression ; therefore, neither ngn2-positive nor dll1-positive cells are initially selected to become neurons. thus, the traditional view of salt - and - pepper pattern formation induced by lateral inhibition should be revised (kageyama., 2008). basal progenitors in the svz, which are derived from radial glial cells by tbr2, retract their apical and basal processes and generally divide only once to generate two neurons (sessa., thus, basal progenitors have a limited proliferation ability. in these cells, hes1 and hes5 expression is down - regulated, suggesting that the notch - hes1/5 pathway is not active in basal progenitors (mizutani. it was recently shown that the developing human neocortex has an expanded outer region in the svz (osvz), and that progenitors in this region (osvz progenitors) divide multiple times and generate a large number of neurons (fietz., 2010 ; hansen., 2010 osvz progenitors have radial glia - like morphology and extend radial fibers to the pial surface. however, they lack apical processes and therefore are not in contact with the ventricular surface. like radial glia, osvz progenitors express hes1, and inhibition of notch signaling by treatment with a -secretase inhibitor induces osvz progenitors to differentiate into neurons or tbr2 basal progenitors (hansen., 2010), suggesting that the notch - hes1 pathway is required for maintenance of osvz progenitors (figure 6). it is possible that the cells that migrate into the svz may become osvz progenitors when notch signaling is active, whereas they may become basal progenitors when notch signaling is inactive. it remains to be determined how notch signaling is regulated in the svz and whether hes1 expression oscillates in osvz progenitors, as observed in radial glia. osvz / ovz progenitors have radial glia - like morphology and extend radial fibers to the pial surface, but lack apical processes. these neurons seem to express notch ligands and activate notch signaling in their sibling osvz / ovz progenitors. similar cells called outer ventricular zone (ovz) progenitors are found in the developing mouse cortex (shitamukai., 2011). ovz progenitors are located outside of the ventricular zone and retain radial fibers reaching the pial surface (basal processes). these cells undergo asymmetric cell division, where each ovz progenitor divides into a daughter cell that inherits the radial fiber (ovz progenitor) and the other that does not. the one that inherits the radial fiber seems to repeat asymmetric cell division multiple times, while the other differentiates into post - mitotic neurons. the former cells (ovz progenitors) express hes1, suggesting that notch signaling is activated in these cells (figure 6). interestingly, these daughter cells (ovz progenitor and neuron) maintain contact with each other for several hours, and neurons seem to express notch ligands and activate notch signaling in their sibling ovz progenitors (figure 6 ; shitamukai., 2011). these observations suggest that asymmetric cell division is required to activate notch signaling in osvz or ovz progenitors by their sibling neurons. not all cells express hes1 in an oscillatory manner : cells in the roof plate, floor plate, and boundary regions, such as the isthmus, express hes1 in a sustained manner (baek., 2006). furthermore, cells in these regions usually do not give rise to any neurons, probably because sustained hes1 expression constitutively represses the expression of proneural genes and cell cycle regulators. the introduction of sustained hes1 expression into neural stem / progenitor cells inhibits their proliferation and neuronal differentiation (baek., 2006). in contrast, when he s genes are inactivated, the cells in the roof plate, floor plate, and boundary regions can differentiate into neurons (baek., 2006). these results suggest that cells with sustained hes1 expression are rather dormant with regard to proliferation and differentiation. this feature is similar to the one observed in fibroblasts, where sustained hes1 over expression leads to reversible quiescence (sang., 2008). the mechanism by which oscillatory versus sustained hes1 expression is regulated remains to be determined. in fibroblasts, jak2 activates stat3 by phosphorylation, and phosphorylated stat3 (pstat3) induces socs3 expression, which in turn inhibits jak2. due to this negative feedback, interestingly, blockade of this pathway with a jak inhibitor inhibits hes1 oscillations by stabilizing the hes1 protein, and hes1 expression becomes steady (yoshiura., 2007). similarly, treatment with a jak inhibitor inhibits hes1 oscillations in neural stem / progenitor cells, suggesting that jak stat signaling is also involved in the regulation of hes1 oscillations in these cells (shimojo., 2008). i d proteins, hlh factors without a basic region, form heterodimers with hes1 through their hlh domains and inhibit hes1 from binding to the n box in the hes1 promoter (bai., 2007), suggesting that i d factors prevent hes1 from negative autoregulation. however, it remains to be determined whether i d factors lead to steady hes1 expression. oscillatory versus sustained hes1 expression leads to different outcomes in neural stem / progenitor cells. when its expression oscillates, neural stem / progenitor cells proliferate actively and differentiate into mature cells. by contrast, when its expression is sustained, neural stem / progenitor cells become dormant. similarly, oscillatory versus sustained ngn2 expression leads to different outcomes. when its expression is sustained, neural stem / progenitor cells differentiate into neurons. by contrast, when its expression oscillates, neural stem / progenitor cells remain undifferentiated. thus, not just the expression but also the dynamics of these genes are very important for the outcomes. the mechanism by which oscillatory versus sustained hes1 expression is regulated remains to be determined. in addition, the dynamics of downstream genes for hes1 and ngn2 oscillations are mostly unknown. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | in neural stem / progenitor cells, expression of the notch effector hes1, a transcriptional repressor, oscillates with a period of 23 h by negative feedback, and hes1 oscillations induce the oscillatory expression of the proneural gene neurogenin2 (ngn2) and the notch ligand gene delta - like1 (dll1). dll1 oscillation leads to the mutual activation of notch signaling between neighboring cells, thereby maintaining a group of cells in the undifferentiated state. not all cells express hes1 in an oscillatory manner : cells in boundary regions such as the isthmus express hes1 in a sustained manner, and these cells are rather dormant with regard to proliferation and differentiation. thus, hes1 allows cell proliferation and differentiation when its expression oscillates but induces dormancy when its expression is sustained. after hes1 expression is repressed, ngn2 is expressed in a sustained manner, promoting neuronal differentiation. thus, ngn2 leads to the maintenance of neural stem / progenitor cells by inducing dll1 oscillation when its expression oscillates but to neuronal differentiation when its expression is sustained. these results indicate that the different dynamics of hes1 and ngn2 lead to different outcomes. |
urinary bladder metastasis from solid tumors is rare and represents 2% of all bladder neoplasms. direct extension from the cervix, prostate and colon is not unusual but metastasis from a distant organ is extremely rare. breast cancer represents as a primary site in about 2.5% cases of all metastatic bladder cancer. breast cancer commonly metastasizes to the lung, bone, liver, lymph nodes and skin ; less frequently it involves the brain, adrenal glands, ovary, spleen, pancreas, kidney, thyroid and heart. there are reports on unusual sites of breast cancer metastases ; the urinary bladder and retroperitoneum is considered one of these unusual sites. a 45-year - old premenopausal female patient presented with two months history of recurrent high - grade fever with chills, bilateral pedal edema and oliguria. on examination bilateral pitting pedal edema was present. but due to persistent urinary tract infection and renal failure we removed dj stents and bilateral nephrostomies were kept. contrast enhanced computerized tomography (cect) was suggestive of irregular circumferential moderately enhancing thickening of urinary bladder with hypodense mass lesion in the retroperitoneum with bilateral hydroureteronephrosis [figure 1 ]. cystoscopy showed thick irregular bladder wall with no definitive mass lesion, so random bladder wall biopsies were taken. biopsy was suggestive of undifferentiated adenocarcinoma [figure 2].immunohistochemistry was positive for cytokeratin, ck 7 and er and negative for ck 20 and pr. contrast enhanced computerized tomography suggestive of irregular circumferential moderately enhancing thickening of urinary bladder histopathological examination of bladder biopsy (h and e, x 100) shows metastatic undifferentiated adenocarcinoma, arrow shows tumor cells subsequently, during a search for primary site of bladder metastasis, we found patient having right breast mass. true - cut biopsy from right breast mass was suggestive of invasive lobular carcinoma of breast (nuclear grade ii). after two cycles of chemotherapy patient started passing urine per urethra and nephrostomy output decreased. after nephrostomy removal patient 's serum creatinine remained static at 1.6 mg% and usg showed no hydronephrosis. metastases to urinary bladder are rare, accounting for less than 2% of all bladder tumors, these are mostly found in advanced stages with peritoneal dissemination. information pertaining to bladder metastases is derived largely from autopsy studies, and known primary sites of origin in descending frequency are gastric cancer, malignant melanoma, breast and lung. potential mechanisms contributing to the appearance of secondary bladder tumors could be due to minute viable tumor emboli that pass through the pulmonary circulation without establishing a lung metastasis and subsequently reach the urinary bladder by hematogenous transport. other possible routes are extension from retroperitoneal involvement or dissemination through the lymphatic or arterial circulation. the relative infrequency of primary adenocarcinoma of the bladder causes the dilemma whether bladder adenocarcinoma represents a primary or secondary process. if the adjacent mucosa contains polypoid formation, brunn 's nests, or glandular or mucous metaplasia, a primary bladder lesion is likely. cytokeratin, ck-7, ck-18, ck-19, ck-20 are useful screening markers for the recognition of epithelial differentiation. other specific markers that are commonly used are er/ pr for endometrial and breast carcinoma, ca 19 - 9 for pancreatobiliary malignancy, prostate specific antigen (psa) for prostate, thyroglobulin for thyroid, uroplakin iii for urothelium, and heppar i for hepatocellular. in a retrospective study bates and baithun found 282 secondary urinary bladder metastases in a series of 6289 bladder tumors (about 4.5% of all bladder tumors detected). seven cases of primary breast cancer were found ; bladder metastases were detected post - mortem in six of these seven cases and all of them had metastasized widely. postobstructive renal failure in breast cancer patients can be treated easily by endoureteral catheterization or percutaneous nephrostomy which allows rapid normalization of renal function in most cases and further administration of effective systemic chemotherapy. survival after the onset of distant metastases is relatively short, poulakis. in 2001 reported a patient with breast cancer and urinary bladder involvement still alive at five years from diagnosis. hence, appropriate treatment and follow - up may improve the prognosis of patients with bladder metastases. | breast carcinoma is the most common nondermatologic cancer diagnosis in women. common metastatic sites include lymph nodes, lung, liver, and bone. breast carcinoma metastatic to the bladder has been reported only sporadically. most patients were symptomatic breast cancer with evidence of disseminated disease at the time of diagnosis. metastasis usually occurred many years after diagnosis, and the prognosis was poor. we report a case of breast caricinoma metastasizing to the urinary bladder and retroperitoneum, which presented initially with acute renal failure. patient was treated with bilateral per cuteneous nephrostomies and chemotherapy. starting from this clinical case we review the available literature on this issue. patients with breast cancer presenting with urinary symptoms should be examined for possible bladder metastasis. |
the events of 11 september 2001, and the subsequent mailings of anthraxladen envelopes within the usa, forever changed the way americans view public health and national security. as recently confirmed (the wmd terrorism research center, 2011), despite the investment of considerable financial and human resources since 2001, the usa does not have the range of medical countermeasures (mcms) or established systems to rapidly and effectively respond to a deliberate chemical, biological, radiological or nuclear (cbrn) attack, or to a naturally occurring infectious disease outbreak. mcms are the drugs, vaccines and medical devices (including diagnostic tests, equipment and supplies) that will be needed to respond to a public health emergency, including products to prevent and respond to anthrax, smallpox, radiological / nuclear agents, pandemic influenza and other emerging diseases. in an effort to fill this gap in mcms, product developers and the us government face particular challenges, most of which fit into two major categories : (i) problems facing eager but relatively inexperienced companies conducting mcm research and development and (ii) unique scientific and regulatory issues and uncertainties facing developers and the government arising from the fact that many mcms can not ethically or feasibly be tested in humans, meaning that much, if not all, efficacy data must be derived from animal experimentation. the federal government has a crucial role to play in facilitating mcm development and acquisition and, therefore, in addressing the associated regulatory and scientific challenges. within the department of health and human services (hhs) the office of the assistant secretary for preparedness and response (aspr) leads the public health emergency medical countermeasures enterprise (phemce or enterprise), a collaboration of agencies, such as the centers for disease control and prevention (cdc), national institutes of health (nih), food and drug administration (fda), department of homeland security (dhs) and department of defense (dod), which is working to support and encourage the development, procurement and stockpiling of mcms. in its role as regulator, evaluating medical products for their safety and efficacy, fda has a unique and critical part to play. in 2010, hhs secretary sebelius released the findings of an extensive review of the enterprise and articulated a new strategic mcm vision (hhs, 2010). the review 's recommendations contributed to the august 2010 establishment of fda 's medical countermeasures initiative (mcmi) to facilitate development and availability of highpriority mcms and strengthen the mcm enterprise. implementation of the mcmi is being coordinated by the office of counterterrorism and emerging threats (ocet) in fda 's office of the commissioner. however, the mcmi involves close collaboration, both internally, among the medical product centres (i.e. center for biologics evaluation and research, center for devices and radiological health, center for drug evaluation and research) and externally, between fda and its federal partners and other relevant stakeholders. fda has taken a threepillar approach to fulfilling its overall mcmi mission : pillar i : enhance the mcm regulatory review process ; pillar ii : advance regulatory science for mcm development and evaluation ; and pillar iii : modernize the legal, regulatory and policy framework for an effective public health response (fda, 2011a). this review presents a summary of the key scientific and regulatory challenges facing mcm development, approval and use. it also describes the approaches fda is taking through the mcmi to address these key challenges. the challenges confronting mcm development and availability are more complex than the already complicated process for developing medical products. generally, mcm development and approval must follow fda 's rigorous product review process. yet, in many cases, such as when limited human efficacy data are available, the scientific and regulatory hurdles may be greater than the challenges inherent in typical drug development. in addition, medical product development is very resourceintensive, with estimates of the costs of developing a new medical product ranging from $ 0.8 to more than 1.0 billion (dimasi and grabowski, 2007). although the project bioshield act funded the creation of a government market in 2004 to acquire certain mcms, including those that are not yet licensed or approved, the funds that are available are relatively small compared with the possible return on investment from a blockbuster drug. these challenges and the lack of a commercial market for many cbrn countermeasures have left most companies with extensive experience in meeting the complex regulatory requirements and a successful track record reluctant to take on mcm development. stepping into the void have been smaller biotechnology companies (often startups), which, although often technically strong, have little or no experience advancing drug development through the fda review and approval process (cohen, 2011). it is therefore critical that fda provide more regulatory and scientific guidance to these companies earlier in and throughout the development process than might be the case with larger, more regulatoryexperienced pharmaceutical companies. however, it is extremely resource intensive, and the mcm scientific and technical expertise at fda who can provide this type of assistance and ensure equity in assistance among mcm sponsors is limited. this situation is exacerbated by the unprecedented scientific challenges and uncertainties fda faces when reviewing and evaluating mcm submissions. it is on these scientific challenges that fda has focused initial efforts during the first year of the mcmi. since the inception of mcmi in 2010, fda has increased the human and fiscal resources it devotes to formal and informal meetings related to mcm development, including increasing preinvestigational new drug (ind) meetings and expanding the number of internal scientific and technical consultations on mcmrelated issues., 2007 ; nbsb, 2010) that fda medical product centres may not be sufficiently consistent in their interpretation and implementation of the agency 's various regulations and policy for example in interpreting fda 's 2002 regulation new drug and biological drug products ; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible (animal rule) and its companion draft guidance, essential elements to address efficacy under the animal rule (fda, 2009). in other instances, issues that may often be misidentified as solely regulatory in nature are actually gaps in key scientific knowledge that are hindering fda 's regulatory guidance and decisionmaking abilities. to address these issues and uncertainties, fda has created crossdisciplinary, crossfunctional public health and security action teams (action teams), comprising expertise from all fda medical product centres, including staff from the centre review divisions. the action teams have already increased intraagency collaboration and informational exchange, fostering more uniformity and consistency where possible. action teams are identifying and classifying the types of hurdles and gaps that are impeding mcm product development while providing a vehicle for harmonizing agency communications with federal partners and stakeholders. action team analysis has identified some impediments as primarily scientific knowledge gaps or statutory, regulatory, or policy limitations. scientific gaps can include limited knowledge about a threat agent 's disease process or a proposed mcm (e.g. its safety, efficacy and/or performance) being developed for a particular cbrn indication. as specific scientific gaps are identified, fda is working closely with federal partners in the enterprise to determine the best approach for addressing them, thus informing fda 's mcm regulatory science research agenda. legal, regulatory and policy limitations can include inconsistencies in interpretation and implementation of existing statutes, regulations or policies, or the lack of an appropriate framework for developing innovative mcm products and technologies. as specific limitations are identified, fda is working internally or with hhs, congress and other relevant partners to resolve them. fda has established a number of action teams, based on the highest research and development priorities determined by the enterprise. areas of focus include in vitro diagnostics, acute radiation syndrome, trauma and the warfighter, advanced manufacturing and development, and paediatric and maternal issues. fda is also developing an animal model qualification program to enable a productneutral evaluation and qualification of animal models within a context of use. the process for animal model qualification will be consistent with the process described in the guidance for industry, qualification process for drug development tools, once it has been finalized (fda, 2010c). in 2010, fda launched the advancing regulatory science initiative, and fda product centres established their regulatory science agendas and priorities to support more active participation in scientific research and to advance development of all fda regulated products (fda, 2010a). regulatory science is the science of developing new tools, standards and approaches to assess the safety, efficacy, quality and performance of fdaregulated products. mcm regulatory science focuses on the development and approval of mcms for cbrn threats and emerging infectious diseases (e.g. pandemic influenza, sars). mcmi 's pillar ii is providing oversight and direction for fda 's overall mcm regulatory science portfolio and serves as the conduit for obtaining stakeholder input to shape the research agenda. initially, pillar ii activities focused on addressing centrespecific priorities identified in their respective agendas as well as on mcm priorities determined by fda 's office of the chief scientist. areas were broadly identified as research related to animal models, biomarkers, and product quality and associated assay development, among others. pillar ii has already significantly strengthened and increased fda 's intramural mcm regulatory science research portfolio. first, at mcmi 's launch, fda cosponsored with the institute of medicine a workshop designed to provide a broad overview of existing regulatory science efforts, review the state of the science regarding mcm product development, and identify opportunities for regulatory science collaborations (iom, 2011). next, a steering committee was established to peer review centrespecific regulatory science research proposals seeking fda mcmi funding. the steering committee comprises the fda chief scientist, a scientific lead from each of fda 's three medical product centres, and representatives of enterprise partners (dod, cdc, nih and aspr). proposals are assessed for significance, alignment with enterprise priorities, scientific feasibility and collaborative environment. it is important to note that the collaborative environment assessment includes consideration of current intraagency collaborations, current collaborations with enterprise partners and assessment of opportunities for potential collaboration, thereby leveraging synergies across proposals. third, building on these efforts, fda released a request for information that solicited further stakeholder input to enhance and refine the current mcm regulatory research agenda and shape the pillar ii regulatory science programme (fda, 2011b). fda 's comprehensive regulatory science programme is intended to address the scientific challenges that are slowing the progress of mcms in the development pipeline and generate the data needed to advance products towards approval and availability. as already noted, one of the primary scientific challenges to mcm development is the infeasibility in many cases of conducting human efficacy studies either because there are insufficient or sporadic natural occurrences of a condition or because of ethical concerns (e.g. associated morbidity / mortality). in such cases, product sponsors must pursue approval through nontraditional regulatory pathways, specifically using the animal rule. although it provides an alternative path to approval, the animal rule raises complicated scientific and regulatory questions as animal data are applied in a new way. the animal rule created a need for robust and relevant animal models for product development and evaluation, but in many cases, models do not exist. once models are developed that adequately represent the human condition for specific diseases, the models may need productspecific adaptation and, in some cases, may not be suitable for all products due to speciesspecific differences (e.g. physiology, immunology, pharmacokinetics / pharmacodynamics) or productspecific differences (e.g. immune modulator targeting receptors not present in all species). targeted regulatory science research is needed to bridge interspecies gaps, identify acceptable correlates of protection or develop new methods, such as in vitro or in silico modelling, that will aid in advancing mcm product development. three products have had indications approved under the animal rule : pyridostigmine bromide, hydroxocobalamin and levofloxacin. however, in these cases, the animal rule approval was facilitated by prior approval for another indication or information available in another country. thus, sufficient safety and efficacy data had been developed to augment animal efficacy and other data submitted to fda for approval under the animal rule, underscoring the fact that substituting animal data for human data is not intended to be an easier route towards approval (gronvall., 2007). in fact, experience has proven the contrary to be true : reliance on animal data exponentially increases the scientific complexities involved in mcm development, leading to increased regulatory uncertainties. the federal food, drug, and cosmetic act (fd&c act) gives fda various legal and regulatory authorities and mechanisms that can facilitate mcm development and regulatory review, and even allow emergency use of certain unapproved products under certain conditions. mcm development and approval must follow fda 's rigorous product regulatory review processes : for drugs and biologics, through the investigational new drug application phase and the new drug application or biologics licence application phase ; or, in the case of a device, through premarket approval or notification 510(k). however, in certain situations, accelerated processes or special mechanisms (e.g. priority review ; special protocol assessments ; and the animal rule, as described in the previous section) are needed. additionally, during or in anticipation of an actual emergency, fda can facilitate use of a needed mcm through expanded access mechanisms (fd&c act, 561, 21 u.s.c. 360bbb) or through an emergency use authorization (eua) (fd&c act, 564, 21 u.s.c. some of the special legal and regulatory mechanisms for mcm development, approval, availability and use were established through emergency preparedness legislation enacted after the 2001 anthrax attacks. the animal rule was established in the public health security and bioterrorism preparedness and response act (2002) ; the emergency use authorities are provided for in the project bioshield act (2004) ; and technical assistance teams in the event of mcm shortages are provided for in the pandemic and allhazards preparedness act (2006). as part of fda 's mcmi, legal and regulatory issues are coordinated and addressed through pillar iii activities, the goals of which are to support mcm development and availability by ensuring that us laws, regulations and policies enable the application of advances in regulatory science to the regulatory review process and adequately support us preparedness for and response to cbrn agents and emerging infectious disease threats. in addition, pillar iii staff have been assessing the strengths and weaknesses of the current legal, regulatory and policy environment regarding mcm development, distribution, availability and use. where changes are needed to better protect public health, fda is working with federal partners and relevant stakeholders to develop and propose new approaches to improve and modernize fda 's legal, regulatory and policy framework for effective public health emergency responses. most recently, fda proposed changes as part of the reauthorization of the pandemic and allhazards preparedness act, which would (i) provide enhanced clarity and flexibility for euas prior to a cbrn event to enhance rapid deployment, (ii) better facilitate preevent planning and positioning of medical products, (iii) clarify fda 's authority to extend the shelf life of stockpiled mcms and (iv) clarify that certain actions taken in preparation for or during an emergency will not violate fda laws. also, to support anthrax preparedness and response efforts based on stakeholder needs, fda collaborated with federal partners to issue a mass dispensing eua in july 2011 (fda, 2011c) and to amend the postal model eua in october 2011 (fda, 2011d). in december 2010, fda, in collaboration with federal partners, sponsored a legal and regulatory preparedness meeting to (i) inform state public health preparedness officials and legal counsel on fda 's legal authorities for mcm responses and (ii) become better informed about response challenges state and local public health officials and responders face. as already mentioned, fda is working to clarify and expand the agency 's interpretation and implementation of the animal rule and the companion draft guidance for industry, animal models essential elements to address efficacy under the animal rule (fda, 2009). fda has created a crosscentre, multidisciplinary team that is carefully considering the numerous comments received during the public comment period following publication of the draft guidance. additional comments were received during and after a subsequent public meeting on the draft guidance in november 2010 (fda, 2010b). in response to the significant revisions requested by the community and expansive scope of the comments, fda intends to publish the guidance as a revised draft, enabling a second comment period. the revised and expanded guidance should provide additional scientific and regulatory information to support a better understanding of the specific expectations for animal data intended to support approval across the agency. since the inception of mcmi in 2010, fda has increased the human and fiscal resources it devotes to formal and informal meetings related to mcm development, including increasing preinvestigational new drug (ind) meetings and expanding the number of internal scientific and technical consultations on mcmrelated issues., 2007 ; nbsb, 2010) that fda medical product centres may not be sufficiently consistent in their interpretation and implementation of the agency 's various regulations and policy for example in interpreting fda 's 2002 regulation new drug and biological drug products ; evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible (animal rule) and its companion draft guidance, essential elements to address efficacy under the animal rule (fda, 2009). in other instances, issues that may often be misidentified as solely regulatory in nature are actually gaps in key scientific knowledge that are hindering fda 's regulatory guidance and decisionmaking abilities. to address these issues and uncertainties, fda has created crossdisciplinary, crossfunctional public health and security action teams (action teams), comprising expertise from all fda medical product centres, including staff from the centre review divisions. the action teams have already increased intraagency collaboration and informational exchange, fostering more uniformity and consistency where possible. action teams are identifying and classifying the types of hurdles and gaps that are impeding mcm product development while providing a vehicle for harmonizing agency communications with federal partners and stakeholders. action team analysis has identified some impediments as primarily scientific knowledge gaps or statutory, regulatory, or policy limitations. scientific gaps can include limited knowledge about a threat agent 's disease process or a proposed mcm (e.g. its safety, efficacy and/or performance) being developed for a particular cbrn indication. as specific scientific gaps are identified, fda is working closely with federal partners in the enterprise to determine the best approach for addressing them, thus informing fda 's mcm regulatory science research agenda. legal, regulatory and policy limitations can include inconsistencies in interpretation and implementation of existing statutes, regulations or policies, or the lack of an appropriate framework for developing innovative mcm products and technologies. as specific limitations are identified, fda is working internally or with hhs, congress and other relevant partners to resolve them. fda has established a number of action teams, based on the highest research and development priorities determined by the enterprise. areas of focus include in vitro diagnostics, acute radiation syndrome, trauma and the warfighter, advanced manufacturing and development, and paediatric and maternal issues. fda is also developing an animal model qualification program to enable a productneutral evaluation and qualification of animal models within a context of use. the process for animal model qualification will be consistent with the process described in the guidance for industry, qualification process for drug development tools, once it has been finalized (fda, 2010c). in 2010, fda launched the advancing regulatory science initiative, and fda product centres established their regulatory science agendas and priorities to support more active participation in scientific research and to advance development of all fda regulated products (fda, 2010a). regulatory science is the science of developing new tools, standards and approaches to assess the safety, efficacy, quality and performance of fdaregulated products. mcm regulatory science focuses on the development and approval of mcms for cbrn threats and emerging infectious diseases (e.g. pandemic influenza, sars). mcmi 's pillar ii is providing oversight and direction for fda 's overall mcm regulatory science portfolio and serves as the conduit for obtaining stakeholder input to shape the research agenda. initially, pillar ii activities focused on addressing centrespecific priorities identified in their respective agendas as well as on mcm priorities determined by fda 's office of the chief scientist. areas were broadly identified as research related to animal models, biomarkers, and product quality and associated assay development, among others. pillar ii has already significantly strengthened and increased fda 's intramural mcm regulatory science research portfolio. first, at mcmi 's launch, fda cosponsored with the institute of medicine a workshop designed to provide a broad overview of existing regulatory science efforts, review the state of the science regarding mcm product development, and identify opportunities for regulatory science collaborations (iom, 2011). next, a steering committee was established to peer review centrespecific regulatory science research proposals seeking fda mcmi funding. the steering committee comprises the fda chief scientist, a scientific lead from each of fda 's three medical product centres, and representatives of enterprise partners (dod, cdc, nih and aspr). proposals are assessed for significance, alignment with enterprise priorities, scientific feasibility and collaborative environment. it is important to note that the collaborative environment assessment includes consideration of current intraagency collaborations, current collaborations with enterprise partners and assessment of opportunities for potential collaboration, thereby leveraging synergies across proposals. third, building on these efforts, fda released a request for information that solicited further stakeholder input to enhance and refine the current mcm regulatory research agenda and shape the pillar ii regulatory science programme (fda, 2011b). fda 's comprehensive regulatory science programme is intended to address the scientific challenges that are slowing the progress of mcms in the development pipeline and generate the data needed to advance products towards approval and availability. as already noted, one of the primary scientific challenges to mcm development is the infeasibility in many cases of conducting human efficacy studies either because there are insufficient or sporadic natural occurrences of a condition or because of ethical concerns (e.g. associated morbidity / mortality). in such cases, product sponsors must pursue approval through nontraditional regulatory pathways, specifically using the animal rule. although it provides an alternative path to approval, the animal rule raises complicated scientific and regulatory questions as animal data are applied in a new way. the animal rule created a need for robust and relevant animal models for product development and evaluation, but in many cases, models do not exist. once models are developed that adequately represent the human condition for specific diseases, the models may need productspecific adaptation and, in some cases, may not be suitable for all products due to speciesspecific differences (e.g. physiology, immunology, pharmacokinetics / pharmacodynamics) or productspecific differences (e.g. immune modulator targeting receptors not present in all species). targeted regulatory science research is needed to bridge interspecies gaps, identify acceptable correlates of protection or develop new methods, such as in vitro or in silico modelling, that will aid in advancing mcm product development. three products have had indications approved under the animal rule : pyridostigmine bromide, hydroxocobalamin and levofloxacin. however, in these cases, the animal rule approval was facilitated by prior approval for another indication or information available in another country. thus, sufficient safety and efficacy data had been developed to augment animal efficacy and other data submitted to fda for approval under the animal rule, underscoring the fact that substituting animal data for human data is not intended to be an easier route towards approval (gronvall., 2007). in fact, experience has proven the contrary to be true : reliance on animal data exponentially increases the scientific complexities involved in mcm development, leading to increased regulatory uncertainties. the federal food, drug, and cosmetic act (fd&c act) gives fda various legal and regulatory authorities and mechanisms that can facilitate mcm development and regulatory review, and even allow emergency use of certain unapproved products under certain conditions. mcm development and approval must follow fda 's rigorous product regulatory review processes : for drugs and biologics, through the investigational new drug application phase and the new drug application or biologics licence application phase ; or, in the case of a device, through premarket approval or notification 510(k). however, in certain situations, accelerated processes or special mechanisms (e.g. priority review ; special protocol assessments ; and the animal rule, as described in the previous section) are needed. additionally, during or in anticipation of an actual emergency, fda can facilitate use of a needed mcm through expanded access mechanisms (fd&c act, 561, 21 u.s.c. 360bbb) or through an emergency use authorization (eua) (fd&c act, 564, 21 u.s.c. some of the special legal and regulatory mechanisms for mcm development, approval, availability and use were established through emergency preparedness legislation enacted after the 2001 anthrax attacks. the animal rule was established in the public health security and bioterrorism preparedness and response act (2002) ; the emergency use authorities are provided for in the project bioshield act (2004) ; and technical assistance teams in the event of mcm shortages are provided for in the pandemic and allhazards preparedness act (2006). as part of fda 's mcmi, legal and regulatory issues are coordinated and addressed through pillar iii activities, the goals of which are to support mcm development and availability by ensuring that us laws, regulations and policies enable the application of advances in regulatory science to the regulatory review process and adequately support us preparedness for and response to cbrn agents and emerging infectious disease threats. in addition, pillar iii staff have been assessing the strengths and weaknesses of the current legal, regulatory and policy environment regarding mcm development, distribution, availability and use. where changes are needed to better protect public health, fda is working with federal partners and relevant stakeholders to develop and propose new approaches to improve and modernize fda 's legal, regulatory and policy framework for effective public health emergency responses. most recently, fda proposed changes as part of the reauthorization of the pandemic and allhazards preparedness act, which would (i) provide enhanced clarity and flexibility for euas prior to a cbrn event to enhance rapid deployment, (ii) better facilitate preevent planning and positioning of medical products, (iii) clarify fda 's authority to extend the shelf life of stockpiled mcms and (iv) clarify that certain actions taken in preparation for or during an emergency will not violate fda laws. also, to support anthrax preparedness and response efforts based on stakeholder needs, fda collaborated with federal partners to issue a mass dispensing eua in july 2011 (fda, 2011c) and to amend the postal model eua in october 2011 (fda, 2011d). in december 2010, fda, in collaboration with federal partners, sponsored a legal and regulatory preparedness meeting to (i) inform state public health preparedness officials and legal counsel on fda 's legal authorities for mcm responses and (ii) become better informed about response challenges state and local public health officials and responders face. as already mentioned, fda is working to clarify and expand the agency 's interpretation and implementation of the animal rule and the companion draft guidance for industry, animal models essential elements to address efficacy under the animal rule (fda, 2009). fda has created a crosscentre, multidisciplinary team that is carefully considering the numerous comments received during the public comment period following publication of the draft guidance. additional comments were received during and after a subsequent public meeting on the draft guidance in november 2010 (fda, 2010b). in response to the significant revisions requested by the community and expansive scope of the comments, fda intends to publish the guidance as a revised draft, enabling a second comment period. the revised and expanded guidance should provide additional scientific and regulatory information to support a better understanding of the specific expectations for animal data intended to support approval across the agency. as identified in the 2010 enterprise review, fda has a crucial role to play in ensuring the success of the us enterprise mission and vision to create a nimble and flexible system to produce the mcms that will be needed quickly, should an attack occur. fda 's mcmi was launched to help address key challenges associated with the regulatory review process, the gaps in mcm regulatory science, and hurdles in the legal, regulatory and policy framework that may be slowing mcm development. filling the nation 's mcm gap is a longterm, complex effort that will require substantial collaboration among governmental entities at all levels, academia, industry and health professionals. in particular, success will require an appreciation of the long timelines, risks and high costs associated with developing mcms, a significant and ongoing investment of resources, and the commitment of our national leadership. | summarydespite substantial investments since the events of 2001, much work remains to prepare the nation for a chemical, biological, radiological or nuclear (cbrn) attack or to respond to an emerging infectious disease threat. following a 2010 review of the us public health emergency medical countermeasures enterprise, fda launched its medical countermeasures initiative (mcmi) to facilitate the development and availability of medical products to counter cbrn and emerging disease threats. as a regulatory agency, fda has a unique and critical part to play in this national undertaking. using a threepillar approach, fda is addressing key challenges associated with the regulatory review process for medical countermeasures ; gaps in regulatory science for mcm development and evaluation ; and issues related to the legal, regulatory and policy framework for an effective public health response. filling the gaps in the mcm enterprise is a huge national undertaking, requiring the collaboration of all stakeholders, including federal partners, current and prospective developers of medical countermeasures, relevant research organizations, and state and local responders. especially critical to success are an appreciation of the long timelines, risks and high costs associated with developing medical countermeasures and the systems to deliver them and the requisite support of all stakeholders, including national leadership. |
hiv-1 associated neurocognitve disorders (hand) commonly follow progressive virus infection in the infected human host.(1) clinical disease features range in severity from subtle deficits to incapacitating dementia and include asymptomatic neurocognitive impairment, mild neurocognitive disorder, and hiv - associated dementia (had).(2) severe dementia is now rare since the use of antiretroviral therapy (art).(3) notably, cognitive impairment is associated, in measure, with immune suppression and a vicious cycle of viral replication, ingress of perivascular macrophages, and neuroinflammation.(4) substantive neuropathological findings, in the era of art, are diminished and described as neuronal dysfunction without loss.(5) the control and diagnosis of hand remains poorly understood despite considerable research efforts.(6) recent research demonstrated a role for adaptive immunity and specifically cd4 + t cells in neurodegenerative diseases. works performed by others and in our laboratories, both in murine models of hand and parkinson s disease (pd), demonstrated that cd4+cd25 + treg serve to attenuate brain mononuclear phagocyte (mp ; macrophage and microglia) inflammation and sustain neuroprotection. nonetheless, the mechanism underlying such treg - associated neuroprotective functions remains incompletely understood.(12) primary roles for treg rests in the cells abilities to provide self - tolerance.(13) acting as a suppressor, its role in hiv disease remains highly controversial. indeed, it has been suggested that treg play detrimental roles since they suppress the hiv - specific immune response and inhibit virus clearance. in contrast, others posit a beneficial role for treg through suppression of immune hyperactivation and control of viral load. in support of the latter is treg s known role in simian immunodeficiency virus (siv) infection in sooty mangabeys and its function as an elite suppressor to human immunodeficiency virus (hiv) infection. the similarity shared in these reports rests in maintenance of treg frequency and function to limit generalized immune activation commonly seen during advanced hiv disease.(26) we now demonstrate a multifaceted role for treg for hiv-1-infected mp. such a role serves a range of regulatory functions acting to control disease such as suppression of viremia and modulation of hiv-1-infected macrophage neurotoxic activities. in support of such claims, we used isobaric tag for relative and absolute quantitation (itraq) of proteins to identify differential protein expression between hiv-1-infected bone marrow - derived macrophages (bmm) with and without treg. we found changes consistent with enhanced antiretroviral immunity through treg - induced upregulation of interferon (ifn)-induced gene products. this was seen among a broad range of related protein expression changes linked to antiviral responses. second, we showed that treg utilizes caspase-3 and granzyme / perforin pathways to kill virus - infected cells. third, treg reduced neurotoxic secretions in vitro through its abilities to transform hiv-1-infected macrophages from an m1 to an m2 phenotype. these observations made in murine treg - bmm cocultures were replicated separately in human treg - monocyte - derived macrophages (mdm) cocultures. taken together, these data demonstrate that treg serve as effectors for virus - infected macrophages and suppressors for inflammation and as such, exert immune surveillance functions relevant to ongoing hiv infection and neuroaids. c57bl/6j male mice (810 wk old) were purchased from the jackson laboratory and used for bmm and t cell isolations. all animal procedures were in accordance with the national institutes of health guidelines and were approved by the institutional animal care and use committee of the university of nebraska medical center. femurs of the mice were excised and flushed with dulbecco s phosphate buffered saline (dpbs) to obtain bone marrow - derived mononuclear cells. cells were passed through a 40 m cell strainer to remove the clumps and then centrifuged. erythrocytes were removed using ack lysis buffer (gibco, grand island, ny). after washing twice with dbps, cells were resuspended and plated in 6-well plates at 1 10 cells / ml in 3 ml complete medium [rpmi 1640 supplemented with 10% fetal bovine serum (fbs), 2 mm l - glutamine, 10 mm hepes, 1 nonessential amino acids, 50 m 2-mercaptoethanol, 100 u / ml penicillin, 100 g / ml streptomycin, and 2 g / ml macrophage colony stimulating factor, mcsf (pfizer, cambridge, ma) ]. after 7 days differentiation, cells were > 98% cd11b as determined by flow cytometry (supplemental figure 1a). vsv pseudotyped hiv-1, yu2 (hiv-1/vsv) was used to circumvent the required cellular receptors necessary for hiv-1 to infect mouse cells. the hiv-1yu2/vsv pseudotypes were generated by cotransfection of 3 g of pyu2 and 1 g of phit / g into 293 t cells (per 1 10 cells) using the fugene 6 transfection reagent (roche diagnostics, indianapolis, in). pseudotyped virus stock was collected after 72 h post - transfection. because the pseudotyped virus only contains hiv-1yu2 genes, but not the glycoprotein gene of vsv, they could only enter the mouse bmm once. the concentration of the virus stock was determined by hiv-1p24 elisa kit (perkinelmer, boston, ma). we used a range of infective viral doses including 1, 2, and 3 pg of hiv-1p24/cell to infect bmm. this was done to ensure a data set independent of multiplicity of infection (moi). after 24 h, the percentage of p24 positive cells was determined by immunohistochemistry. as shown in supplemental figures 1b and 1c at 1 pg of hiv-1p24/cell > further, the level of hiv-1p24 staining increased in parallel to the infective dose. in subsequent experiments, virus was placed into the media for 24 h then removed by vigorous washings with phosphate buffered saline (pbs).(27) cd4 + t cell subsets were isolated using previously described techniques. briefly, immune cells were isolated from the spleens and lymph nodes (inguinal, brachial, axillary, cervical and mesenteric) by dissection, followed by mechanical dissociation by pushing the organs through a 70 m mesh filter. cells were washed with dpbs, erythrocytes were removed by the ack lysis buffer (gibco) and cells washed twice more with pbs. the processed cells were then passed through a mouse t cell enrichment column (r&d systems, minneapolis, mn) for t cell negative selection, followed by passage of the purified t cells through a mouse t cell cd4 subset column (r&d systems) for negative selection of cd4 + t cells. cd4 + t cells were then labeled with monoclonal antimouse cd25-pe antibody, then reacted with anti - pe microbeads (miltenyi biotec, auburn, ca) and passed through the macs column in a magnetic field for positive selection of cd25 + cells. the flow through containing cd4+cd25- t cells was used in these experiments and propagated as cd3/cd28-activated conventional t cells (tcon) (see below). the isolated cd4+cd25 + cells retained on the column and then eluted were used as treg (supplemental figure 2a, supporting information). +) pure (supplemental figure 2bd, supporting information), respectively. treg and tcon were expanded ex vivo using cd3/cd28 t cell expander dynabeads (invitrogen dynal, oslo, norway). the dynabeads were added to cells plated in 24 well plates at 1 10 cells / ml in complete rpmi along with 1000 u / ml mouse ril-2 (r&d systems) for treg cultures ; whereas, 100 u / ml mouse ril-2 was used for tcon cell cultures. cells were expanded in culture for 10 days with half - media changes every 23 days. treg remained > 83% pure after expansion as determined by cd4+foxp3 + staining (supplemental figure 2c, d, supporting information). the function of treg was evaluated by lymphocyte proliferation suppression assay as described below (supplemental figure 2e, f, supporting information). mouse and human t cells were cocultured only with same species mouse and human macrophages. peripheral blood mononuclear cells (pbmc) from hiv-1, hiv-2, and hepatitis seronegative human donors were obtained by leukophoresis in full compliance and approval of the university of nebraska medical center institution review board. monocytes were purified by countercurrent centrifugal elutriation.(30) cells were cultured in complete rpmi medium and maintained in a 37 c and 5% co2 atmosphere. on days 2 and 5, half of the medium was exchanged. after seven days in culture, mdm were infected with hiv-1ada (a macrophage tropic viral strain) at a moi of 1 or left untreated (controls) in media devoid of mcsf. on day 1 after infection, a full medium exchange was performed followed by a half medium exchange every 23 days thereafter in medium devoid of mcsf. on day 7 after infection, cells were cocultured with activated tcon or treg (1:1) for an additional 24 h. the 1:1 ratio of t cells to macrophages was used based upon our prior data from effector cell optimization. moreover, further additions of tregs in virus - infected macrophage cocultures would eliminate all virus - infected cells and preclude any subsequent proteomic testing (data not shown). cd4/cd8 t cell ratios from donor blood were determined prior to starting all experiments with human cells. human tregs were isolated from pbmc using the cd4+cd25 + regulatory t cell isolation kit (miltenyi biotec). briefly, human tregs were isolated by first depleting all non - cd4 + cells by indirect magnetic labeling of cells with a cocktail of biotin - conjugated monoclonal antihuman antibodies against cd8, cd14, cd16, cd19, cd36, cd56, cd123, tcr/ and cd235a ; adding antibiotin magnetic microbeads ; and then passing cells through a magnetic column and collecting the cd4 + enriched fraction. then cd4+cd25 + t cells were isolated by positive selection using magnetic microbeads conjugated to monoclonal anti - cd25 antibody and passing the cells over the magnetic column. the eluted cd4+cd25- cells were used as tcon, and the retained cd4+cd25 + cells were used as treg in these experiments. purity was determined by cd25-pe staining and detection by flow cytometry (supplemental figure 3a, supporting information). treg and tcon cells were expanded and activated ex vivo using human treg expander dynabeads (invitrogen, carlsbad, ca). the function of expanded treg was evaluated by lymphocyte proliferation suppression assay (supplemental figure 3b, supporting information). cells (1 10) were labeled with fluorescently labeled antibodies to cd3, cd4, cd8, cd11b (ebioscience, san diego, ca), cd25 (bd pharmingen, san diego, ca), and cd39 (r&d systems). for cd39 staining, alexa fluor 488 donkey antisheep secondary antibody (invitrogen) was used. for intracellular staining, cells were fixed, permeabilized (foxp3 staining kit, ebioscience, san diego, ca) and stained for hiv-1p24 (beckman coulter, brea, ca), mouse foxp3 (ebioscience), granzyme a (santa cruz biotechnology, santa cruz, ca), granzyme b (ebioscience) and perforin (ebioscience). all the data were acquired on facscalibur flow cytometer and analyzed with cellquest software (becton dickinson, franklin lakes, nj). for lymphocyte proliferation suppression assay (supplemental figures 2e, f, and 3b, supporting information), tcon were incubated with 1 m carboxyfluorescein diacetate, succinimidyl ester (cfse) per 10 10 cells for 15 min and washed twice with rpmi 1640 culture medium. excluding the control group, tcon were cultured alone or with treg at 2-fold escalating ratios in flat - bottom 96-well plates with addition of dynabeads mouse t - activator cd3/cd28. after 72 h, cells were collected, dynabeads were removed by dynamag15 magnet (invitrogen), and cfse was measured by flow cytometry. after 7 days differentiation, bmm were replated at 2 10 cells per well in 6-well plates. hiv-1-infected bmm were cocultured with dynabead - activated tcon or treg at a ratio of 1:1. twenty - four hours later, culture supernatants were collected as conditioned medium 1 (cm1), which contained soluble molecules from macrophages and/or t cells. t cells were removed by pbs washing and 2 ml fresh medium were added to each well. after another 24 h, culture supernatants were collected as conditioned medium 2 (cm2), which contained soluble molecules from macrophages only. the cytokines and virions in conditioned medium were detected by cytometric bead array mouse inflammation kit (bd biosciences, san jose, ca) and alliance hiv-1p24 elisa kit (perkinelmer, boston, ma), respectively, according to manufactures protocols. for the cell lysates, macrophages were washed using pbs and then air - dried in a laminar flow hood. for each plate, cells were lysed by 500 l ice - cold lysis buffer containing 0.05% sodium dodecyl sulfate (sds, bio - rad, hercules, ca) and 0.5 m triethylammonium bicarbonate (teab, sigma - aldrich, st. lysates were clarified by centrifugation at 14 000 g at 4 c for 45 min. for hiv-1p24 immunochemistry staining, mouse bmm or human mdm were fixed and permeabilized with acetone : methanol (1:1, precooled to 20 c) for 15 min at room temperature (rt). cells were washed 3 times with dpbs for 5 min each, then blocked (10% normal goat serum in dpbs) for 30 min at rt. cells were incubated with mouse anti - hiv-1 p24 antibody (1:20, dako, carpinteria, ca) in blocking buffer at 4 c overnight. cells were washed with dpbs and incubated with secondary antibody according to envision kits (dako). briefly, samples were incubated with horse radish peroxidase (hrp) conjugated polymer for 30 min at rt. after washing with dpbs, samples were incubated with hrp substrate 3,3-diaminobenzidine (dab) substrate - chromogen for 510 min at rt, which resulted in brown - colored precipitate in hiv-1/vsv infected groups. pictures were taken with a light microscope (nikon eclipse ts100). for detection of intracellular proteins after treatment, bmm were fixed by 4% paraformaldehyde (pfa) at 37 c for 15 min and permeabilized by 0.2% triton x-100 in dpbs at 4 c for 30 min. nonspecific activity was blocked by incubation in 5% bovine serum albumin (bsa) in dpbs at room temperature for 1 h. cells were incubated with fluorescence labeled antibodies (granzyme b and perforin) and unlabeled primary antibodies (tubulin and granzyme a) at rt for 3 h followed by staining with alexa fluor 488 goat antimouse igg or alexa fluor 546 goat antimouse igg (invitrogen), respectively, at rt for 2 h. as for actin staining, we used alexa fluor 546 phalloidin. after washing, cells were mounted with prolong gold antifade reagent (invitrogen) with dapi and analyzed by zeiss 510 meta confocal laser scanning microscope. human mdm detection of apo2.7 was also stained similarly using a phycoerythrin (pe) conjugated primary antibody (1:10, beckman coulter, brea, ca). for t cell intracellular staining of granzyme a, b and perforin, 2 10 cells were sedimented on shandon microscope slides (thermo fisher, waltham, ma) by centrifugation at 1200 rpm for 5 min then covered with prolong gold antifade mounting solution (invitrogen). for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay, tunel and flica caspase assays, cells were replated at 2 10 cells / well in 8-well polystyrene culture slides for 24 h. cells were infected with hiv-1/vsv for 24 h, then cells were washed and cocultured with t cells for 24 h before adding probes. the cytotoxic effects of tcon versus treg secreted factors on hiv-1/vsv infected bmm after 24, 48, and 72 h in transwell cocultures were determined by mtt (sigma) assay.(10) mitochondrial membrane integrity was assessed by jc-1 (calbiochem, san diego, ca) cell stainings following the manufacturer s suggested protocol. caspase 1 and caspase 3/7 activities were measured using the carboxyfluoroescein flica assay (immunochemistry technologies, bloomington, mn) according to the manufacturer s protocol. tunel was performed using the in situ cell death detection kit, ap (roche applied science, indianapolis, in) according to the manufacturer s protocol. briefly, bmm were washed by dpbs, fixed by 4% pfa and permeabilized by 0.1% subsequently, cells were incubated with tunel working solution, which contains terminal deoxynucleotidyl transferase (tdt) and fluorescein - dutp, at 37 c for 1 h. apoptotic cells were identified as green fluorescent tunel positive cells by fluorescence microscopy and were normalized to total number of cells as determined by bright field images. protein concentrations of the cell lysates were quantified with 2-d quant kit (ge healthcare, piscataway, ma). proteins were further reduced, alkylated, digested and itraq labeled using itraq reagents multiplex kit (applied biosystems, carlsbad, ca) according to the manufacturer s protocol. briefly, proteins were reduced with 5 mm tris-(2-carboxyethyl)phosphine (tcep) at 60 c for 1 h and blocked with 10 mm methyl methanethiosulfonate (mmts) at rt for 10 min. then, the proteins were digested by 10 g trypsin at 37 c for 1618 h. peptides were then labeled with itraq reagent (114 for control, 115 for hiv-1/vsv, 116 for hiv-1/vsv cocultured with tcon, 117 for hiv-1/vsv cocultured with treg) at rt for 1 h, followed by adding 100 l milli - q water to each group to stop labeling reaction. the labeled peptides from the four groups were combined and dried in a speed vac (thermo, waltham, ma) to obtain a brown pellet, indicating efficient labeling. for isoelectric point (pi)-based peptide separation, we used the 3100 offgel fractionator (agilent technologies, santa clara, ca, usa) with a 12-well setup. prior to electrofocusing, pooled peptides were desalted using a mixed mode cation exchange cartridge (waters, milford, ma). peptides were resuspended in a final volume of 1.8 ml 1 offgel sample solution. the ipg gel strip (agilent technologies, santa clara, ca) with a 310 linear ph range was rehydrated with rehydration solution for 15 min according to the protocol of manufacturer. then after fractionation, samples were cleaned up using c-18 spin column (pierce, rockford, il). finally, samples were dried in a speed vac and stored at 80 c until ready for analysis by matrix - assisted laser desorption / ionization tandem mass spectroscopy (maldi - ms / ms). peptide samples were spotted using the off - line tempo c maldi spotting system (applied biosystems, carlsbad, ca). then, ms and ms / ms were performed using the 4800 maldi - tof / tof (time of flight) mass spectrometer (applied biosystems). the tempo lcmaldi robotic spotting system equipped with a c18 reversed phase capillary column (ab sciex, foster city, ca) was used to further fractionate peptides from offgel fractions, followed by data acquisition using 4800 maldi tof / tof (ab sciex, foster city, ca) as previously published.(31) briefly, after using an in - house packed c18 column to separate fractions by hplc gradient based on hydrophobicity, lc fractions were spotted onto maldi 1232-spot format plates, with a spotting interval of 24 s and applying 2.8 kv plate voltage. data was acquired from lcmaldi spot fractions using 4800 maldi tof / tof equipped with a 200 hz repetition rate nd : yag laser. spectrum from a total of 800 laser shots was accumulated for each tof ms spectrum between 8004000 m / z. data dependent ms / ms mode was operated using cid gas and 2 kv collision energy ; programmed laser stop conditions were employed for the accumulation of ms / ms spectra from 8004000 laser shots. eighteen - day - old embryonic fetuses were obtained from terminally anesthetized pregnant c57bl/6j mice.(9) cerebral cortices were dissociated and digested into a single - cell suspension, which was then cultured at a density of 2.0 10 cells / well in poly - d - lysine - coated 24-well plates with complete neurobasal medium supplemented with 2% b27, 1% penicillin / streptomycin, and 0.5 mm l - glutamine (invitrogen, carlsbad, ca). twenty - four hours later, neurons were fixed / permeabilized and then stained for tubulin. dendrite morphology was observed by fluorescent microscopy (zeiss, thornwood, ny). for western blots, 10 g of protein from each total cell lysate was electrophoretically separated on nupagenovex 412% bis - tris gels (invitrogen) and electrotransfered to polyvinylidene membranes (bio - rad, hercules, ca). membranes were incubated with respective primary antibodies at 4 c overnight and hrp - conjugated secondary antibodies (1:2000, santa cruz biotechnology, santa cruz, ca) at rt for 1 h. hrp activity was visualized by using a supersignal west pico chemiluminescent substrate (pierce, rockford, il). band intensity was measured using imagej and normalized to -actin (1:1000, santa cruz biotechnology). antibodies used for western blot assays included murine anti - inos (1:500) and arg-1 (1:1000, bd transduction laboratories, franklin lakes, nj), caspase-1 (1:500, santa cruz biotechnology), caspase-3 (1:1000, cell signaling, danvers, ma), ndufs3 (1:1000, invitrogen, carlsbad, ca, usa), phospho - p38 (1:500, santa cruz biotechnology), perforin (1:500, ebioscience, san diego, ca, usa), stat1 (1:2000, santa cruz biotechnology), isg15 (1:1000, cell signaling, danvers, ma, usa), and hiv-1p24 (1:500, dako, carpinteria, ca). detection of hiv-1p24 by elisa from culture supernatants was used to assay viral particle release according to the manufacturer s protocol (perkinelmer, boston, ma). intra- and extracellular camp was quantified using the direct cyclic amp enzyme immunoassay (assay designs, plymouth meeting, pa). no was measured in culture supernatants using the griess reagent system (promega, madison, wi). statistical analyses were performed by one - way anova with bonferroni posttest and student s t test by using graphpad prism version 4.0c for macintosh (graphpad software, san diego, ca). strict filtering of itraq raw data was accomplished by log - transforming the raw data from 3 independent experiments then normalizing using a two - stage linear model macros written by mayo clinic.(32) protein / peptide records were excluded from further analysis if the corresponding confidence values were less than 50, or less than 10 copies of peptide records across all three experiments per corresponding proteins were available. a one - way anova with unequal variances was fitted to the corresponding normalized data for each protein. the associated p - values from ratios of labeled samples (from t test) were adjusted controlling for false discovery rate (fdr) via a method proposed by benjamini and hochberg(33) and those less than 0.05 were reported as differentially regulated proteins (strictly filtered data). treg subverts brain mp functions and elicits neuroprotective activities in a hiv-1/vsv murine model of hiv encephalitis (hive) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of parkinson s. to determine the protein composition up- or down - regulated in infected bmm cocultivated with treg we used itraq assays followed by offgel fractionation and maldi tof / tof mass spectrometry. hiv-1/vsv infected bmm in solitary culture and with tcon were also used for comparisons (supplemental table 2, supporting information). analyses of proteomic changes in cocultures were done using comparisons to hiv - infected bmm alone in order to distinguish changes in the proteome that occur in the setting of hiv due to t cell interactions / responses from those that occur solely because of hiv infection. a combination treatment with treg and tcon was not used as this would represent total cd4 + t cell populations and preclude analyses of cd4 + t cell subsets. after strict data filtering (described in materials and methods) we identified 293 proteins changed upon hiv-1/vsv infection compared to uninfected bmm, 21 proteins changed in treg cocultures compared to hiv-1/vsv infection alone, and 20 proteins were differentially regulated in tcon cocultures compared to hiv-1/vsv infection alone. proteome alterations due to treg treatment were strongly associated with antiviral immune responses and apoptosis. in addition, proteins involved in cell shape / motility and metabolism were also uncovered (table 1 and supplementary table 2). these changes pointed to an underappreciated role for treg as effector cells and as such led us to explore aspects of antiviral immunity and induced apoptosis for the infected bmm as the proteome changes suggested. protein quantitation was made using itraq as performed on cell lysates of bmm from three separate experiments. these experiments compared hiv-1/vsv infected murine bmm that were cocultured with murine treg (117 label) to hiv-1/vsv infected murine bmm cultured alone (115 label). p - value after benjamini and hochberg fdr adjustment. to elucidate the mechanisms by which treg could alter functional outcomes for hiv-1-infected macrophages, we first focused on validating the enhanced antiviral immune response supported by protein changes observed after strict filtering of the itraq data. treg coculture resulted in upregulation of bmm expression of signal transducer and activator of transcription 1 (stat1) and isg15 (figure 1a, b) found in type i ifn antiviral signaling.(34) since isg15 was reported to inhibit release of hiv-1 virions, we assayed whether the increased isg15 expression observed resulted in reduced virion release.(34) hiv-1p24 levels in bmm cell lysates and culture supernatants were measured by western blotting and elisa. no changes in hiv-1p24 were observed between hiv-1/vsv infected bmms and hiv-1/vsv infected bmms cocultured with cd3/cd28-activated treg or tcon in cell lysates (figure 1c). this reflected that the pseudotyped virus is not capable of spreading viral infection, so the level of hiv-1p24 is dependent only on the initial infective dose (supplemental figures 1b, c, supporting information). however, elisa tests of culture supernatant fluids showed both tcon and treg inhibited virus release by reductions in hiv-1p24 concentrations (figure 1d). moreover, treg more than tcon (the inhibition rate is 79 versus 45%, respectively) inhibited hiv-1p24 release in culture fluids. we posit that such findings could result from stimulation of autocrine / paracrine pathways linked to innate ifn antiviral immunity regulated by treg (figure 1e). treg inhibition of virus release from infected bmm is interferon - stimulated gene 15 pathway associated. (a) western blotting showed increased stat1 phosphorylation in hiv-1/vsv infected bmm cocultured with treg group. (b) western blotting showed dramatically increased free isg15 and isgylated proteins in hiv-1/vsv infected bmm cocultured with treg group. (c) western blotting showed no change of hiv-1 p24 expression between hiv-1/vsv infected bmm group, hiv-1/vsv infected bmm group cocultured with tcon group and hiv-1/vsv infected bmm group cocultured with treg group. in this study the pseudotyped virus we used could enter the mouse bmm once, so the level of p24 staining is dependent only on the infective dose. data shown are representative of three independent experiments. (d) both tcon and treg could inhibit hiv-1 release determined by hiv-1 p24 elisa. data shown are representative of three independent experiments. p < 0.001. (e) proposed pathways involved in initiating antiviral immune response in hiv-1-infected macrophages, which was enhanced by treg and tcon. in macrophages, tlr3 induces a trif - dependent pathway, which recruits kinases (tbk1, ikk and ikk) that mediate activation of the transcription factors irf3 and nf-b. together with irf7, irf3 and nf-b translocate into the nucleus and bind to positive regulatory domains on the ifn genes promoter, leading to ifn transcription. secreted ifns signal through binding to their cognate receptors (ifnar1 and ifnar2) and then recruit kinases (tyk2 and jak1) leading to translocation of phosphorylated transcription factors (stat1 and stat2) and irf9 to the nucleus where binding to the enhancer region of the ifn promoter and isg promoter occurs, which leads to the activation and nuclear transport of isgf3 and induction of isre to produce ifns and isgs. both ifns and isgs could inhibit ubiquitination of hiv gag and tsg101, which results in less virus release. with unknown mechanisms, treg exhibit much greater capacity to enhance the antiviral immune response than tcon. in the past decade following treg discovery, substantive research has focused on the mechanisms for immune tolerance by treg through its inhibitory molecule expression and secretion, such as ctla-4, gitr, il-10 and tgf- (reviewed by zhu and paul(35)). however, tregs also exhibit perforin - dependent cytotoxicity against autologous target cells(36) indicating that treg can act as natural cytotoxic cells, like natural killer cells and cytotoxic t lymphocytes. based on such prior findings and our observations of granzymes in the infected bmm lysates treated with treg by mass spectrometry, we hypothesized that treg may induce hiv-1-infected macrophage death to deplete the cell source of neurotoxic factors. to test this hypothesis, we used terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) assay to assess the cytotoxicity of treg against hiv-1/vsv infected bmm. greater than 50% of bmms were tunel positive in treg treated cocultures (figure 2a, b). cytotoxicity of treg was < 10% in uninfected bmm (data not shown) supporting the notion that treg are killers of activated or pathogen - altered bmm. results were replicated in hiv-1ada infected human mdm cocultured with human treg (supplemental figure 4ac, supporting information). in these assays human mdm were shrunken and detached following treg cocultivation (supplemental figure 4c, supporting information). taken together, the reductions seen in hiv-1p24 in macrophage culture supernatant fluids by treg likely reflect both antiviral innate immunity and the killing of infected cells. (a) treg induces hiv-1/vsv - infected bmm death shown by tunel assay plus confocal immunofluorescence microscopy and trypan blue staining plus light microscopy. for tunel assay, bright field is shown in the top panel and the corresponding fluorescent field is shown in the middle panel. trypan blue staining is shown in the bottom panel (trypan blue positive cells are indicated by yellow arrows). (b) graphs represent the mean percentage of tunel positive bmm and trypan blue positive bmm counted and calculated from five different fields (data were pooled from three independent experiments). each bar represents a mean sd p < 0.05, p < 0.01, p < 0.001. based on these observations of treg - induced control of viral infection, we next investigated molecular mechanisms underlying the treg induced cytotoxic responses. immunoblots for cleavage of procaspases-1 and 3 demonstrated that treg induced hiv-1/vsv infected bmm apoptosis is caspase-3 specific. in contrast, tcon induces infected bmm pyroptosis through caspase-1 (figure 3a, b). hiv-1/vsv infection itself triggered caspase-1 activation in bmm and was enhanced by tcon but was reduced by treg (figure 3a). densitometric analysis of blots for caspase-1 subunits failed to reveal differences between caspase-1 p20 in hiv-1/vsv infected bmm alone versus infected bmm cocultured with tcon (16 to 18% when compared to actin). however, there were differences in caspase-1 p10 observed between hiv-1/vsv infected bmm alone and hiv-1/vsv infected bmm cocultured with tcon (11 to 17% when compared to actin). futhermore, procaspase-1 was present in cell lysates from hiv-1/vsv infected bmm at 30% compared to actin, while 42% compared to actin from hiv-1/vsv infected bmm cocultured with tcon. in addition, faint bands were observed in these blots for caspase-1 in the treg treatment group and for caspase-3 in the tcon treatement group, suggesting much limited activation. apoptosis induction by treg and pyroptosis by tcon was detected independently using a fluorochrome inhibitor of caspases assay (flica) utilizing binding of fluorescent inhibitors to specific active caspases (figure 3c). replicate observations were also seen in human cells (supplemental figure 4a, supporting information). (a) western blotting showed hiv-1/vsv infection triggers caspase-1 activation in bmm, which was enhanced by tcon coculture while attenuated by treg coculture. (b) western blotting showed treg coculture activated caspase-3 pathway in hiv-1/vsv infection bmm. white scale bars represent 50 m. caspase-3 and 7 activation was measured by flica assay plus immunofluorescence microscopy. (d) proposed mechanisms treg and tcon use to modulate hiv-1-infected macrophage functions. upon infection, hiv-1 activates nf-b and the inflammasome, which results in il-1 release. in addition, those proinflammatory cytokines increase mitochondrial activity, which could lead to energy exhaustion and pyroptosis. however, treg use their anti - inflammatory cytokines to extinguish the inflammation resulting from virus infection. excessive atp is an important inflammatory molecule and could be hydrolyzed by ecto - nucleoside triphosphate diphosphohydrolase cd39 and cd73 expressed on treg. for example, m1 induce th1 and th17, while m2 induce th2 and itreg. (e) comparison of cd39 expression on tcon and treg was determined by flow cytometric analysis. briefly, upon infection, hiv-1 activates nuclear factor - kappa beta (nf-b) and the inflammasome in macrophages resulting in inflammatory cytokine release. in addition, as demonstrated, excessive activation commonly leads to energy exhaustion and pyroptosis. when compared to apoptosis, pyroptosis leads to increased adenosine triphosphate (atp) release. low concentration of atp is a critical apoptotic cell find - me signal for scavenger recruitment. on the contrary, excessive atp incites inflammation. thus, enzymatic removal of excessive atp is one strategy to resolve such inflammation, which could be achieved by treg through its surface ectonucleoside triphosphate diphosphohydrolase, cd39 and cd73.(37) indeed, treg was found to have significantly greater cd39 expression than tcon (figure 3e). previously, grossman,. reported that activated human naturally occurring treg utilized the perforin - granzyme a pathway to kill autologous target cells.(36) meanwhile, they demonstrated that activated mouse naturally occurring treg cells use perforin - granzyme b pathway to kill target cells. to compare the expression profile of granzymes in tcon and treg before adding them to the infected bmm, we used confocal microscopy and flow cytometry to evaluate granzyme a and b and perforin intensity. as shown in supplemental figure 5 (supporting information), both tcon and treg exhibited cytoplasmic granular staining of granzyme a, b and perforin. treg showed larger size and more abundant granules than tcon (supplemental figure 5a, supporting information). flow cytometry results confirmed those observations (supplemental figures 5b, c, supporting information). to verify that treg cells inserted perforin into the hiv-1-infected cell membrane, an important step in transferring granzymes to target cells, we used immunoblotting assays to detect inserted perforin. a very clear band was observed in treg treated cell groups, while a diminished one was in tcon treated macrophages (figure 4a). next, we used intracellular staining and confocal microscopy to visualize granzymes and perforin in bmm. both control and hiv-1/vsv infected bmm showed no staining, indicating bmm do not produce granzyme and perforin de novo. however, cytoplasmic granular staining of granzymes and perforin were seen in the treg treated cells and less so in tcon bmm cocultivations (figure 4b). (a) western blotting demonstrating increased p38 phosphorylation (upper panel) and perforin insertion (middle panel), which were normalized to -actin (bottom panel) in hiv-1/vsv infected bmm cocultured with treg compared with hiv-1/vsv infected bmm cocultured with tcon groups. (b) confocal immunofluorescences of bmm with specific monoclonal antibodies against granzyme a (upper panel), granzyme b (middle panel) and perforin (bottom panel) show cytoplasmic granular staining of granzyme a (red), granzyme b (red) and perforin (red) in tcon and treg treated groups. both control and hiv-1/vsv infected groups showed no staining of those antibodies, which means bmm did not produce granzyme and perforin. higher intensity of granzyme and perforin were seen in the treg treated group compared with the tcon treated group. white scale bars represent 5 m. (c) cleaved ndufs3, the 30 kda subunit of mitochondrial complex i and the important substrate of granzyme a, was shown in hiv-1/vsv infected bmm cocultured with treg group by western blotting. (d) membrane potential - dependent staining of mitochondria in hiv-1/vsv infected bmm by jc-1 visualized by fluorescence microscopy showed the loss of red jc-1 aggregates fluorescence and cytoplasmic diffusion of green monomer fluorescence following coculture with treg. it is reported that granzyme a accesses the mitochondrial matrix to cleave the novel identified substrate, nadh dehydrogenase [ubiquinone ] ironsulfur protein 3 (ndufs3), through perforin to initiate caspase - independent cell death. together with other subunits, ndufs3 forms complex i of the electron transport chain in mitochondria. in addition, faint bands were observed on immunoblots of hiv-1/vsv infected bmm lysates with / out tcon, indicating that nudfs3 might be cleaved by other enzymes (figure 4c). we also detected increased p38 phosphorylation in treg treated bmm (figure 4a). since altered nudfs3 and phosphorylated p38 suggest that mitochondrial function was affected, we used 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl - benzimidazolylcarbocyanine iodide (jc-1) staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay to evaluate its membrane potential and activity. fluorescence microscopy showed the loss of red jc-1 aggregates fluorescence and cytoplasmic diffusion of green monomer fluorescence in bmm following treg coculture (figure 4d). light microscopy showed reduced color intensities / cell and overall levels of mtt in treg treated bmm. in contrast, for infected bmm cocultured with tcon, mtt staining intensities were increased in infected cells (supplemental figure 6a, supporting information). such treg induced cytotoxicity on infected bmm was cellcell contact dependent as demonstrated by transwell cocultures (supplemental figure 6b, supporting information). we noted that among all cell death assays we used in this study, only around 50% of cells are killed by treg. in addition, greater than 98% of bmm were infected as determined by p24 immunoflurescence staining (supplemental figure 1b, supporting information). we observed that the privileged bmm underwent remarkable cytoskeletal rearrangement (figure 5a), which may indicate its polarization switching.(40) as shown in figure 5a, treg induced changes in infected bmm morphology from a polarized to a rounded appearance with contracted processes showing redistribution of actin from focal adhesions to a cortical distribution. such alterations paralleled functional outcomes in the bmm proteome (table 1 and data not shown). we hypothesized that a polarization switch in this model could be due to either direct effects of treg or in response to cytotoxicity induced by treg. to test this, we used western blotting to detect expression of inducible nitric oxide synthase (inos) and arginase-1 (arg-1), which are putative markers for classical activation macrophage (m1) and alternative activation macrophage (m2), respectively. we found that treg downregulated inos 35-fold, with a concomitant 18-fold upregulation of arg-1 (figure 5b). the decreased no production by hiv-1/vsv - infected bmm cocultured with treg supports this transformation. this is in stark contrast to the significantly increased no production by infected bmm in the presence of tcon (figure 5c). (a) treg induces hiv-1/vsv infected bmm morphology change from elongation to roundness shown by confocal immunofluorescence microscopy (zoomed part is shown at the bottom panel). white scale bars represent 20 m. (b) western blotting showed treg downregulated inos, while upregulated arginase-1. treg inhibited no production from hiv-1/vsv infected bmm, p < 0.001. (d) concentrations of camp in the lysate (l) and supernatant (s) of tcon or treg were detected by elisa, p < 0.05 (e) concentrations of camp in the lysate and supernatant of bmm with different treatments. the camp in the supernatant was undetectable, while its concentration in lysate increased in treg treated group. p < 0.001. to decipher the mechanisms underlying this phenotypic switch in a strictly cell contact - dependent manner, we measured the levels of cyclic adenosine monophosphate (camp) in the culture supernatants and cell lysates, based on two important findings : camp is reported to modulate inos and arg-1 expression in macrophages;(41) and treg is reported to transport camp to target cells via gap junctions.(42) indeed, we found no detectable camp in t cell or bmm culture supernatants (figure 5d, e). however, compared with tcon, treg harbors higher levels of camp. and most importantly, coculture of infected bmm with treg resulted in increased intracellular camp (5-fold) but not with tcon (figure 5e). since camp in supernatants is undetectable, we believe it may have traversed membranes via gap junctions. an alternative hypothesis is that production of adenosine as a result of coculture of treg expressing cd39 likely contributed to the increased intracellular camp production through adenosine receptor signaling. both mechanisms could be involved and would be up to future studies to determine. given the multiple mechanisms of treg presented herein used to modulate hiv-1/vsv infected macrophages (supplemental figure 7, supporting information), we expected that treg must have neuroprotective roles due to their abilities to inhibit viremia and resolve inflammation. indeed, our lab recently showed the neuroprotective properties of treg in an hive model.(9) to confirm this anticipation, we used conditioned medium to treat primary neurons in vitro. neuroprotection is readily observed in treg - treated bmm groups (figure 6). comparisons between neurons treated with conditioned media from hiv-1/vsv infected bmm in the absence or presence of treg and tcon are shown (figure 6 and supplemental table 1, supporting information). furthermore, addition of media from uninfected macrophages was not observed to result in any neurotoxicity (figure 6 and data not shown). tubulin staining shows persevered neuronal dendrites in groups cultured with both cm1 (upper panel) and cm2 (bottom panel) from treg treated hiv-1/vsv - infected bmm, which was not seen in tcon derived cm. moreover, cm2 from the tcon treated group exhibits more toxicity than the infected group, which suggests that the proinflammatory cytokines are more destructive than viral proteins. broad functions of treg were identified in the current report and beyond what is known for immune suppression. for the hiv-1-infected macrophage this includes enhancement of innate antiviral immunity, polarization in control of inflammatory activities and toxic secretory responses, and immunosurveillance in the killing of virus - infected macrophages. for the latter treg utilize several mechanisms including granzyme a, b and perforin. in addition, treg also altered redox biology to lower levels of oxidative stress.(9) these results, taken together, demonstrate a much broader range of functions of these regulatory cells than previously appreciated and help explain their control of disease. this of course includes hand which is rarely seen during subclinical disease and immunocompetence.(43) the abilities of art in lowering hand severity have long been appreciated.(44) the mechanisms described revolve around the number and function of cd8 + ctls resulting in the elimination of infected cells. importantly, th1, th2, th17 and treg are heterogeneous in function based, in large measure, on their cytokine secretion and specific transcription factor expression. indeed, cytokine profiles, tissue environment, microbial infection or injuries affect the cells differential fates.(35) it has long been appreciated that treg control immune responses to infections, but whether the ultimate outcome from treg actions in disease is protective or destructive remains unresolved. for the better part of its existence treg serve to limit damage caused by excessive virus - induced inflammatory responses.(47) however, for hiv-1 infection, its role can be seen as a double - edged sword. treg - mediated suppression can lead to cessation of antiretroviral immune responses and in so doing allow productive viral replication. it is possible that effector t cell responses could prevent treg - mediated suppression of antiviral responses ; especially those mediated through soluble factors, but could still allow treg killing of infected macrophages. on the other hand, secondary end organ effects of virus that include kidney, liver, lung, and brain pathologies could be attenuated by treg through control of inflammatory responses. it is of interest to note that changes in numbers and function of treg in lymphoid tissues and blood during hiv-1 infection are evolutionary. commonly, an increased frequency of treg is observed in hiv-1-infected individuals and notably in those with readily detectable plasma viremia.(52) thus, whether treg are ultimately beneficial for hiv disease still remains an active source of discussion and debate. for hand, the roles of treg are likely better defined.(9) indeed, our previous works in murine models of hive using infected bone marrow - derived macrophages showed that following adoptive transfer of treg astrogliosis and microgliosis were attenuated with substantive neuroprotection.(9) such observations parallel our laboratory s results in an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of pd. the current study clearly illustrates control of inflammatory effector functions by cd4+cd25 + treg for hiv - infected macrophages. furthermore, treg are effective in inhibiting hiv-1 replication and release.(9) indeed, we recently demonstrated treg abiltity to migrate to sites of inflammation in the brains of hive mice resulting in decreased hiv-1p24 and gliosis, with a concomitant increase in neurotrophins.(55) these functions support a multifaceted and beneficial role of treg in hiv infection and help further explain the neuroprotection provided, notably during art where adaptive immunity is largely preserved.(56) the observations that treg induce interferon - mediated antiviral immunity and kill hiv-1-infected mps by caspase-3 and granzyme / perforin pathways, depleting cellular sources of neurotoxic factors is of importance. indeed, treg are capable of killing a variety of autologous target effector cells, including cd4 + and cd8 + t cells, cd14 + monocytes, and dendritic cells.(36) furthermore, treg preferentially kill activated t cells.(36) our results demonstrate that hiv - activated m1 macrophages are also killed more so than uninfected cells. the phenotypic switch from m1 to m2 for the macrophages is attributed to treg in the setting of hiv infection but the mechanism by which this occurs is yet to be determined, but may involve both cytokine dependent and independent pathways.(57) indeed, our work suggests that direct camp transfer to infected cells and/or adenosine signaling are responsible, in part, for the phenotypic switch. adenosine signaling has been shown in macrophages to reduce the production of pro - inflammatory cytokines including tnf-, il-12, mip-1, and no, as well as increase production of il-10. furthermore, adenosine signaling can increase intracellular camp and activate p38 pathways, consistent with our findings.(62) thus, killing of hiv-1-infected bmm by treg may be a result of both the antiviral response itself and the bmm activation state. regardless of the mechanism, a treg directed phenotypic switch from m1 to m2 phenotype is advantageous for neuronal survival. the m1 phenotype is pro - inflammatory and is associated with neurodegeneration and increased viremia. an m2 phenotype however, is associated with tissue repair, anti - inflammatory cytokine production, and decreased ros production. thus an m2 phenotypic transformation in the setting of an inflammatory environment such as in hand can contribute to resolution of inflammation and neuroprotection observed with treg treatment.(9) furthermore, some evidence suggests that m2 macrophages retain their phagocytic activity and have enhanced intracellular killing capability.(66) as major cns apcs and a source of cytokines and chemokines, perivascular macrophages and microglia strongly influence the activation state of astrocytes, and influence the trafficking of adaptive immune cells into the brain.(67) astrocytes also can contribute to hiv-1 induced cns damage as a potent source of cxcl10 production in hiv-1-infected individuals, especially with tnf- and ifn- production by mps. importantly, treg are able to decrease the production of these cytokines that could ultimately diminish astrocyte cxcl10 production.(9) furthermore, hiv-1-infected macrophages induce astrocyte cxcl8 secretion, a chemokine that enhances chemotaxis of neutrophils and monocytes in had.(68) these findings illustrate the interaction between glia and immune cells that can contribute to neuroinflammation or, when influenced by treg, resolution of inflammation. recognition of the multifaceted functions of treg is an important first step toward a complete understanding of the broad roles of adaptive immunity in the setting of hiv infection and particularly that of the cns. this is certainly a vital part of the complex cellular and viral interplay present during hand. indeed, treg also have the capacity to attenuate inflammation in a range of neurodegenerative diseases and may be harnessed for therapeuptic gain.(69) | regulatory t cells (treg) induce robust neuroprotection in murine models of neuroaids, in part, through eliciting anti - inflammatory responses for hiv-1-infected brain mononuclear phagocytes (mp ; macrophage and microglia). herein, using both murine and human primary cell cultures in proteomic and cell biologic tests, we report that treg promotes such neuroprotection by an even broader range of mechanisms than previously seen including inhibition of virus release, killing infected mp, and inducing phenotypic cell switches. changes in individual treg - induced macrophage proteins were quantified by itraq labeling followed by mass spectrometry identifications. reduction in virus release paralleled the upregulation of interferon - stimulated gene 15, an ubiquitin - like protein involved in interferon - mediated antiviral immunity. treg killed virus - infected macrophages through caspase-3 and granzyme and perforin pathways. independently, treg transformed virus - infected macrophages from an m1 to an m2 phenotype by down- and up- regulation of inducible nitric oxide synthase and arginase 1, respectively. taken together, treg affects a range of virus - infected mp functions. the observations made serve to challenge the dogma of solitary treg immune suppressor functions and provides novel insights into how treg affects adaptive immunosurveillance for control of end organ diseases, notably neurocognitive disorders associated with advanced viral infection. |
exposed root surfaces in the anterior region may have several consequences : impaired aesthetics, increased risk of root caries, and hypersensitivity. whereas circularly denuded root surfaces (facial / oral and interproximally recessions) due to periodontitis respond neither completely nor predictively to surgical coverage attempts, there are several techniques that are successfully used to treat facial recessions : for example, coronally advanced and lateral positioned flaps, free gingival and connective tissue grafts (ctg), and guided tissue regeneration. ctg according to the envelope technique is an established method with favorable long - term stability. although a decent number of clinical studies investigating and comparing the efficacy of different root coverage techniques do exist [3, 6 ], data on patient - centered outcomes are scarce. however, for a technique aiming at aesthetical improvement patient - centered outcomes, that is, the question whether a patient actually notices aesthetic improvement should be regarded as main outcome variables. thus, structured reviews addressing root coverage techniques have explicitly stated that clinical trials addressing patient - centered outcomes (aesthetics and postsurgical morbidity) are required [3, 6 ]. the concept of oral health - related quality of life (ohqol) may be an approach to address patient - centered outcomes. the ohip (oral health impact profile) questionnaire is one of several instruments which have been developed to measure ohqol. is the most comprehensive questionnaire to assess ohqol and able to measure patients ' problems and symptoms. it is well suited to characterize oral problems and symptoms because the questionnaire items were developed through interviews with patients. as a result, it is a standardized, internationally accepted instrument which was translated into several languages (e.g., german : ohip - g49). hence, the objectives of this prospective longitudinal clinical pilot study were the evaluation of the effect on the oral health impact profile (ohip) and patient - centered results of the envelope technique for ctg. all patients, for whom root coverage in miller class i and ii recessions was arranged at the dental office of dr. the following inclusion criteria had to be fulfilled : denuded root surface of miller class i or ii, absence of probing pocket depths (pd) 5 mm at the recession tooth and its adjacent teeth, oral hygiene instructions and effective individual oral hygiene (interproximal space plaque index [api ] < 35%) prior to surgery, written informed consent. exclusion criteria : miller class iii and iv, presence of probing pocket depths (pd) 5 mm at the recession tooth and its adjacent teeth, hemorrhagic disease, anticoagulative therapy. the study had been approved by the institutional review board for human studies of the medical faculty of the johann wolfgang goethe - university frankfurt am main (approval number 320/07). immediately prior to surgical therapy the following parameters were assessed at 6 sites per tooth : plaque (present / absent) and probing pocket depths (pd) to the nearest 0.5 mm using a manual periodontal probe (pcpunc15, hufriedy, chicago, usa). at the facial aspect of the test tooth the following parameters were measured to the nearest 0.5 mm using the periodontal probe : recession depth (rd), measured from the gingival margin to the cemento - enamel junction (cej). the periodontal probe was oriented horizontally and located at the most apical convexity of the cej. then, the horizontal distance between the mesial and distal gingival margin was assessed. gingival width (gw), after staining with 3% iodine solution from the gingival margin to the mucogingival border. immediately prior and 3 months after surgery images of the facial aspect of the test teeth were taken (figures 13). immediately prior to surgery patients were asked about their personal reason for undergoing surgical root coverage : immediately prior to and 3 months after surgical therapy patients were asked to complete the ohip questionnaire (ohip - g49 clinic version). one week 1 day after surgery patients were asked about postsurgical pain. pain intensity after surgery for ctg donor and recipient site were assessed separately using a visual analogue scale (vas). three months after surgery all patients were questioned as of their opinion and impression of the performed ctg would the patient undergo this kind of surgery again (yes / no). by how much has the stated reason of surgery (ctg) improved (school grades : a [very good ] to f [insufficient ]). satisfaction with the result (grade a [very good ] to f [insufficient ]). the surgical technique that was examined in this study has been described in detail before, therefore only a brief description is provided. local anesthesia was rendered as major palatinal nerve block at the palatal donor region and as infiltrations (ultracain d - s forte, sanofi / aventis, hoechst, deutschland) at the buccal aspect of the recession tooth. the denuded root surface was scaled and planed thoroughly until the surface was hard and smooth as probed by an explorer. by removing a tissue collar of 0.5 mm width around the recession the sulcus epithelium was excised. the resulting wound should facilitate vascularisation of the ctg particularly in the gingival margin area. thereafter a pouch (envelope) was prepared using a no.15c or mb69 blade : gingiva and mucosa were separated from the periosteum to provide nutrition for the ctg from the underlying periosteum and covering soft tissue. at the palate an incision was made 2 mm paramarginally in the premolar and first molar region. a second parallel incision was made 1 to 2 mm apart from the first, both their mesiodistal length measuring twice the recession width. both incisions converged into the palatal tissue to meet at the periosteum and, thereby, providing a tissue wedge that was removed from the palate. thereafter, the epithelium was removed and the ctg was placed within the pouch covering the denuded root surface totally, while being at least 50% submerged within the pouch at the same time. the ctg was fixed with tissue adhesive (histoacryl, b. braun melsungen ag, melsungen, germany), and the surgical site was covered by periodontal dressing (coe - pak, gc america inc., the harvesting site at the palate was sutured (permilene 6/0 dsmp13, b. braun melsungen ag, melsungen, germany). patients were instructed to refrain from mechanical plaque control at the surgical sites for 1 week after surgery. to prevent postsurgical infection, they rinsed with a 0.2% chlorhexidine gluconate solution (corsodyl, fink gmbh, herrenberg, germany) for 2 minutes, twice daily for this period. patients were not provided with prescriptions for pain medications and additional anti - inflammatory drugs were not recommended. if more than 1 recession was treated, the recession with the largest depth was included in all cases. the primary end point in this study was chosen to be the change of oral health impact profile (ohip - g49) from baseline to 3 months after surgery. absolute reduction of recession height in mm (difference of pre and postsurgical recession height). relative root coverage in % (rd reduction divided by baseline rd multiplied by 100). amount of recession defects showing 100% root coverage postsurgically (number of recession defects with total coverage divided by total number of defects multiplied by 100). further secondary end points were 2 variables, used to describe patient - centered outcomes : (i) postsurgical pain, and (ii) patients ' judgment of treatment results. prevalence of plaque at facial sites was assessed as number and amount of all sites in per cent. mean standard deviation of baseline and postsurgical pd, recession depth, width, and width of gingiva as well as their difference were calculated and compared using a paired t - test with p <.05 allowing for statistically significant difference (two - tailed tests). relative root coverage in % was calculated for each recession defect and mean standard deviation for the total sample. means standard deviations were calculated also for patient - centered parameters at baseline, 3 months after surgery and change after 3 months (ohip - g49) and 1 week after surgery (pain prevalence, intensity [vas ], and duration [days ] at donor and recipient site). comparisons were made using the paired t - test (ohip - g49) and wilcoxon sign rank test (pain parameters) (two - tailed tests). a number of individuals who judged improvement of the cause for treatment and general satisfaction with the treatment result were compared between the group who would and would not undergo surgery again using a test. statistical analysis was performed using a pc program (systat for windows version 10, systat inc., a total of 16 patients (42.6 11.1 years) participated in this study : 5 males with a mean age of 38.0 5.2 years, 11 females with a mean age of 44.7 12.6 years. the majority of recessions included into the study were miller class i (14). most patients aimed for root coverage due to root sensitivity (11) and aesthetics (9). two patients asked for root coverage due to caries, 1 of whom gave root sensitivity as the only reason. only 1 patient was treated due to occurrence of recession in the course of orthodontic therapy. mean presurgical recession depth of 2.5 0.8 mm (1.54 mm) was statistically significantly reduced by 1.2 0.9 mm (p <.001) to postsurgical 1.3 1.3 mm. mean presurgical recession width of 4.3 0.9 mm (3.57 mm) was statistically significantly reduced by 1.6 1.7 mm (p =.003) to postsurgical 2.7 2.1 mm. mean baseline width of gingiva was 3.3 1.7 mm (06 mm) which was statistically significantly increased by 1.5 1.4 mm (p <.001) to postsurgical 4.8 1.2 mm. clinical re - examination revealed a mean root coverage of 48 39%. in 5 of 16 defects (31%) complete root coverage (figures 1 and 3) had been achieved 3 months after surgery (table 1). eight patients (50%) reported postsurgical pain at the donor site that averaged to an intensity of 2.1 2.3 vas. pain at the donor site lasted on average of 1.4 2.3 days. at the recipient site only 6 patients (38%) reported postsurgical pain averaging to an intensity of 1.1 1.9 vas and duration of 0.8 1.4 days. pain experience at the donor site was more pronounced than at recipient site regarding all assessed parameters : prevalence (p =.007), intensity (p =.016), and duration (p =.042) (table 2). at baseline a mean ohip - g49 of this mean value was statistically insignificantly (p =.139) decreased by 3.6 8.5 (1018) 3 months after surgery to 12.1 10.0 (135). only 3 of the total 16 (19%) patients required pain medication after surgery. all of them took just one dose of ibuprofene (200 mg) and reported postsurgical pain either at the donor and the recipient site. the 3 patients taking pain killers reported the highest scores for intensity (donor : 57, recipient : 26.5) and duration (donor : 38 hours, recipient : 25 hours) of pain. thirteen of 16 patients (81%) would undergo ctg surgery for similar reasons again. within the group, who would not undergo surgery again, the number of individuals who did observe less improvement of the cause for treatment (p =.139 ; table 3) or are generally less satisfied with the treatment result (p =.085 ; table 4) is larger than that in the group who would undergo surgery again. compared to most studies on root coverage after ctg the reported 1.2 0.9 mm of absolute and 48% of relative coverage are less favorable. reported between 2.2 and 3.47 mm absolute and between 64.7 and 94.58% relative root coverage after ctg. however, the observation period has to be considered when comparing the results of the present study. the studies referred to by rocuzzo. had to report observation periods of at least 6 months by definition of inclusion criteria. the present study reports results 3 months after surgery, which may be looked upon as too early. harris had reported a mean creeping attachment of 0.8 mm from 4 weeks after surgery to the final postoperative visit. creeping attachment occurred in 21 of 22 defects and resulted in complete root coverage in 17 of 22 defects. it may be speculated that root coverage may further increase from 3 to 6 or 12 months after surgery. however, despite incomplete root coverage 3 months after surgery patient - centered parameters (e.g., ohip) are already improved. more patients reported postsurgical pain at the donor site (50%) than at the recipient site (38%). further, those, who reported postoperative pain, described it as more intense and longer lasting at the donor (2.1 2.3 vas ; 1.4 2.3 days) than at the recipient site (1.1 1.9 vas ; 0.8 1.4 days). this was reported also in a retrospective analysis, which found none of the 20 patients remembering discomfort after surgery at the ctg recipient site, whereas 4 patients complained about discomfort at the palatal donor site for several days. the however rare occasion of palatal sensory dysfunction at the ctg donor site has been described recently. the results of this present study did show that root coverage using ctg according to the envelope technique improved the oral health impact profile although the improvement was not statistically significant. to our best knowledge this is the first clinical trial using an ohip questionnaire to assess patient - centered result of plastic periodontal surgery. however, the study provides data needed to calculate minimal sample sizes for clinical trials to evaluate the differences of oral health impact of different therapies. to detect a mean difference of 3.5 with a test power of 80% and a type 1 error < 0.05 a minimal sample size of 45 sets of defects shall be recruited. in most cases surgery to attain root coverage is performed to improve aesthetics or to reduce sensitivity. whereas there exists a decent number of clinical studies investigating and comparing the efficacy of different techniques with regard to millimeters or percentages of root coverage [3, 6 ], data on patient - centered outcomes are scarce. however, for a technique aiming to aesthetical or sensitivity improvement patient - centered outcomes, that is, the question whether a patient actually notices aesthetic or sensitivity improvement, should be regarded as main outcome variables. thus, future studies should compare root coverage techniques regarding patient - centered outcomes using questionnaires, for example, ohip. it may be, that patients ' satisfaction is achieved by techniques different from those that most favorably improve millimeters and percentages of root coverage. the method of choice to improve aesthetics might not be the method of choice to cover denuded roots completely. in the present study for most patients root coverage had asked for root coverage to improve aesthetics, only 4 due to hypersensitivity. in this former trial 19 of 20 patients (95%) would undergo ctg surgery for similar reasons again whereas in this trial only 13 of 16 patients (81%) would undergo surgery again. the longer the period that has passed after surgery, the higher the likeliness that the patient 's memory of discomfort and pain has faded. three months after surgery as in this analysis, the memory of surgery may still be quite vivid and, thus, more reluctance present to have surgery again. under the limitations of the present study we draw the following conclusions. root coverage using ctg according to the envelope technique provides improvement of ohip as early as 3 months after surgery. over all, patients were reasonably satisfied with the surgical technique and its results. | purpose. the aim of this prospective longitudinal clinical pilot study was the evaluation of the effect on the oral health impact profile (ohip) and patient - centered results of the envelope technique for connective tissue graft (ctg). methods. sixteen patients (11 females) 24 to 71 years of age (42.6 11.1) received ctg that had been harvested from the palate and grafted using the envelope technique. prior to and 3 months after surgery, all patients were examined clinically, completed the ohip - g49 questionnaire, and were asked to judge the results of surgery. results. mean baseline recession depth of 2.5 0.8 mm was reduced by 1.2 0.9 mm (p <.001). root coverage amounted to 48 39%. in 5 of 16 defects complete root coverage was achieved. pain at the donor site was more pronounced than at recipient site regarding prevalence (8/6 ; p =.007), intensity (2.1 2.3/1.1 1.9 [visual analogue scale ] ; p =.016), and duration (1.4 2.3/0.8 1.4 days ; p =.042). baseline ohip (15.7 12.1) was decreased by 3.6 8.5 three months after surgery (p =.139). thirteen patients (81%) would undergo ctg surgery for similar reasons again. conclusions. root coverage using ctg according to the envelope technique provided improvement of ohip as early as 3 months after surgery. over all, patients were reasonably satisfied with the surgical technique and its results. |
rosette forming glioneuronal tumor (rgnt) a rare slowly growing tumor affecting mainly the young adults which has been included as one of the glioneuronal tumors in the 2007 version of the world health organization (who) classification of central nervous system tumors. the neoplasm is composed of distinct neurocytic rosettes, perivascular pseudorosettes and an astrocytic component mostly pilocytic astrocytoma. the cytomorphology of this rare entity has been described only in a few isolated case reports. here, we are addressing the potential role of cytology for the diagnosis of rgnt as well as the close differential diagnoses which can cause difficulties during squash smear cytological examination. a 15-year - old male presented with complaints of headache and vomiting of 6 months duration. he was diagnosed to have a fourth ventricular lesion by imaging and empirically treated with antituberculous drugs. the patient had no relief of complaints and hence was referred to our institute. on clinical examination, he had no neurological deficits. a magnetic resonance imaging brain was done, which showed a solid cystic lesion near the fourth ventricle. the tumor was hyperintense on t2-weighted images [figure 1a ] and showed minimal enhancement with gadolinium contrast [figure 1b ]. the patient underwent midline suboccipital craniectomy and c1 arch excision with subtotal decompression of the tumor. (a) magnetic resonance imaging t2-weighted axial image showing a welldefined hyper intense solid cystic lesion in the region of 4 ventricle, with focal peripheral iso intense areas (b) sagitial image showing irregular peripheral enhancement of the lesion with central nonenhancing area. (c) smear cytology of a neoplasm with pilocytic astrocytes, rosenthal fibres, eosinophilic granular bodies (h and e, 200) (d) cytology with neurocytic cells around central eosinophilic neuropil (h and e, 200) (inset a rosetie in smear h and e, 400) the intra - operative squash smears were fixed in 95% ethanol and stained with hematoxylin and eosin as well as toluidine blue stains. cytology showed a neoplasm composed of glial cells with oval dark nuclei, long hairlike processes and rosenthal fibers [figure 1c ]. these cells were seen in groups, forming rosettes [figure 1d ] with central eosinophilic fibrillary material and some arranged around vessels forming pseudo rosettes [figure 2a ]. a cytologic diagnosis of ependymoma was considered based on the age and the site of the lesion. (a, b) cytology and histology showing perivascular pseudorosetie patiern of small cells with myxoid stoma. (h and e, 200) histology of the neoplasm with pilocytic astrocytes, rosenthal fibres and eosinophilic granular bodies. (h and e, 200) histology of neurocytic cells around central eosinophilic neuropil (h and e, 200) (inset showing roseties with neuropil showing s-100 positivity (avidin biotin, 200) the paraffin sections showed features of a typical rgnt with small neurocytes forming rosettes and perivascular pseudorosettes [figure 2b and d ]. this was admixed with pilocytic form of glioma having bipolar astrocytes, rosenthal fibers and eosinophilic granular bodies [figure 2c ]. the neurocytic cells were distributed in an abundant myxoid and neuropil background [figure 2b and d ]. the neurocytic rosettes and pseudorosettes with neuropil material showed strong s-100 (monoclonal, novacastra, 1:100) immunopositivity [figure 2d inset ]. these areas were glial fibrillary acidic protein (gfap), synaptophysin and neuron specific enolase (monoclonal, novacastra, 1:100) negative on immunostaining. the neurocytic cells were positive for neurofilament protein (monoclonal, novacastra, 1:100). the postoperative period of the patient was unremarkable, and he is now under regular follow - up. the intra - operative squash smears were fixed in 95% ethanol and stained with hematoxylin and eosin as well as toluidine blue stains. cytology showed a neoplasm composed of glial cells with oval dark nuclei, long hairlike processes and rosenthal fibers [figure 1c ]. also seen these cells were seen in groups, forming rosettes [figure 1d ] with central eosinophilic fibrillary material and some arranged around vessels forming pseudo rosettes [figure 2a ]. a cytologic diagnosis of ependymoma was considered based on the age and the site of the lesion. (a, b) cytology and histology showing perivascular pseudorosetie patiern of small cells with myxoid stoma. (h and e, 200) histology of the neoplasm with pilocytic astrocytes, rosenthal fibres and eosinophilic granular bodies. (h and e, 200) histology of neurocytic cells around central eosinophilic neuropil (h and e, 200) (inset showing roseties with neuropil showing s-100 positivity (avidin biotin, 200) the paraffin sections showed features of a typical rgnt with small neurocytes forming rosettes and perivascular pseudorosettes [figure 2b and d ]. this was admixed with pilocytic form of glioma having bipolar astrocytes, rosenthal fibers and eosinophilic granular bodies [figure 2c ]. the neurocytic cells were distributed in an abundant myxoid and neuropil background [figure 2b and d ]. the neurocytic rosettes and pseudorosettes with neuropil material showed strong s-100 (monoclonal, novacastra, 1:100) immunopositivity [figure 2d inset ]. these areas were glial fibrillary acidic protein (gfap), synaptophysin and neuron specific enolase (monoclonal, novacastra, 1:100) negative on immunostaining. the neurocytic cells were positive for neurofilament protein (monoclonal, novacastra, 1:100). the postoperative period of the patient was unremarkable, and he is now under regular follow - up. rosette forming glioneuronal tumor was first described in 2002 as a distinct variant of mixed glioneuronal tumor by komori. which was later confirmed by preusser. until date, approximately 43 cases of rgnt have been reported. the pathologic features of rgnt can be easily diagnosed in excision biopsies with neurocytic - like cells, forming neurocytic and perivascular rosettes dispersed in a microcystic myxoid matrix admixed with pilocytic astrocytes. approximately, there are eight case reports describing the intra - operative squash smear features of this tumor. initially, komori. and jacques. had described the cytology of this entity as smears showing cells with round nuclei having granular chromatin, inconspicuous nucleoli, and scant cytoplasm admixed with piloid astrocytes. they did not stress the typical appearance of neurocytic rosettes on smears kinno. and ghosal. had described the combination of neurocytic rosettes as well as pilocytic astrocytes in rgnt in their case reports. however, due to sampling error only part of the tumor may be represented in the smear preparation. hence in squash smears, we can easily miss the classical neurocytic rosettes, especially with other elements dominating the cytology. on cytology the main differential diagnosis of rgnt include pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor (dnt), oligodendroglioma, neurocytoma, pineocytoma and ependymoma. pilocytic astrocytoma have an almost similar clinical setting and in smears show long hair such as cytoplasmic processes, rosenthal fibers, eosinophilic granular bodies and occasionally an abundant mucinous background. but they lack the neurocytic rosettes seen in rgnt. before the introduction of the terminology of rgnt dnt in smears may also show sheets of small round oligo like cells and fibrillary cells in an abundant mucinous background. they occur usually as epilepsy associated tumors in the temporal lobe. a few scattered floating neurons, ganglioid cells as well as calcospherites can be seen on cytology of dnt. it is to be emphasized here that any tumor in the fourth ventricle with features of dnt on cytology, the possibility of rgnt should be considered. it is composed of monomorphous round cells with a finely branching capillary network as in rgnt cytology. however, in oligodendrogliomas the smears will be more cellular and crowded, with calcified spherules and have no rossetes or gliofibrillary matrix the neurocytic cells of rgnt are small and round with more fine and uniform nuclear chromatin. neurocytoma and pineocytoma, composed of monotonous population of small cells forming neuropil rosettes, are differentiated from rgnt by the absence of pilocytic astrocytic component, mucoid background and by the specific site of the tumor. the dominant perivascular pseudorosettes and gliofibrillary processes in rgnt can be mistaken for ependymoma of the fourth ventricle in smears. the nuclei of ependymoma when compared with rgnt are larger and round with pale stippled chromatin and have one or more prominent nucleoli. the presence of pilocytic features, mucoid background, neurocytic rosettes with central neuropil and absence of distinct tubular ependymal canals can help in distinguishing rgnt from ependymoma. despite its rarity, rgnt should always be considered in the differential diagnosis of tumors occurring in the fourth ventricle. a review of literature on rgnt cytology highlights that on adequate sampling as well as correlation with the relevant clinical picture including the site it is fairly easy to diagnose this entity on squash smears. | rosette forming glioneuronal tumor (rgnt) is a recently recognized and extremely rare glioneuronal tumor occurring in the fourth ventricle. it is crucial for the cytopathologist to be aware of this entity as it can be easily mistaken for more common neoplasms occurring at this site. we present here the cytology of such a rare case of rgnt that was misdiagnosed as ependymoma. the varying cytological features of this entity, as well as the common diagnostic difficulties encountered in cytology, are highlighted in this report. |
disability due to chronic pain (dcp) results in absence from work and is a major public health concern in japan and many western countries.14 various screening tools have been developed to identify chronic pain subgroups and comorbid factors.57 a widely used powerful tool is the start back tool (start), a 9-item screening tool that was developed as a prognostic indicator of lower back pain (lbp). items 14 evaluate physical factors and items 59 assess psychosocial factors (figure 1).5,8 the start score is often used by primary care physicians in england to make clinical decisions.5 specifically, the start results indicate the subgroup that an lbp patient falls into, which helps determine which treatment strategies may be most effective. the start has been shown to be particularly effective for individual patient management in the physiotherapy setting. patients who underwent start testing and subsequent targeted therapy had higher clinical and cost efficacy than patients who did not undergo start testing and were treated with usual care strategies.5 we previously translated the start into japanese,9 and this version was linguistically validated in a general cross - cultural adaptation process.1012 we also evaluated the reliability and validity of the start into japanese in a large number of japanese patients with lbp.13 the lower back was the most common site of chronic pain and accounted for 65% of all cases of reported chronic pain in a japanese epidemiological study.1 however, chronic pain often originates in places other than the lower back, and a generic screening tool is needed to help effectively manage chronic pain from all sites. one such tool is the generic version of the start back 5-item screening tool (start - g), a modified version of the 9-item start.8 the start 9-item screening tool provides an easy way to stratify patients into three subgroups according to the probability of a poor prognosis or pain chronicity. these categories are defined as low risk, medium risk, and high risk (figure 2).8 on the other hand, the use of start - g (5-item) screening tool has not yet been established. the start - g has also not been validated for evaluating chronic pain in a large group of japanese subjects. therefore, the current study was performed to examine the validity of start - g in such a population using cross - sectional data obtained from start - g surveys administered online. this study was reviewed and approved by the medical / ethics review board of the japan labour health and welfare organization at kanto rosai hospital (kanagawa, japan, approval number : 2012 - 22). written informed consent was not obtained, but submitting the completed questionnaire was considered evidence of consent. before completing the questionnaire, potential participants read an explanation of the survey s purpose and were informed that they should proceed to the questionnaire only if they agreed to participate in the study. as an incentive, participants received online shopping reward points from the internet research company that helped conduct this study (united, inc., participants were recruited from an online panel conducted by an internet research company (united, inc.). the all - japanese study population consisted of ~1.25 million registered research volunteers between the ages of 20 and 64 years. from this volunteer pool, 965,919 individuals were randomly selected and invited by e - mail to complete an online questionnaire on health problems associated with pain. the 5-item start - g tool is a modified version of the 9-item psychosocial subscale that specifically identifies distress in other conditions.5 questions address fear (one item from the tampa scale of kinesiophobia), anxiety (one item from the hospital anxiety and depression scale), pessimistic patient expectations (one item from the pain catastrophizing scale), low mood, (one item from the hospital anxiety and depression scale), and how bothersome pain is.7 the first four items had possible responses of agree or disagree, and the bothersome item had possible responses from 0 to 5 (likert scale). we used the 5-item start back screening tool that is available from the keele university website (march 2013, figure 3).8 the study questionnaire investigated pain experienced over the past month in 20 different anatomical sites. all anatomical sites were illustrated on diagrams to ensure that participants correctly identified each area. examined sites included the head, chin, teeth / mouth, face, throat, neck, shoulder, elbow, wrist / hand, chest, abdomen, back, low back, hip, thigh, knee, lower leg, ankle / foot, genitals, and anus. the degree of chronic pain experienced over the last 4 weeks was assessed using the numerical rating scale (nrs), with scores ranging from 0 (no pain at all) to 10 (the worst pain imaginable). somatizing tendency was assessed using a subset of items from a linguistically validated japanese version of the brief symptom inventory (bsi).14,15 seven somatic symptoms were assessed for severity, including faintness or dizziness, pain in the heart or chest, nausea or upset stomach, difficulty breathing, numbness or tingling in part of the body, weakness in part of the body, and hot or cold spells. all symptoms were assessed on a five - point scale that evaluated how much the participant was bothered by the symptom. participants chose from the following response options : not at all (0), mildly (1), moderately (2), quite a bit (3), and extremely (4). for this test, participants were grouped by the number of somatic symptoms or pain sites. a participant was considered to have a symptom if he / she responded with a 24, which is indicative of somatization.16,17 the presence / absence of a dcp was also investigated. a dcp was considered present when the pain symptoms had continued for at least 6 months and the subject had withdrawn from social activities because of pain. participant demographic and clinical characteristics were summarized using descriptive statistics. to examine floor and ceiling effects, the percentages of respondents with total scores of 0 and 5 were calculated. floor and ceiling effects were considered present when > 15% of respondents had the lowest or highest possible score, respectively.18 to examine start - g reliability, we evaluated internal consistency by calculating cronbach s alpha coefficients. an alpha index > 0.70 indicates a satisfactory internal consistency.19 spearman s correlation coefficients were used to evaluate concurrent validity by examining correlations between start - g and nrs pain scores. correlation coefficients were interpreted using cohens20 criteria for correlation strength in psychometric validation (0.10 = weak, 0.30 = moderate, and 0.50 = strong). the ability of start - g scores to differentiate between participants with known differences (known - group validity) was examined using the jonckheere participants were categorized into the following groups according to the number of somatic symptoms present : no symptoms, one symptom, and two or more symptoms. associations between start - g scores and the presence of a dcp were examined using receiver operator characteristic (roc) curves and the corresponding area under the curve (auc). the following traditional academic point system for auc values can be used as a rough guide for classifying diagnostic test accuracy : 0.901.00 = excellent, 0.800.90 = good, 0.700.80 = fair, 0.600.70 = poor, and 0.500.60 = fail.21 statistical analyses were performed using spss statistical software (version 20.0 ; spss, inc., chicago, il, usa). all reported p values are two - sided, and statistical significance was defined as p 15% of respondents had the lowest or highest possible score, respectively.18 to examine start - g reliability, we evaluated internal consistency by calculating cronbach s alpha coefficients. an alpha index > 0.70 indicates a satisfactory internal consistency.19 spearman s correlation coefficients were used to evaluate concurrent validity by examining correlations between start - g and nrs pain scores. correlation coefficients were interpreted using cohens20 criteria for correlation strength in psychometric validation (0.10 = weak, 0.30 = moderate, and 0.50 = strong). the ability of start - g scores to differentiate between participants with known differences (known - group validity) was examined using the jonckheere, participants were categorized into the following groups according to the number of somatic symptoms present : no symptoms, one symptom, and two or more symptoms. associations between start - g scores and the presence of a dcp were examined using receiver operator characteristic (roc) curves and the corresponding area under the curve (auc). the following traditional academic point system for auc values can be used as a rough guide for classifying diagnostic test accuracy : 0.901.00 = excellent, 0.800.90 = good, 0.700.80 = fair, 0.600.70 = poor, and 0.500.60 = fail.21 statistical analyses were performed using spss statistical software (version 20.0 ; spss, inc., chicago, il, usa). all reported p values are two - sided, and statistical significance was defined as p 0.70.19 however, nunnally and bernstein26 recommend a minimum test reliability of > 0.90 for making clinical decisions. therefore, it is possible that test reliability was overestimated. finally, this cross - sectional study did not assess the ability of the start - g to predict pain consistency. future longitudinal studies are needed to better understand potential associations between risk groups and long - term pain outcomes. these should also examine whether or not the start - g score is predictive of dcp. the start - g scale had acceptable internal consistency, reliability, and validity (concurrent and known groups) in japanese patients with chronic pain. we hope that these analyses of the psychometric properties of start - g will enable japanese clinicians to use this survey as a screening tool for detecting dcps. the start - g is simple, fast, and suitable for use in primary care settings, all of which suggest that the start - g may facilitate screening for dcp in the primary care setting in japan. we hope using the start - g will ultimately ease physical, social, and economical burdens of chronic pain in the japanese population. | objectivethe generic start back 5-item screening tool (start - g) is used to manage chronic pain in the lower back and elsewhere. this study evaluated the validity of the japanese version of this generic screening tool.materials and methodsjapanese participants between the ages of 20 and 64 years completed online surveys regarding pain. survey reliability was assessed with internal consistency, as calculated using cronbach s alpha coefficients. spearman s correlation coefficients were used to evaluate concurrent validity between the start - g score and standard reference questionnaires. associations between start - g scores and the presence of a disability due to chronic pain (dcp) were analyzed using receiver operator characteristic (roc) curves.resultsanalyses ultimately included data obtained from 52,842 japanese participants (54.4% male) with a mean (standard deviation) age of 47.7 (9.4) years. approximately 1.5% of participants had dcp, and the mean start - g score was 1.2 (1.4). the cronbach s alpha coefficient was 0.71, indicating an acceptable reliability. the start - g score moderately correlated with the pain numerical rating scale (nrs) score (spearman s correlation coefficient : r = 0.34). when the start - g threshold was set at 4, the sensitivity and specificity of the dcp predictive model were 65.8% and 82.4%, respectively, and the area under the roc was 0.808.conclusionthe start - g was internally consistent and was able to distinguish between subjects with and without a dcp. therefore, the start - g can reliably be used in the japanese population to identify patients with dcp. |
these comorbidities might lead to the overuse or misuse of antipsychotic drugs, which has been suggested to increase mortality in this patient population. recognizing the increased use of polypharmacy in elderly patients with dementia, the centers for medicaid and medicare services (cms) encourages health care providers to seek alternative approaches in treating these individuals. some of the non - pharmacological treatment approaches include consistency in caregivers, individualization of routine care, and selection of activities that are appropriate for the patient s cognitive abilities. aromatherapy and essential oils have been used medicinally for thousands of years in china, israel, egypt, europe, and ancient greece, and they should be investigated as an additional non - pharmacologic approach in this patient population. lavender (lavandula angustifolia) contains the active compounds linalyl acetate and -linalool which have been used for their sedative, analgesic, and anxiolytic properties as alternative therapy in the treatment of pain, anxiety, and stress [57 ]. lavender may exert anxiolytic effects by inhibiting voltage gated calcium channels primarily in the neurons of the hippocampal region. additional studies indicate that autonomic arousal is suppressed with the use of lavender. a recent meta - analysis conducted by perry. examined 15 randomized controlled trials that evaluated the effects of lavender on anxiety and mood. the authors concluded that due to the lack of conclusive evidence of the effects of lavender on behavioral issues, further studies are warranted to fully evaluate its potential as an alternative therapy. furthermore, a double - blind randomized trial comparing oral lavender to placebo showed that the former was both efficacious and safe for the relief of anxiety disorder not otherwise specified without causing adverse drug events. additionally, a small multi - center study evaluated the effects of lavender aromatherapy on 24 nursing home patients who suffered from anxiety and disturbed sleep patterns. the nursing staff s perceptions of the patient s behaviors including mood, activity, sleep quality, and unrestfulness were recorded as well as any changes in medication use. it was found based on the nursing perceptions that 40% (n = 10) of the patients had a good / very good response to the lavender aromatherapy intervention. while existing evidence shows promising results, data on the use of lavender in treating behavioral issues in the elderly population, especially those with dementia, needs to be elucidated. our study s purpose was to evaluate the effects of diffused lavender in decreasing the occurrence of behavioral issues in a geriatric population with dementia. this was a pre - post quasi - experimental study conducted over a four - month period between june and september of 2013. the primary objective was to determine the impact of diffused lavender on the frequency of behavioral issues [bis ], defined as a composite of restlessness / wandering [rw ], agitation [agt ], anger [ang ], and anxiety [anx ]. inclusion criteria consisted of patients over the age of 65 with a clinical diagnosis of dementia and enrolled in the river garden adult day care center in jacksonville, florida. river garden is a private nonprofit institution that provides elder care services in residential, outpatient, and community based settings. the adult day care program is a center dedicated to assist elderly patients who are able to live at home but require monitoring during daytime hours. patients enrolled in this program must also be able to take all medications prior to attending the daycare program or after. patients that were not enrolled in the day care program were excluded from this study. lavender aromatherapy was implemented immediately after a two - month pre - intervention observation phase. as methods of administration of aromatherapy show wide variation among studies, there is no current gold standard or recommended regimen. in this study, lavender essential oils (young living essential oils, lehi, ut) were diffused using the advanced essential oil diffuser (abundant health, llc., spanish fork, ut) in the day care center common area for 20 min twice a day, once in the morning and once in the mid afternoon during active clinic days. the advanced essential oil diffuser allows the lavender to be diffused directly from the oil bottle. this diffuser, due to the 6.5 psi pump power allows for a saturated area up to 1000 square feet. the common area in the adult day care center is 1000 square feet in size and is a moderately open space. the estimated oil output ranges from 0.75 to 1.3 ml over 15 min. we used this information to estimate that 1 ml of oil would be diffused per 15 min of use for the high setting on the diffuser. since we chose to diffuse for 20 minutes twice a day based on the daily program schedule, one 15 ml bottle of lavender was used in 5 days. during the pre- and post - intervention phases, a single un - blinded observer recorded unique bis using the behavior / intervention monthly flow record (med - pass inc. and heaton resources, dayton, oh) (figure 1). this form allows for the systematic monitoring and documentation of bis as well as interventions needed to address these issues. the observer was a certified nurse assistant with training and experience on using the form and documenting information comprehensively for all patients in the program. bis that occurred during the study period were addressed as per center protocol with either one on one staff time or use of diversional activities appropriate for each behavior. behavior intervention monthly flow record. reprinted with permission from med - pass, inc. and heaton resources. the average number of observations per day was computed for the four bis to adjust for variation in days. the difference between post - intervention and pre- intervention frequencies was calculated and compared with the wilcoxon s signed - rank test. this method was used to evaluate whether the median differences were centered around zero, which would indicate no change from pre - intervention to post - intervention. wilcoxon s rank - rum test was used to analyze the computed differences between age groups and sex. data management and analysis were conducted using sas version 9.3 (sas institute, cary, nc). the study protocol was approved by the institutional review boards of both the university of florida and baptist health jacksonville. twenty - three patients were included in the study. males comprised 34.7% of the study population, and 95.6% were caucasian. of the two age cohorts analyzed, 60.8% and 39.2% fell into the age cohorts of 7085 and 86100, respectively (table 1). although the number of bis was lower in the post - intervention period compared to the baseline observational phase, the frequency of bis did not reach statistical significance (pre - intervention n = 487, post - intervention n = 310 ; p =.06) (table 2). in the analysis of individual bis, there was a statistically significant difference found for the frequency of agt (pre - intervention n = 129, post - intervention n = 25, p =.001) (table 3). there was no difference in the frequency of observations for rw, ang, or anx. there was a significant difference between the age groups for the computed difference of agitation with patients in the 7085 cohort being less agitated then the 86100 cohort (p =.04). there was no significant difference for effects of sex on the computed differences of any of the four bis (table 3). behavioral incidences included the combination of restlessness / wandering, agitations, anger, or anxiety. use of lavender aromatherapy decreased the overall frequency of bis in a group of patients with dementia, yet this change did not reach statistical significance. the use of lavender had the biggest effect on the frequency of agitation episodes for which a statistically significant decrease was observed. these results have to be interpreted cautiously as the sample size was small, and the study might have been underpowered to detect differences in specific bis. nevertheless, this pilot study shows that diffused lavender may be considered as an adjunct to drug therapy to reduce the frequency of agitation in patients 7085 years old with dementia. it is important to note that these effects were seen with no adverse effects reported by patients adding to the potential benefits of this intervention. due to the quasi - experimental study design, lack of randomization this limitation, the pre post study design used the exact same patient sample during both phases. in addition, patients were aware of the intervention and that they were being monitored, which may have produced a hawthorne effect and confounded results. to mitigate the impact of this effect, the observer selected in the study was part of the normal staff and already known to the patients. using a single observer during the entire study period has the advantage of increasing the accuracy of recorded information ; however, errors in misclassification of bis could have occurred based on the subjective assessment of the behavior. this might be evident in the finding of agitation as the only statistically significant change. future studies should aim at having an objective measurement of bis based on validated scales such as cohen mansfield agitation inventory (ccmai) and the pittsburgh agitation scale (pas). alternative causes can not be disregarded in the interpretation of the results of this study. confounding variables include changes in temperature between the two observational periods and variability with support staff which could impact a patient s behavior. a recent cochrane systematic review assessed the role of aromatherapy on patients with dementia and concluded that more well - designed randomized controlled trials are needed. despite the scarcity of current evidence on the use of lavender, there seems to be some potential benefits of its use. a study by sakamoto. showed that lavender olfactory stimulation may reduce falls and agitation in nursing home residents. another study by holmes. showed modest efficacy in treating agitated behaviors in patients with severe dementia. implementing blinding in these studies might be challenging, but perhaps researchers could use artificial fragrances that resemble the oil being tested, without the natural properties believed to be beneficial. to the best of our knowledge, none of the studies reported in the literature have reported adverse effects of lavender aromatherapy. recognizing that polypharmacy might have detrimental effects in the elderly, non - pharmacological approaches such as aromatherapy need to be revised. the use of diffused lavender twice daily has been shown to reduce the frequency of agitation in elderly patients with dementia. although diffused lavender did not show statistical difference in reducing the frequency of other behaviors (restlessness / wander, anger, anxiety), the study population may have been too small to find a difference ; a trend towards decreased bis was seen. future studies on the use of diffused lavender in elderly patients with dementia should include a larger sample size to assess impact on agitation and anxiety and should aim to determine the optimum dose of diffused lavender. this work was supported in part by the amda foundation and the 2012 amda foundation / pfizer quality improvement award. this work was supported in part by the amda foundation and the 2012 amda foundation / pfizer quality improvement award. | abstract objectives : to evaluate the effects of diffused lavender on the frequency of behavioral issues [bis ], defined as a composite of restlessness / wandering [rw ], agitation [agt ], anger [ang ], and anxiety [anx ] in an adult day care center. secondary objectives evaluate systematic differences on the frequency of bis between age cohorts, gender, and individual behaviors. design : pre - post quasi - experimental study. setting : private nonprofit adult day care center for patients with dementia. participants : elderly patients older than 65 years of age with a clinical diagnosis of dementia, who require daytime monitoring. intervention : lavender aromatherapy twice a day for 20 min during a two - month period during active clinic days. measurements : behavioral issues were recorded using the behavior / intervention monthly flow record during the pre- and post - intervention periods. results : there was no significant difference on frequency of bis between pre - intervention and post - intervention periods (p =.06). there was a significant difference between pre - intervention and post - intervention total number of agt occurrences (129 vs. 25 ; p value <.01). there was no significant difference between age cohorts for computed difference of rw, ang, and anx issues. there was a significant difference between age cohorts for computed difference of agt (p value =.04) as the 7085 age cohort showed less agitation compared to the 85100 age cohort. conclusion : the use of diffused lavender twice daily has shown to reduce the frequency of agitation in elderly patients with dementia, especially in the 7085 age cohort. though diffused lavender did not show statistical differences in the frequency of other behaviors (restlessness / wander, anger, anxiety), the study population may have been too small to find a difference. |
the main public health strategy for containing influenza is annual vaccination, which is recommended for the elderly and others belonging to risk - factor categories, which present the highest morbidity and mortality, as reported by the world health organization (who) recommendations. influenza viruses are constantly changing, mainly as a result of so - called " antigenic drift ", which consists of the continuous, spontaneous modification of viral surface composition, and regards hemagglutinin (ha) and neuraminidase (na) proteins. for this reason, the vaccine composition has to be adapted annually to integrate viral strains as similar as possible to the epidemic strains. the degree of similarity or difference between the circulating viruses and the viruses included in the vaccines is often referred to as " vaccine match " or " vaccine mismatch ". vaccine effectiveness, i.e. the ability to prevent influenza cases, is determined both by the degree of vaccine matching and by the characteristics of the subjects immunized, such as their age and health status. the degree of antigenic drift and the frequency of drifted viruses in circulation can change from one season to another, in comparison with each of the strains included in the seasonal flu vaccine. since 1973, surveillance systems have enabled the who to issue recommendations for the composition of influenza vaccines. careful analysis of epidemiological data based on the antigenic identification of strains, pathogenic potential and transmissibility is a valuable means of evaluatevaluating the persistence and dissemination of new influenza strains [2 - 4 ]. since 1999, the who has issued two different sets of recommendations every year : one for the northern and one for the southern hemisphere ; these recommendations are issued several months before the influenza season begins, in order to allow timely production of the upcoming seasonal influenza vaccine in conformity with the manufacturers ' recommendations. even when circulating influenza viruses are mildly or moderately drifted in comparison with the vaccine, available evidence suggests that people may still receive some protective benefit from vaccination. two main types of influenza vaccine are currently available : inactivated vaccine and live attenuated vaccine. the first inactivated influenza vaccine (iiv) was monovalent and was protective against the a (h1n1) strain. in 1940, however, a different influenza virus was isolated (influenza b) and the first bivalent vaccine was subsequently tested in healthy adults. current inactivated vaccines are mostly produced by means of propagation in embryonated hens ' eggs. however, the availability of embryonated hens ' eggs is a limiting factor in vaccine production, and global production is not expected to be able to meet the increased demand for doses in the pandemic season. at the end of the 1970s, a new strain of influenza a with different ha and na was identified. since then, two influenza a strains (h1n1 and h3n2 subtypes) and one influenza b (victoria or yamagata lineages) strain have been included in most influenza vaccines, called trivalent influenza vaccines (tiv). the first trivalent live attenuated influenza vaccine (laiv) was licensed in russia in the late 1970s and in north america in 2003. the aim of vaccination with a live attenuated virus is to induce a secretory and systemic immune response that more closely resembles the immune response detected after natural infection. however, the immunological mechanisms of action and correlates of protection remain largely unclear. in more recent years, improvements were made, primarily in production technologies and use of adjuvants, while innovative formulations were based on two principles : the production of reassortant strains between wildtype viruses (for their antigenic properties) and cultureadapted strains (for their replication properties). alternative routes of delivery have been also investigated, in particular intradermal (i d) administration. an i d tiv received marketing authorization in the eu in february 2009, and was licensed by the european medicines agency (ema) for adults older than 60 years in the 2010/11 season in europe, and in canada in september 2010. in the us, the same vaccine was approved by the food and drug administration (fda) on 10 may 2011 and has been available in the us since the 2011/2012 influenza season for subjects older than 64 years. in 2013, the who recommendations included a second influenza b strain in the vaccine composition, allowing member countries to make their own decision on the possibility to recommend a tiv or a quadrivalent (qiv) influenza vaccine in their immunization programs. who recommendations define the criteria for identifying risk groups and other groups targeted for vaccination. age is considered a risk factor for flu infection, as the elderly are at high risk of complications such as morbidity, hospitalization and mortality. vaccination is recommended for the elderly worldwide, though age specifications differ from one country to another. in the last decade, research has focused on increasing the protection of elderly subjects and improving their immune response, which has been shown to be lower than that of younger adults. a number of studies have demonstrated that mf59-adjuvanted vaccine and i d influenza vaccine confer greater immunogenicity than non - adjuvanted vaccines in the elderly [10 - 14 ]. for this reason, it is advisable to immunize these vulnerable subjects with non - conventional vaccines. other categories of at - risk subjects have been identified, and, on the basis of the latest clinical evidence and guidelines from scientific societies, it is recommended that they should be vaccinated against influenza every year. in this regard, it has been demonstrated that influenza - vaccinated patients with rheumatoid arthritis or systemic lupus erythematosus are less likely to contract pneumonia, acute bronchitis or viral infections than unvaccinated patients. in most studies, neither dmards nor tnf inhibitors have hampered humoral immune responses to influenza vaccination, while rituximab has been seen to do so severely. moreover, a large meta - analysis revealed that the occurrence of adverse events following influenza vaccination was comparable in patients with autoimmune inflammatory rheumatic diseases (aiird) and in healthy controls. on the basis of this evidence and expert opinions, in 2011 the evidence - based european league against rheumatism (eular) formulated recommendations for annual influenza vaccination in patients with aiird. it is well established that the immunological response to the seasonal tiv influenza vaccine is also attenuated in cancer patients. rates of seroprotection and seroconversion vary by malignancy type and are higher in patients with solid tumors, unlike in those with hematologic malignancies or in allogeneic hematopoietic stem cell recipients. recent literature has reported that the use of myeloablative chemotherapy regimens and biologics is correlated with decreased immunogenicity to influenza vaccines. moreover, in cancer patients, influenza infections not only result in acute illness but can also lead to delay in vital treatments for the malignancy, such as subsequent dosing of chemotherapy or biologics. in order to avoid these complications, vaccination remains the principal way to boost immunity against seasonal influenza, and therefore prevent infection. the use of systematic influenza vaccination in patients with coronary heart disease prevents cardiovascular morbidity and all - cause mortality, as reported in various cohort studies and randomized clinical trials. on the basis of this evidence, since 2006 the american heart association and american college of cardiology has recommended influenza immunization with inactivated vaccine as part of comprehensive secondary prevention in persons with coronary and other atherosclerotic vascular diseases (class i, level b). in the us, recommendations for routine use of vaccines in children, adolescents and adults are issued by the advisory committee on immunization practices (acip). routine annual influenza vaccination is currently recommended for all persons aged 6 months who do not have contraindications. no preference is expressed for laiv or iiv for any person aged 2 through 49 years for whom either vaccine is appropriate, but some indications are given for laiv, which should not be used in particular conditions : confirmed severe allergic reactions, asthma, long - term aspirin use and most forms of altered immunocompetence. in the case of specific immunocompromising conditions, the infectious diseases society of america (idsa) has published detailed guidance for the selection and timing of vaccines in persons with congenital immune disorders, stem - cell and solid - organ transplantation, anatomic and functional asplenia, and cochlear implants. in europe, the european centre for disease prevention and control (ecdc) publishes periodic reports of national recommendations for the upcoming influenza season and of vaccination coverage rates in all 31 member states. at present, there is no consensus among european countries regarding the routine seasonal influenza vaccination of children, although this recommendation is now standard in the united states, and the who recommends vaccinating children aged from 6 to 59 months. the reluctance of some countries to adopt this measure may reflect a lack of evidence regarding cost - effectiveness and risk perception. live intranasal vaccines not requiring injection were licensed by the european medicines agency in 2010 and may, in the near future, increase the acceptance and delivery of annual vaccination among those eu / eea countries recommending vaccination for children. as yet, however, the immunization rate in this age - group is still very low. since the 2010/11 pandemic season, the number of countries recommending seasonal influenza vaccination for pregnant women has increased, although there are some differences between countries with regard to the period in which vaccination is recommended. a body of literature has demonstrated the safety and effectiveness of vaccine in this group, including benefits for the fetus and the newborn child [27, 28 ]. in all 31 member states, seasonal influenza vaccination is recommended for patients with immunosuppression due to disease or treatment and those with metabolic disorders or chronic pulmonary, cardiovascular and renal diseases. in other chronic conditions, such as hepatic disease, hiv / aids and morbid obesity, vaccination influenza vaccination is also offered to healthcare workers (hcws) in most european countries. in some cases, recommendations also extend to other professional categories, such as military personnel, poultry industry workers, laboratory staff, police, firefighters, veterinary service workers and educational staff. member states are encouraged to adopt and implement national, regional or local action plans or policies, as appropriate, aimed at improving seasonal influenza vaccination coverage, with the aim of reaching a vaccination coverage rate of 75% in ' older age groups ' as soon as possible, and, if possible, in all the other risk groups. in italy, representatives of the ministry of health, regional health authorities, the national institute of health and scientific societies constitute the national committee on immunizations, which annually updates a document indicating vaccine composition and recommendations for groups at risk. the vaccination coverage target is established in each year at 75% for all subjects aged over 64 years. influenza vaccination is also recommended for high - risk individuals 65 years had the greatest impact on the icer : for all these reasons, vaccination with qiv in the us is predicted to reduce morbidity and mortality. in europe, eichner. obtained similar results on using an individual - based simulation tool to connect people in a dynamically evolving, age - dependent contact network based on the polymod matrix. in accordance with international recommendations, vaccination providers and immunization programs should work to achieve the target of 75% vaccine coverage in at - risk groups, with a view to reducing influenza - related morbidity and mortality. this goal can be reached by expanding access to immunization services and extending vaccination campaigns to other target populations, on the basis of the most recent scientific evidence available. while the introduction of new vaccines is desirable, their use must be supported by strong evidence, in terms not only of higher immunogenicity, but also of greater effectiveness, in order to combat the growing phenomenon of vaccine hesitancy. indeed, public debate over vaccine effectiveness, which largely depends on matching between circulating influenza strains and vaccine strains, can negatively impact on vaccination coverage. for this reason, it is crucial to improve systems of surveillance of the most likely circulating strains and to ensure greater and broader vaccine effectiveness, which is expected to be achieved in the near future through the use of qiv vaccine. moreover, switching from tiv to qiv is expected to be a cost - effective strategy that will further reduce the burden of influenza, as reported in several recent analyses worldwide. the evolution of manufacturing processes will see the development of new technologies able to produce large quantities of vaccine rapidly in each influenza season, and new vaccines will be introduced. however, the production of a universal vaccine that is long - lasting and not subject to antigenic modifications still remains the ultimate goal. | summarythe main public health strategy for containing influenza - related disease is annual vaccination, which is recommended for the elderly and others belonging to risk - factor categories, who present the highest morbidity and mortality, as reported by the world health organization (who) recommendations.the availability of different influenza vaccine formulations makes the choice of the best immunization strategy a challenge for stakeholders and public health experts.heterogeneity in at - risk categories included in national influenza vaccine recommendations still exists, in particular among european countries. broader consensus is expected, which should positively impact on influenza vaccination coverage.the availability of quadrivalent vaccines, containing both influenza b lineages, offers the potential to improve protection by overcoming the drawbacks of wrongly predicting which b lineage will predominate in a given year. |
twelve wistar strain male rats, aged 120 days and with a mean weight of 250 to 300 g, were obtained from the breeding center of the anatomical and biological sciences center, shahid beheshti medical university (sbmu) tehran, iran. the animals were maintained under natural light and humidity conditions (temperature 22 ; 12-h light / dark cycles) in individual cages cleaned daily, fed once a day, and offered water ad libitum. the study was approved by the institutional medical ethics committee of shahid beheshti medical university. on day 0, the rats were anesthetized by the intramuscular injection of 50 mg / kg ketamine hydrochloride (rotex, made in germany) and 5 mg / kg diazepam (tpico, made in iran). their dorsal hair was shaved, and their skin was cleaned with povidone - iodine. the rats were equally divided into experimental and control groups randomly (six rats in each group). four incisional wounds measuring 20 mm in length were made in the dorsolateral regions of each experimental rat and one incisional wound in each control rat. all the incisions were performed in the skin and subcutaneous cell tissue using a scalpel. the depth of the surgical incisions was controlled by removing the epithelial tissue to the extent that the dorsal muscular fascia was exposed. the wounds in the experimental rats were treated with : aloe vera gel (av), thyroid hormone cream (tc), silver sulfadiazine 1% (s), or vehicle (v). all the therapies were initiated on day 0 and repeated daily for 14 consecutive days. an aloe vera plant was obtained from a medical plant garden and sent to the laboratory of anatomical and biological sciences center, shahid beheshti medical university (sbmu) for the extraction of aloe vera gel. after washing the leaves thoroughly, the base, apex and margins were cut off carefully to facilitate the slicing of the aloe vera leaves, revealing the transparent mucilage. a greenish gel - like liquid was obtained which was further refined through the use of a strainer. we also prepared a topical t3 cream by dissolving 150-ng of t3 (sigma, made in spain) in 20 l of ethanol and then mixed it into the vehicle. the tensile strength of the wounds was measured in both the experimental and control groups on day 14 after surgically inducing the wounds. we cut standardized 5mm - wide skin strips perpendicularly to the incisions with a double - bladed cutting instrument. the specimens were placed in 0.9 percent saline after necropsy and were evaluated within one hour. from each wound, we obtained one strip specimen and placed it in a material testing machine (zwick, made in germany) using two griping clamps. the distance between the edges of the clamps was 5 mm and the specimens were loaded uniaxially while the deformation rate was kept constant at 15 mm a minute so that failure and complete load - deformation curves were recorded by transducers coupled to bridges. from these curves, we analyzed the parameters of (1) ultimate tensile stress (newtons / square millimeter), which was derived from the ultimate load divided by the original unstrained cross - sectional area ; (2) ultimate tensile strength (fmax newtons), the load that causes a structure to fail ; and (3) the area under the load - deformation curve (newton millimeters), the amount of work done by the deforming load. we used a one - way analysis of variance and t tests to compare the data related to ultimate tensile stress, fmax, and area under load - deformation curve. an aloe vera plant was obtained from a medical plant garden and sent to the laboratory of anatomical and biological sciences center, shahid beheshti medical university (sbmu) for the extraction of aloe vera gel. after washing the leaves thoroughly, the base, apex and margins were cut off carefully to facilitate the slicing of the aloe vera leaves, revealing the transparent mucilage. a greenish gel - like liquid was obtained which was further refined through the use of a strainer. we also prepared a topical t3 cream by dissolving 150-ng of t3 (sigma, made in spain) in 20 l of ethanol and then mixed it into the vehicle. the tensile strength of the wounds was measured in both the experimental and control groups on day 14 after surgically inducing the wounds. we cut standardized 5mm - wide skin strips perpendicularly to the incisions with a double - bladed cutting instrument. the specimens were placed in 0.9 percent saline after necropsy and were evaluated within one hour. from each wound, we obtained one strip specimen and placed it in a material testing machine (zwick, made in germany) using two griping clamps. the distance between the edges of the clamps was 5 mm and the specimens were loaded uniaxially while the deformation rate was kept constant at 15 mm a minute so that failure and complete load - deformation curves were recorded by transducers coupled to bridges. from these curves, we analyzed the parameters of (1) ultimate tensile stress (newtons / square millimeter), which was derived from the ultimate load divided by the original unstrained cross - sectional area ; (2) ultimate tensile strength (fmax newtons), the load that causes a structure to fail ; and (3) the area under the load - deformation curve (newton millimeters), the amount of work done by the deforming load. data were summarised as meansem. we used a one - way analysis of variance and t tests to compare the data related to ultimate tensile stress, fmax, and area under load - deformation curve. since an increase in the synthesis of collagen occurs in the wound healing process, we used a biomechanical method to examine and compare among the treatments for tensile stress, fmax, and area under load - deformation curve (table 1). as table 1 indicates, the tensile stress was not significantly different among tc, av and s. averaged ultimate tensile stress was greater in the av, tc and s groups than in the v and control groups, but this difference was not statistically significant. no difference in fmax was observed among the tc, s, v, and control groups. the area under the load - deformation curve was significantly greater in the av group than in the other groups and was not significantly greater in the s and tc groups than in the v and control groups. skin wound healing is a dynamic response to injury that has three overlapping phases : inflammation, granulation tissue formation and remodeling. during the wound healing process, especially the transition from granulation tissue to scar tissue formation, collagen remodeling occurs, that is, the degradation of collagen with the formation of larger collagen bundles and an increase in the number of intermolecular cross - linkages. this process is controlled by several proteolytic enzymes called matrix metalloproteinases that are discharged by fibroblasts, macroghages, epidermal cells and endothelial cells. the tensile strength of a wound can be related to its collagen formation and maturation. on the other hand, the strength of the repaired wound tissue is the result of the remodeling of collagen and the formation of stable intra - and inter - molecular cross - linkages. this study examined and compared the effects of aloe vera leaf gel, thyroid hormone cream, and silver sulfadiazine cream on the healing of skin incisional wounds in rats by biomechanical methods to assay the tensile stress of the wounds. according to the results of the study, fmax and the area under the load - deformation curve significantly increased in the wounds treated with aloe vera as compared with the wounds treated with thyroid hormone and silver sulfadiazine, suggesting that aloe vera was more effective in healing the wounds, which has been observed in other studies. a possible explanation for this increase is that the aloe leaf gel extract infiltrates into the wounds, contributes to the increase of fibroblasts, macrophages, and epidermal cells activities, subsequently facilitates enzyme activity during collagen remodeling, and possibly even aids in the formation of cross linkages as the collagen matures. in other words, aloe vera may have a direct effect on the wound healing process as a whole, which is manifested as an increase in fmax and the area under the load - deformation curve, suggesting an improvement in the tensile strength of the wounds. the results are also in agreement with those of chithra. that attributed this improvement in tensile strength to an increase in the aldehyde groups of collagen fibres responsible for forming cross - linkages. subramanian. also confirmed the effect of aloe vera on increasing wound contraction and collagen synthesis and attributed this to the mannose-6-phosphate known to be present in aloe vera leaf gel. mannose - containing products have been shown to increase macrophage activity and therefore stimulate fibroblast activity and collagen synthesis. the results indicate that thyroid hormone did not have any significant effect on the healing of the incisional wounds. thus, the results do not provide evidence for safer. who found that topical triiodothyronine stimulated epidermal proliferation, dermal thickening, and hair growth. one possible explanation for this discrepancy is the difference in the type of wound between the two studies ; in the present study incisional wounds were under investigation while in the other, excisional wounds. an incision is a cut made into the tissue, mainly for operation, therefore topical medication must infiltrate into the tissue to accelerate the healing process. on the other hand, an excision is a removal skin tissue and topical medication is put on the wound directly, which may contribute more to the healing process. another possible explanation is the difference in the methods of assessing wound healing : safer. used a histological approach to assess whether topical triiodothyronine stimulates epidermal proliferation, dermal thickening, and hair growth while in the present study we drew on a biomechanical assessment method to explain the physical characteristics of the skin tissue. the findings of the current study indicate that ssd was not significantly effective on skin incisions, providing more evidence for the studies conducted by mccauley. and cooper., in that they explained the inhibitory and cytotoxic effects of ssd and mafenide acetate on human keratinocyte and fibroblast growth [25 - 28 ]. leitch. also found inhibitory effects of antimicrobial agents on wound contraction in an acute rat wound model. muller. compared the impact of ssd with or without aloe vera, nystatin with and without ssd, and placebo on time to achieve 50% and 90% wound healing in excisions of sprague - dawley rats. no difference in the acceleration of wound healing was observed between the control and ssd treatment lesions. in addition, the combination of aloe vera with ssd has been suggested to improve wound healing. such inhibitory effects may be related to the cytotoxic characteristics of ssd on cells synthesizing collagen, proteoglycan, and other products. the results of this study indicate that topical application of aloe vera can lead to significantly rapid wound healing and stronger repaired tissue. we can conclude that aloe vera can effectively be applied as a topical treatment to accelerate the wound healing process of surgically induced incisional wounds in rats as compared with other topical applications such as : thyroid hormone, silver sulfadiazine, and vehicle. | many research studies report the healing effects of aloe vera, thyroid hormone cream and silver sulfadiazine. however, the effects of these therapeutic agents are not well understood and have not been compared in one study. this study aimed at investigating the effects of topical application of an aloe vera gel, a thyroid hormone cream and a silver sulfadiazine cream on the healing of skin wounds surgically induced in wistar rats for determining the treatment of choice. in a randomized controlled trial, twelve male rats, aged 120 days and with a mean weight of 250 to 300 g, were divided randomly into 5 groups based on drug treatments : aloe vera gel (av), thyroid hormone cream (tc), silver sulfadiazine 1% (s), vehicle (v) and control. to evaluate the efficacy of each treatment technique, a biomechanical approach was used to assess tensile stress after 14 days of treatment. tensile stress was significantly improved in the aloe vera gel group as compared with the other four groups (p0.05). while the other treatment options resulted in better healing than the control group, this difference was not significant. we conclude that aloe vera topical application accelerated the healing process more than thyroid hormone, silver sulfadiazine and vehicle in surgically induced incisions in rats. |
antibiotics are well known to alter neuronal functioning, with penicillin being the best studied on not only a clinical level but on a basic science level as well. after multiple reports emerged on the probable relation between high dose penicillin and seizure, detailed animal studies emerged that showed toxic doses of penicillin given intravenously could induce myoclonic jerks at the spinal level, and could be abolished by penicillinase administration. given the wide spread use of structurally related antibiotics that share the beta lactam ring structure of penicillin, clinicians need to be aware of the potential for secondary neurotoxic effects. this report describes reversible frequent myoclonic jerks of the extremities during intravenous administration of nafcillin with piperacillin. a 66-year - old male who underwent a repeat knee prosthesis surgery developed pain and swelling at the operative site shortly afterwards with fever and elevation of the white blood count to 13,700. for the treatment of staphylococcus warneri isolated from the operative site, intravenous antibiotics were given, with vancomycin started initially at a dose of 1.25 gm q8 hours, but then switched to a combination of nafcillin 2 grams 18 hours iv, plus piperacillin / tazobactam 4.5 grams q6h iv. shortly after starting this regimen, the patient noted the onset of random myoclonic jerks of the extremities, which would be an isolated limb but at other times be all four limbs at once as a single isolated myoclonic jerk. in addition, the patient reported something is wrong with my time clock and felt surprised that long periods of time had passed, and also had a spell of getting up to go to the bathroom, and feeling there was a gap in time. approximately 40 random myoclonic jerks were estimated to occur over the course of each day of the six week course of the nafcillin / piperacillin combination by the patient and his wife, who stayed in the room, and noticed random jerks of the extremities also during sleep, which would awaken the patients at times. electrolytes were stable with a sodium of 140 on the first day of myoclonic jerking with potassium normal at 3.8, calcium normal at 8.9, and magnesium normal at 2.10. a non - contrast computed tomography of the head was negative (figure 1). past medical history was negative for any neurological problems, and there was no personal or family history for seizures or myoclonic jerks. neurological exam revealed the patient to be fully oriented with accurate 4 of 4 complex object recall at 5 minutes, with normal deep tendon reflexes and normal muscle tone and strength. the patient reported complete cessation of the myoclonic jerks after discontinuation of antibiotics, and has been free of this problem afterwards. the presented case clearly illustrates the potential for patients to develop myoclonic jerks as a secondary adverse effects of piperacillin and/or nafcillin. given the underlying pathophysiology of interaction of penicillin with the gamma - aminobutyric acid type a (gaba - a) receptor chloride channel, it is likely that these two antibiotics may interact in a similar way, and possibly reduce inward inhibitory chloride fluxes into both spinal and supra - spinal neurons. piperacillin has been noted to not only induce an encephalopathy, but can also produce seizures as shown by lin., who rapidly reversed this by high - flux hemodialysis in a 57 year old end stage renal disease patient who developed 2 generalized tonic - clonic seizures after five 250 mg doses of piperacillin / tazobactam an excellent study by sugimoto. defined the pathophysiology of the ability of penicillin to produce seizure activity by reducing chloride currents across the gaba - a receptor gated channel. by inducing a selective mutation of tyrosine to phenylalanine at position 256 within the gene encoding the gaba - a receptor gated channel, this selective mutation of the beta 2 subunit rendered the altered gaba - a receptor insensitive to the pro - convulsant medication picrotoxin. detailed voltage clamp study on frog oocytes transfected with these altered channels showed a dramatic inability for penicillin to reduce inhibitory currents. in summary, clinicians need to be aware of the potential for triggering myoclonic activity with piperacillin and nafcillin. as they are structurally similar to penicillin, these two antibiotics may place patients at risk as well for developing seizure activity. the pathophysiology for these effects appear to be related to reducing inhibitory hyper - polarizing chloride currents across gaba - a receptor gated channels within the brain | a 66-year - old male receiving intravenous piperacillin and nafcillin for a post - surgical wound infection developed intermittent myoclonic jerks of all four extremities that disappeared after discontinuation of these two medications. in addition there was a mild yet definite intermittent encephalopathic effect ; head computed tomography examination as negative and there was no prior history for seizure or myoclonus. these two beta lactam ring antibiotics are structurally similar to penicillin, which is well known to induce not only myoclonus but also seizure activity by reducing the gamma - aminobutyric acid (gaba) induced inhibitory currents by inducing an open chloride channel block of the gaba type a receptors within the brain. clinicians need to be fully aware of the potential epileptogenic effects of piperacillin, nafcillin, and related antibiotics. |
pain is defined as an unpleasant sensory and emotional experience arising from any part of the body. it is associated with actual or potential tissue damage or described in terms of such damage by international association for the study of pain. it is an experience and in this respect, it differs from nociception. nociception is called a neural process that provides transduction and transmission of a noxious stimulus to the brain via pain pathways. the pain arises from a complicated interaction between signaling systems, modulation of higher centers, and individual perception. the whole human population experiences pain in varying degrees and daily routine is affected negatively. pain may be occurred acutely or chronically related with various disturbances such as lesions, traumatic injury, tumors, inflammatory diseases, parkinson 's disease, and diabetes. since different mechanisms involve in the pathophysiology of acute and chronic pain and even nociceptive and neuropathic pain, the management strategies and current drug classes also vary. although there are too many analgesic agents, there are certain problems such as tolerability, tolerance, abstinence syndrome, insufficiency, possible drug interactions, and side effects. thereby, the development of new analgesic compounds is still going on. in this respect, the development and the use of imidazoline receptor ligands have gradually drawn attention since the role of imidazoline receptors in pain modulation was identified. for instance, various ligands which bind to imidazoline-2 (i2) receptors, the imidazoline receptor subtype which is predominantly involved in pain modulation, have been synthesized 2-(4,5-dihydroimidazole-2-il) quinoline hydrochloride (bu224), 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-bfi), 4-chloro-2-(imidazoline-2-yl) isoindoline (rs-45041), etc., in last decades and all of them have been reported to exhibit antinociceptive properties as discussed in this review. whereas the single use of imidazoline receptor ligands is effective in tonic and chronic pain, combined usage of other analgesic drugs such as morphine and clonidine is also effective in the potentiation of both acute and chronic pain conditions such as neuropathic pain. in fact, it is known that the i2 receptor agonism is one the mechanisms of neuropathic pain control, and the ligands that use this mechanism are in the phase 2 and phase 3 studies. in this review, we document the role of i2 receptors and ligands in antinociception and the relevant experimental studies performed by various researchers. although the term imidazoline receptor has not yet been adopted by major professional societies including international union of pharmacology committee on receptor nomenclature and drug classification since these receptors have not yet been cloned and the signaling pathway not characterized, this term is widely used in the literature. therefore, it is also frequently called as imidazoline binding sites ; however, the term imidazoline receptor is employed in this review. the presence of imidazoline receptors, with high affinity for imidazoline subdivision containing compounds, first became apparent in the mid-1970. the hypotensive effect induced by clonidine, 2-adrenoceptor agonist, and microinjection into the rat brainstem was not mimicked by norepinephrine. the imidazoline receptors are broadly located in the mammalian cells of the central nervous system (cns) and peripheral nervous system and contribute to cardiovascular system activity, gastric acid secretion, insulin release, antinociception, alzheimer 's, and parkinson 's disorders. according to a general opinion, there are three main classes of heterogeneous imidazoline receptors, as seen in figure 1. imidazoline-1 (i1) receptors constitute a family of nonadrenergic high - affinity binding sites for some ligands such as clonidine and idazoxan. they are located in the plasma membranes in the brain, heart, kidney, liver, and pancreas. the i2 receptors bind imidazolines and guanidines and have a lower affinity for 2-aminoimidazolines such as clonidine. the i2 receptors are mitochondrial, not g - protein coupled, and allosteric binding sites, possibly with a modulatory function, on monoamine oxidase - a and -b (mao - a and -b). these enzymes are found in the neurons and astroglia cells and have a critical role in the inactivation of neurotransmitters. thereby, the i2 receptors may be useful as a therapeutic agent in various neurological diseases in which neurodegeneration is observed. besides, it is known that these subtypes contribute to reduce body temperature, exert control over central noradrenergic and hypothalamic - pituitary - adrenal axis activity, and regulate the small intestinal motility. more remarkably, it has been shown that the i2 receptors take part in the antinociception in several acute and chronic pain models and the ligands acting on the i2 receptors may be assessed as novel analgesics. the imidazoline-3 (i3) receptors whose biological importance investigated further, are present in the pancreatic beta islet cells and modulate insulin secretion. we focus on the i2 receptor subtypes in this educational forum because involvement of this subtype in antinociception is well determined. pain modulation is a highly complex process, including numerous interacting peripheral and central mechanisms. the activation of the peripheral nociceptors or mediators that are released by the damaged tissue is required for the perception of pain. the activation - triggered signal is conveyed with the afferent transmission to the spinal cord with a and c nerve fibers and transmission parts through the dorsal horn (dh) to the higher centers via parallel ascending pain pathways. the impulses originated from the brain stem nuclei, descend to the spinal level and affect the transmission of pain signals at the dh. the relative balance between descending inhibition and facilitation can be changed by the type and intensity of the stimulus and also by the time following an injury. this complex process is regulated by the interaction of various chemicals and receptors over an extensive network from the periphery to the cns. the rate of participation of the chemicals and receptor types in the modulation depends on the types of pain and noxious stimulus. the imidazoline receptors that are taken part in this extensive receptor networks have gradually gained important in the recent years. when the roles of imidazoline receptors are evaluated in terms of pain, the i2 receptors are distributed ubiquitously throughout the brain regions such as the arcuate nucleus, caudate nucleus, putamen, globus pallidus, substantia nigra, interpeduncular nucleus, area postrema, mammillary peduncle, ependyma, lateral mammillary nucleus, and the pineal gland. low to moderate densities are found in the cerebral cortex, thalamus, hippocampus, amygdala, inferior olivary nucleus, ependymal, and various periphery regions. the i2 receptors are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines. also, they have two subtypes as i2a and i2b, which differ in terms of their sensitivity to amiloride. the i2 receptor is first located on the outer membranes of mitochondria and as allosteric sites on enzyme mao - a and mao - b. however, the imidazoline binding site on mao is separate from the active domain of the enzyme that recognizes the mechanism - based inhibitors, and it is not equally available in all the tissues. the i2 receptors have the same molecular weight as mao, and the amino acid sequencing of purified i2 receptors is similar to mao. two mao isoforms play a fundamental role in the metabolism of monoamine neurotransmitters such as serotonin, noradrenaline, and dopamine. as the i2 receptor is thought to be a modulatory site on the mao protein, the activity of i2 receptors may involve in several neurological disorders. another fascinating development with i2 receptors is that its effect on nociception since these monoamines appear to play a significant role in specific cns structures implicated in pain modulation spinal cord, cerebral cortex, etc., and are involved in the antinociception of several drugs such as antidepressants which are commonly used for the management of pain. the ligands binding to the i2 receptors can be considered as inhibitors of mao - a and mao - b since they inhibited monoamine oxidation, and this inhibition could explain some biological effects of imidazoline receptor ligands such as pain modulation. in fact, it is well identified that increased levels of monoamines such as serotonin and noradrenaline contributes to pain control, especially in the cns regions related to pain. the drugs such as mao inhibitors, serotonin noradrenaline reuptake inhibitors, and selective serotonin reuptake inhibitors that provide the enhancement of levels of these neurotransmitters are successfully being used to relieve various pain conditions. some evidence that shows the i2 receptors contribute to the modulation of pain [figure 1 ] reports that the ligands that affect i2 receptors are effective for tonic pain, neuropathic pain, but little effective for acute pain. moreover, the activation of i2 receptors has been suggested to be a way of enhancing opioid analgesic actions. when they are combined with opioids, the i2 receptor ligands increase the analgesic effects of opioids in both acute and chronic pain. in chronic use, tolerance and addiction to the opioids can develop. however, the i2 receptor ligands can reduce the development of this opioid tolerance or prevent from deprivation syndrome that is triggered by the antagonists in animals. when all of these findings are considered together, ligands that affect the i2 receptors can be beneficial in monotherapy or combination therapy with opioids to overcome pain. therefore, various new selective ligands are being developed for this receptor types. although the i2 receptors and their signaling pathways have not yet been characterized, the majority of the i2 receptors are widely accepted as being allosteric sites on mao. therefore, the i2 receptor ligands are described as allosteric modulators. allosteric modulation is the modulation of a protein via binding a ligand to the domain that is distinct from the active site of the protein. ligands that improves the activity of the protein are referred as allosteric activators or agonists, whereas those that decrease the activity of the protein are referred as allosteric inhibitors or antagonists. in principle, it is not precisely known whether a ligand is an i2 receptor agonist, an antagonist, or an inverse agonist in a constitutively active system. each and every ligand has two properties (i.e., affinity and efficacy) that govern its effects related to certain receptors in a bioassay. it is possible to have a knowledge of the relative efficacy of these ligands by systematic comparison. ligand efficacy values may vary depending upon assays, however, the rank order of their efficacy remains unchanged with few exceptions (e.g., functional selectivity). idazoxan, agmatine, and clonidine are ligands that bind to the i2 receptors as well as 2 and/or i1 receptors with varying affinities. due to the proven effect of the i2 receptors in pain, further studies are required to discover selective analgesic i2 receptor ligands and to elucidate their physiological functions. over the last few decades, investigators have attempted to synthesize and recognize selective ligands for the i2 binding sites. for instance, the i2 receptor ligand 2-bfi (pki = 8.47 for i2 in rabbit kidney membrane with [h ] 2-bfi) which has high affinity for both i2a and i2b sites has been used to characterize these sites in several species, including humans. it has been proved that quinolone compound bu224 has high affinity (pki = 8.43 for i2 in rabbit kidney membrane with [h ] 2-bfi) for the i2 receptors over i1 receptors and 2-adrenoceptors, among all the compounds of bu series. rs-45041 - 190 is also a selective ligand with a high affinity (pki = 9.37 for i2 in rabbit kidney membrane with [h ] idazoxan) for the i2 site which exhibits comparable data to that observed using 2-bfi in membrane - binding and autoradiographic studies. as mentioned before, the imidazoline receptor ligands are declared to modulate certain processes which involve mao activities in cns. the studies reveal that among all of the imidazoline receptor ligands those mentioned in this review ; 2-bfi, bu224, 2-phenyl-6-(1h - imidazol-1il) quinazoline (cr4056), idazoxan, and rs-45041 more selectively inhibit mao - a while lsl60101 and lsl61122 inhibit mao - b. after this part of the review, several studies will be conducted with the mentioned ligands [figure 2 ] that have a higher affinity for the i2 receptors and the provided results will be included. the imidazoline imidazoline-2 receptor ligands in pain it should first be noted that clonidine that mediated the discovery of the presence of imidazoline receptors, is a 2-adrenoceptor agonist that binds not only to 2 adrenoceptors but also to imidazoline receptors in particular to i1 receptors compared to i2 receptors ; however, its antinociceptive activity occurs via 2-adrenoceptors rather than imidazoline receptors. agmatine is a nonselective and most extensively studied endogenous imidazoline receptor agonist, and has a moderate affinity to 2-adrenoceptors as well as all subtypes of imidazoline receptors (pki < 5 for i2 in rabbit kidney membrane with [h ] 2-bfi). it is indicated that this endogenous substance prevents reflex respond to the noxious stimulus through nonadrenergic receptors in mice. it does not show a significant effect on acute phasic pain while it is vice versa in the acute tonic (differs from acute phasic pain in terms of using chemicals to induce noxious stimuli), inflammatory, and neuropathic pain. although agmatine showed weak effectiveness in a few studies, in many others, it was not efficient when administered systemically in acute phasic pain. spinal and supraspinal agmatine administration also does not reduce the thermal threshold, similar to systemic agmatine treatment. (supraspinal) agmatine administration did not produce analgesic effects in the tail - flick test in mice. the acute systemic agmatine treatment markedly decreased mechanical allodynia induced by complete freund 's adjuvant (cfa), chronic pain model, in mice. agmatine injections by systemic and supraspinal route have also been demonstrated to be effective in relieving hyperalgesia and/or allodynia in several neuropathic pain models. systemic administration of the i2 receptor ligands as phenyzoline, 2-bfi, 2-(2-benzofuranyl) imidazole hydrochloride (lsl 60101) ; the analog of 2-bfi, 2-styryl-2-imidazoline ; valldemossine or tracizoline (lsl 61122) also did not produce antinociception in the acute pain model. in addition, researches showed that the i2 receptor ligands such as rs-45041 - 190, cr4056, 2-bfi, bu224 were effective in inflammatory and neuropathic pain. rs-45041 - 190, the first selective and high - affinity ligand, showed significant results on the carrageenan - induced thermal and mechanic hyperalgesia tests when administered systemically (i.p.), not spinally (i.t.) in rats. cr4056 (moderate affinity i2 receptor ligand (ic50=596 nm, in rat whole brain membrane with [h ] 2-bfi), is a new, highly selective i2 receptor ligand which inhibits mao - a activity more selectively than mao - b. in a study, cr4056 was found active in cfa - induced model of inflammation. in acute capsaicin - evoked pain model this effect was antagonized by idazoxan (pki = 7.22 for i2 in rabbit kidney membrane with [h ] 2-bfi), which is both a 2-adrenoceptor and i2 receptor antagonist. similarly, in another study, cr4056 oral administration dose - dependently reversed the allodynia in bortezomib - induced peripheral neuropathy model that is painful., have investigated the antinociception induced by cr4056 in a rat model of postoperative pain and caselli. acute administration of cr4056 was found effective in relieving postoperative pain and joint pain as well as inflammatory and neuropathic pain. even naproxen showed low and nonremarkable antinociception compared to antinociception induced by cr4056 in these pain models. in addition, in joint pain model driven by both nociceptive and neuropathic mechanisms, the rats treated for 7 days (from days 14 to 21) after the induction of cartilage degeneration with cr4056, showed a significant reduction of both basal pain behaviors (allodynia and hyperalgesia). this result demonstrates either a long lasting effect or even an actual symptom modifying effect. the joint pain model is related to osteoarthritis that is driven by both nociceptive and neuropathic mechanisms. shortly, these studies present a new opportunity for the management of inflammatory, neuropathic, postoperative, and joint pain based on the selective interactions with the central i2 receptors. the phase ii trials on neuropathic pain for cr4056 are available and still going on. the effect of bu224, a high affinity selective i2 receptor ligand, has been assessed electrophysiologically on the nociceptive neurons responses in the spinal dh. route attenuated the nociceptive responses of dh neurons, creating a dose - dependent inhibition of c - fiber - induced responses, a-fibre - evoked responses, post discharge, and winding - up of the cells. idazoxan (i.t.) completely and significantly antagonized these effects while the nonselective 2-adrenoceptor antagonist yohimbine and the highly selective 2-adrenoceptor antagonist atipamezole only partially antagonized. when we consider the results, it is possible to say that bu224 has a high affinity for the spinal i2 receptors, as well as it has an insignificant action at the spinal 2-adrenoceptor receptors. it is obvious that the i2 receptor ligands are more effective in chronic pain models than acute phasic pain models as mentioned with agmatine. chronic pain usually means a persistent pain lasting 3 months or more, and pharmacotherapy of chronic pain always comprises repeated dose. a possible result of repeated doses of analgesics is a gradual decrease in analgesic effect, in other words, development of analgesic tolerance. from this point of view, in a research performed by li., antihyperalgesic effects of 2-bfi and cr4056 have also been tested in repeating treatments, in addition to single doses in chronic constriction injury (cci)-induced neuropathic pain and cfa - induced inflammatory pain models. the antinociceptive tolerance did not develop against repeated administration (daily for 79 days) of 2-bfi or cr4056 in cfa - treated or cci rats. it is possible to say that the repeated dose regimen for the i2 receptor ligands in chronic pain treatment may be useful as monotherapy or adjunctive therapy without tolerance and addiction. it has been also shown that three high selective i2 receptor ligands ; 2-bfi, bu224, and lsl 61122 possess antihyperalgesic effects in the rat models of cfa evoked inflammatory pain in this study. also, the i2 receptor antagonist idazoxan antagonized the antihyperalgesic effects of 2-bfi in cfa - treated and cci rats. these data also support the beneficial roles of the i2 receptor ligands in pain control. although most parts of preclinical pain studies focus on sensorial relieving pain, the sensorial and emotional compounds should be studied together. for instance, the effects of the i2 receptor ligands such as 2-bfi, bu224, and cr4056 on escape / avoidance behaviors on cfa injected rats were studied connectedly to the affective pain in the same study. this method is suitable to measure the dissociable components of effective pain which is different from the sensory pain. it was observed that 2-bfi, bu224, and cr4056 increase the escape / avoidance behavior in the hyperalgesia increasing doses. these results exhibit that the i2 receptor ligands may be effective against the affective components of pain. more recently, thorn. have studied the antinociception induced by 2-bfi and phenyzoline, high affinity ligand for i2 receptors (pki = 8.60 in rabbit kidney membrane with [h]-idazoxan), using the von frey filament test in rats with cfa - induced inflammatory pain. providing antinociception by 2-bfi was not surprising, however, phenyzoline also produces antinociception in this chronic model as in acute phasic pain under weak noxious stimulus, as reported previously in a study by sampson. even some i2 receptor ligands as agmatine are not effective alone in acute phasic pain, they potentiate morphine antinociception. in a study, performed in the warm water, tail withdrawal procedure in rats by using selective i2 receptor ligand 2-bfi along with agmatine, it was observed that these two ligands increase the antinociceptive effects of morphine and tramadol. in contrast, another selective i2 receptor ligand bu224 failed to increase the antinociceptive efficacy but prevented agmatine and 2-bfi ligands to increase the morphine and tramadol - induced antinociception. the reason that bu224 acts differently from other imidazoline receptor ligands is its lower efficacy in spite of its high affinity. this contradictory situation may be confusing, but it should be noted that affinity and efficacy are distinct terms from each other. a ligand that shows low efficacy may bind its binding site with high affinity. in yet another study, the combination of 2-bfi and morphine produced additive effects on mechanical hyperalgesia in cfa - treated rats. these results suggest that the combination of the i2 receptor ligands and opioids may be effective in chronic pain treatment. however, the previous studies indicated the i2 receptors to contribute potentiation mechanism, and i1 and i2 receptors contribution of potentiation mechanism was not clearly understood. more recently, a study which has been done to understand which subtype contributes to potentiation mechanism showed that a significant oxycodone - induced antinociceptive respond could not be reversed by efaroxan (i1 receptor antagonist) but could be reversed by bu224 (i2 receptor antagonist). as a similar way, endothelin eta receptor antagonist 5-(dimethylamino) -n-(3,4-dimethyl-5- isoxazolyl)-1-naphthalenesulfonamide - induced potentiation of oxycodone antinociception was reversed by bu224 but not efaroxan. so, it is thought that the i2 receptors participate in the potentiation, the i1 receptors do not. in yet another study performed for understanding the importance and mechanisms of potentiation, agmatine, high selective and more powerful substances such as 2-bfi, lsl 60101 (pki = 6.45 for i2 in rat cerebral cortex with [h ] idazoxan), lsl 61122 (pki = 8.74 for i2 in rabbit kidney with [h ] idazoxan), and aganodine have been tested. it was observed that central (i.c.v.) or peripheral (s.c.) administration of the i2 receptor ligands (but not i1 or 2 adrenoreceptor) potentiate the morphine - evoked supraspinal antinociception in mice. the enhanced morphine antinociception via the i2 receptor ligands was reversed by idazoxan, bu224, and isothiocyanatobenzylimidazoline, an irreversible i2 antagonist. in the same study, it has also been shown how the augmentation of morphine antinociception by the i2 agonists changes in mice with pertussis toxin impaired guanosine triphosphate - binding gi - go proteins. the potentiation ability effect was blocked. therefore, the contribution of gi - go transducer proteins in the modulation of morphine antinociception induced by the i2 receptors can not be disrespected. most recently, thorn. studied 2-bfi and phenyzoline with oxycodone as combinations, separately. 2-bfi and oxycodone produced additive interactions while phenyzoline and oxycodone produced synergistic interactions for their effects on mechanical hyperalgesia in cfa - treated rats. the imidazoline receptors are also important for the tolerance developed with opioids as well as improvement of opioid analgesia as mentioned before. for instance, agmatine prevents or decreases the tolerance development against morphine or other opioids. additionally, -difluoromethylornithine and aminoguanidine, which may affect the metabolism of endogenous agmatine, were found effective in the inhibition of acute morphine tolerance in tail - flick test. similarly, boronat., have assessed the role of imidazoline receptors in opioid (morphine and pentazocine) tolerance in rats by the administration of idazoxan. idazoxan completely prevented the morphine tolerance, but 2-methoxy - idazoxane and rs-15385 - 197, selective 2-adrenoceptor antagonists, did not and it remarkably reduced tolerance to pentazocine. in contrary, su., showed that idazoxan promoted the development of tolerance to morphine and induced the abstinence syndrome in morphine - dependent mice and rats similar to naloxone. the chronic concurrent administration of 2-bfi, lsl 60101, and lsl 61122, selective and potent i2 receptor ligands, and morphine, also prevented or attenuated morphine tolerance. in the light of the positive outcomes, it is supported that the i2 receptor ligands as promising therapeutic coadjuvants in the management of chronic pain with opiate drugs since these agents prevent tolerance development and enhance opioid analgesia. all the studies show us that the i2 receptors are also steady, new drug targets for analgesics. even if the mechanism of the i2 receptor is not well known in the modulation of pain, it is known that it plays a role in tonic and chronic pain but not in the acute phasic pain. additionally, when they are combined with opioids in both acute and chronic pain, the i2 receptor ligands increase the antinociceptive actions of opioids. the development of tolerance and addiction induced by chronic administration of opioids is one of the major problems in the clinic. however, the i2 receptor ligands can reduce the opioid tolerance development or prevent from deprivation syndrome in the combination therapy. they are valuable for the chronic pain treatment and also valuable as therapeutic coadjuvants of opiates, because of the attenuation of opioid tolerance and addiction. | pain is an unpleasant experience and effects daily routine negatively. although there are various drugs, many of them are not entirely successful in relieving pain, since pain modulation is a complex process involving numerous mediators and receptors. therefore, it is a rational approach to identify the factors involved in the complex process and develop new agents that act on these pain producing mechanisms. in this respect, the involvement of the imidazoline receptors in pain modulation has drawn attention in recent years. in this review, it is aimed to focus on the imidazoline receptors and their ligands which contribute to the pain modulation. it is demonstrated that imidazoline-2 (i2) receptors are steady new drug targets for analgesics. even if the mechanism of i2 receptor is not well known in the modulation of pain, it is known that it plays a role in tonic and chronic pain but not in acute phasic pain. moreover, the i2 receptor ligands increase the analgesic effects of opioids in both acute and chronic pain and prevent the development of opioid tolerance. so, they are valuable for the chronic pain treatment and also therapeutic coadjuvants in the management of chronic pain with opiate drugs due to the attenuation of opioid tolerance and addiction. thus, the use of the ligands which bind to the imidazoline receptors is an effective strategy for relieving pain. this educational forum exhibits the role of imidazoline receptors and ligands in pain process by utilizing experimental studies. |
shafer in 1983 introduced the term ameloblastic carcinoma (ac) to describe ameloblastomas in which there had been histological malignant transformation. it is currently defined as a rare odontogenic malignancy that combines the histological features of ameloblastoma with cytological atypia, even in the absence of metastases. although this lesion represents a separate entity, differentiating it from ameloblastoma has been often challenging to pathologists. recent study targets the different expression pattern of immunohistochemical markers in order to differentiate a case of ac from ameloblastoma. a wide range of epithelial - associated factors are implicated in the relative aggressive biological behaviour of the odontogenic epithelium while only a few studies have investigated non - epithelial factors. myofibroblasts and cancer - associated fibroblasts are important components of the tumour stroma. in fact the presence of stromal myofibroblasts has been linked to the biological behaviour of both benign and malignant tumours. in a recent case study, we attempted to differentiate ac from ameloblastoma on the basis of difference in expression pattern of alpha smooth muscle actin (alpha - sma). a 27 year old male patient presented to the department of oral and maxillofacial surgery with a chief complaint of the pain and swelling over the left lower back side of the face since last 4 months. the swelling was insidious in onset and was associated with moderate degree of pain. the skin over the swelling was normal in colour but there was slight increase in temperature. intraoral examination revealed expansion of the lingual cortical plate in the anterior aspect and buccal cortical plate in the posterior region. two ulcers were seen with respect to the swelling, one on the anterior lingual aspect measuring about 12 x 8 mm and second one measuring about 5 x 4 mm (figure 1a, b). intraoral view showing buccal (a) and lingual (b) cortical expansion and overlying mucosal ulceration. computed tomographic image of the mandible showed an ill - defined radiolucent mass with respect to the left ramus / body area of size about 2 x 3 cm (figure 2). the differential diagnosis included aggressive odontogenic tumour, intraosseous squamous cell carcinoma, and metastatic carcinoma. incisional biopsy was performed and the findings were suggestive of ac. as the patient did not give any history of previous surgery, so the primary variant of ameloblastic carcinoma was considered. patient underwent hemi - mandibulectomy along with excision of the tumour mass under general anaesthesia, following which he was suggested radiation therapy (figure 3). computed tomography image showing mandibular cortical plate expansion and erosion of the bony plates. the surgically excised mandibular specimen showing the extent of the lesion. the excisional specimen underwent routine tissue processing and samples were used for haematoxylin and eosin staining and immunohistochemical (ihc) staining for alpha - sma. sections of 3 thicknesses were cut and mounted on organo - silane coated slides (biogenex). after dewaxing in xylene, sections were dehydrated in ethanol, rinsed in distilled water, placed in 3% h2o2 for 10 min and rinsed in distilled water for 15 min. for antigen retrieval procedure, slides were placed in citrate buffer solution, ph = 6, in a microwave at 92 c for 10 min. after cooling at room temperature for 20 min, slides were exposed to primary alpha - sma mouse anti - human antibody (biogenex), dilution 1:100, for 60 min at room temperature. sections were rinsed in pbs for 10 min, reacted with aec substrate - chromagen kit, rinsed in pbs for 2 min, counterstained in harris hematoxylin (nice chemicals), and covered with dpx mounting medium. tissue sections of a specimen of follicular variant of solid multicystic ameloblastoma were also examined for alpha - sma to determine whether these diagnostic tests could be used to differentiate ac (primary) from ameloblastoma. the hematoxylin and eosin stained sections of ac showed odontogenic epithelial islands of highly irregular shape spread in a scanty fibrous connective tissue stroma. the central cells in few areas showed pleomorphism with an attempt of malignant keratin pearl formation. necrosis, vascular and neural invasion were not observed (figure 4). based on the findings of cellular pleomorphism, presence of mitotic figure and irregular shaped islands of odontogenic epithelial cells, a diagnosis of ac was made. haematoxylin and eosin stained section of ameloblastic carcinoma showing cellular atypia (original magnification x400). ihc stained slide of ac showed strong immunoreactivity to alpha - sma in the stroma surrounding the tumour islands (figure 5). additional finding was that few cells within the tumour island also exhibited faint positivity towards alpha - sma (figure 6a, b). while in case of follicular ameloblastoma, alpha - sma positive cells were seen only in the stroma and that too in reduced number. positive cells in the wall of endothelial vessels were taken as internal positive control (figure 7). increased expression of alpha smooth muscle actin in ameloblastic carcinoma (original magnification x100). expression of alpha smooth muscle actin (indicated by arrows) in the epithelial islands of ameloblastic carcinoma : a = original magnification x200 ; b = original magnification x400. expression of alpha smooth muscle actin in the stroma of follicular ameloblastoma (original magnification x200). two types of typical ameloblastoma must also be considered in the differential diagnosis of ac. first being the acanthomatous ameloblastoma which exhibits varying degrees of squamous metaplasia and even keratinization of the stellate reticulum portion of the tumour islands ; however, peripheral palisading is maintained and no cytologic features of malignancy are found. the other being kerato - ameloblastoma, which is a rare variant of ameloblastoma that contains prominent keratinizing cysts that may cause some alarm and distract the pathologist from the otherwise ameloblastomatous features. an additional consideration in the differential diagnosis of ameloblastic carcinoma is squamous cell carcinoma arising in the lining of odontogenic cyst. histologically, this lesion tends to more closely resemble oral squamous cell carcinoma than what we have described for ac. however, it is of interest that ac can apparently arise from a cystic lining. posterior portion of the mandible is the most common site of involve with maxillary involvement being less frequent. the lesion most commonly presents with swelling with or without associated pain, rapid growth, trismus and dysphonia. both, the epithelium and the stroma, makes up an ecosystem in which there is a continuous molecular cross - talk between the participating cells. the appearance of myofibroblasts in the adjoining stroma is secondary to the neoplastic changes in the adjacent epithelium. tgf1 and pdgf released by neoplastic cells, even at a pro - invasive state, are responsible for emergence of myofibroblast. tumour progression occurs within a microecosystem, where cancer cells and myofibroblasts exchange proteinases and cytokines that promote growth directly through stimulation of proliferation and survival, as well as invasion through local proteolysis of the extracellular matrix and stimulation of motility. studies on oral squamous cell carcinoma demonstrated the increased stromal myofibroblasts as assessed by alpha - sma immunoreactivity is associated with poor prognosis. in the present study we investigated the expression of alpha - sma in ac and compared the findings to that of a case of follicular amelobalstoma. increased expression of alpha - sma positive cells was seen in the stroma of ac (figure 5 and figure 6a, b). the expression of alpha - sma in the epithelial islands of ac was minimal. similar study was done by kamath. and bello., and they also reported increased expression of alpha - sma in the stroma of ac and few areas of epithelial islands were also positive for alpha - sma. they suggested the use of alpha - sma in differentiating ac from ameloblastoma and expression of alpha - sma within the epithelial islands is highly predictive of ac. the role of myofibroblast in tumour progression is an important area of current research and has emerged as a potential target for therapeutic intervention. these cells are recruited by the tumour cells and infiltrate the tumour microenvironment to support tumour growth and progression by the secretion of growth factors, extracellular matrix proteins, and by stimulating angiogenesis. recent reviews have emphasized the advantages of therapeutic targeting of the tumour - associated stroma as the stromal cells is presumably critical for the growth of nearby neoplastic cells and these are stable genetically in contrast to carcinoma cells, which accumulate adaptive mutations during the course of therapy in order to acquire drug resistance. at present, there are relatively few studies in support of the expression of alpha - sma in odontogenic carcinomas to correlate it with the diagnosis and prognosis of these lesions. study involving larger sample size and survival analysis is needed to validate this conclusion. pathologist still find it difficult to differentiate a case of ameloblastoma from ameloblastic carcinoma. immunohistochemical expression of alpha smooth muscle actin may help in establishing an early diagnosis and chemotherapeutic agents against stromal myofibroblasts can be used as an adjunct to the surgery in planning the treatment for these locally aggressive and infiltrating lesions. | abstractbackgroundthe aim of the present article is to report a case of ameloblastic carcinoma and use a marker alpha smooth muscle actin as a tool to differentiate cases of ameloblastic carcinoma from that of ameloblastoma.methodscase study reporting a case of ameloblastic carcinoma (ac) with expression of alpha smooth muscle actin (alpha - sma) as a marker for emergence of stromal myofibroblasts. the expression of myofibroblasts was also compared with that of ameloblastoma.resultsdifference between the two lesions in the pattern of expression of alpha smooth muscle actin was also observed. there was increase in the number of myofibroblasts in the stroma of ac while in ameloblastoma, it was comparatively less. secondly, few areas of the carcinomatous ameloblastic island also exhibited a mild positivity towards alpha smooth muscle actin.conclusionsincrease in number of stromal myofibroblast may be taken as a predictor for carcinomatous transformation. further studies with greater sample size can validate the use of alpha - sma as a marker to differentiate ameloblastic carcinoma from ameloblastoma. |
near - infrared spectroscopy (nirs) offers a new monitoring tool to be used in critically ill patients. nirs measures the saturation of hemoglobin in the skeletal muscle and serves as an index of perfusion. we have studied the value of tissue hemoglobin oxygen saturation (sto2) in a number of settings, including intensive care unit (icu) shock resuscitation, predicting outcomes in the emergency department (ed), and to better understand massive transfusion (mt). in the 1980s william shoemaker and colleagues wrote a series of papers addressing the use of physiologic monitoring to predict outcome and to assist in clinical decision - making [1 - 3 ]. in one observational study, shoemaker and colleagues tracked these variables in preoperative and postoperative periods of high - risk patients undergoing elective surgical procedures. following operative intervention, survivors will increase their do2 to above 600 ml / min / m. similarly, normal oxygen consumption is approximately 120 ml / min / m ; following an operation, survivors increase their oxygen consumption to supernormal levels of above 150 ml / min / m. likewise, nonsurvivors attempt to increase their oxygen consumption in this range but are not able to achieve it. based on these observations, shoemaker and colleagues concluded that survivor parameters include cardiac index > 4.5 l / min / m, do2 > 600 ml / min / m, and oxygen consumption > 150 ml / min / m. they proposed that the survivor parameters should be adopted as resuscitation goals and popularized the term supranormal oxygen delivery. in the late 1980s shoemaker and colleagues published the results of a prospective trial where there were three study groups who were managed by three different strategies. the first group received a central venous line, and central venous pressures were used at the discretion of clinicians to guide resuscitation. pulmonary artery (pa) catheters were placed in the second group, and monitored variables (pa wedge pressure and cardiac index) were again used at the clinicians ' discretion to direct resuscitation. pa catheters were also placed in the third group of patients ; however, these patients were managed with a resuscitative protocol that emphasized early volume loading followed by inotropic support with dobutamine. the results of this trial showed a dramatic reduction in mortality from 23% and 35% down to 4% between the central venous pressure and pa control groups and the pa protocol - treated group, respectively. based on these observations and others, shoemaker and colleagues proposed that unrecognized flow - dependent oxygen consumption contributed to the development of multiple organ failure (mof). at this time, mof was the leading cause of late icu deaths and its pathophysiology was unclear. this theory attributed myocardial dysfunction that occurs in patients in shock as an important cause of subsequent mof. this concept that unrecognized flow - dependent oxygen consumption could be corrected by maximizing do2 became a popular strategy. meanwhile, new technology was being introduced into the icu - including continuous venous oximetry and continuous cardiac output monitoring with pa catheters. this permitted widespread use of oxygen transport variables to guide resuscitation. at the denver general hospital in the early 1990s, surgical intensivists developed a bedside clinical protocol that involved identification of patients who were at risk for postinjury mof. upon arrival in the icu, a pa catheter was presumptively placed in high - risk patients and a series of escalating interventions was utilized to maximize the patient 's do2. based on the 12-hour response to these interventions, the intensivists could predict who would develop mof. a number of prospective randomized trials were performed in the 1990s to test whether supernormal resuscitation truly reduced mortality in critically ill patients. the results of these studies were varied and suggested that there are subgroups of patients who do benefit from this strategy. benefits of hemodynamic optimization were most readily observed in acutely ill patients who had not succumbed to end - organ failure. in the late 1990s at the university of texas houston medical school, a team of surgical intensivists collaborated with bioengineers and health information experts to further refine the logic for traumatic shock resuscitation and implemented it with a computerized clinical decision support application. patients meeting specific criteria (evidence of major torso trauma, evidence of shock as documented by base deficit > 6 meq, and anticipated blood transfusions > 6 units in 12 hours) had pa and peripheral artery catheters inserted upon icu arrival. they were resuscitated to a do2 goal of 600 ml / min / mwith a series of escalating interventions to achieve this goal in nonresponders. this became the standard of care in the shock trauma icu at the memorial hermann hospital in houston, texas. the protocol provided a unique opportunity to prospectively collect data on how patients responded to interventions and to further refine the existing protocol to optimize resuscitation. abg, arterial blood gas ; art, arterial ; bd, base deficit ; ci, cardiac index ; do2, oxygen delivery ; hb, hemoglobin ; icu, intensive care unit ; lr, lactated ringer 's solution ; ng, nasogastric ; pa, pulmonary artery ; pcwp, pulmonary wedge pressure ; prbc, packed red blood cells ; prco2, regional carbon dioxide tension measured by gastric tonometry. reproduced with permission from. this protocol also provided the opportunity to test the utility of various monitors in this process of care. to evaluate skeletal and subcutaneous sto2 changes as surrogates for do2 i changes and to compare these variables with other commonly used indices of shock resuscitation, we conducted a prospective study using sto2 monitors in shock resuscitation. figure 2a, b represents the variables tracked over the first 24 hours of icu admission. these included at 6 mm (subcutaneous), sto2 at sto2 20 mm (skeletal muscle), mixed venous hemoglobin oxygen saturation (svo2) (derived from the pa catheter), and serial lactate levels and base deficit levels (both obtained from serial blood testing). these variables were monitored simultaneously in severely injured patients undergoing protocol - directed shock resuscitation. upon arrival in the icu, do2 averaged approximately 400 ml / min / mand was driven by the protocol intensivists to a maximum of roughly 700 ml / min / mby 24 hours. throughout this resuscitation, skeletal muscle sto2 (at 20 mm) appears to be quite responsive to changes in systemic do2. changes in do2 resulted in a predictable decrease in lactate and base deficit levels, signifying effective shock resuscitation. subcutaneous (at 6 mm) was generally unresponsive to the sto2 resuscitation intervention, while svo2 derived from the pa catheter shows only a small rise from roughly 70 to 78% during the resuscitation process. changes in sto2 (20 mm) showed a strong correlation with changes in do2, base deficit, and lactate (r = 0.95 vs. 0.83 vs. 0.82, respectively) but only modest correlation with svo2 (r = 0.55). shock resuscitation variables during shock resuscitation (first 24 hours) and the following 12 hours. (a) tissue hemoglobin oxygen saturation (sto2), deltoid skeletal muscle and subcutaneous sto2 saturation, monitored non - invasively using a prototype near - infrared spectrometer (biospectrometer - nb oximeter ; hutchinson technology, inc., (b) mixed venous hemoglobin oxygen saturation (svo2) monitored invasively using a pulmonary artery catheter with fiberoptic oximetry capability. bd, base deficit ; lactate, serum lactate concentration. reproduced with permission from. the observation that the sto2 is an index of profusion that tracks do2 during active resuscitation led laboratory investigators to investigate the role of sto2 monitors in the setting of hemorrhagic shock. greg beilman and colleagues at the university of minnesota looked at the use of sto2 as an early determinate of irreversible shock. monitored pigs were subjected to a hemorrhagic shock protocol involving removal of 35% of the pig 's blood volume over a 90-minute period. resuscitation was done in a stepwise fashion, with administration of a 20 ml / kg bolus of lactated ringer 's solution every 30 minutes, for a total of four boluses. eighteen out of 20 animals survived 90 minutes of hemorrhagic shock : 12 of the animals survived resuscitation (resuscitatable) and six animals developed irreversible shock (nonresuscitatable). hemodynamic and nirs measurements were compared between the groups (resuscitatable vs. nonresuscitatable) at each step of resuscitation. all animals had a decrease in cardiac output, svo2 and do2 and an increase in lactate. a decrease in skeletal muscle, liver and stomach sto2 was also observed. beyond 30 minutes of resuscitation, reversible shock patients were seen to have a steady increase in cardiac output, while irreversible shock patients demonstrated a steady decrease. more notably, the drop in skeletal sto2 was significantly greater in animals who did not survive resuscitation. measurements of hind limb sto2 in each group diverged within 30 minutes of shock, such that by the end of the 90-minute period the sto2 value for the nonresuscitatable group remained low despite resuscitation. animals destined to survive shock and resuscitation did not exhibit an irreversible decline in sto2. these findings demonstrate skeletal sto2 as a reliable, non - invasive means for early differentiation between resuscitatable and nonresuscitatable animals. another investigative group from the university of miami, led by stephen cohn, was also interested in the use of non - invasive sto2 to guide fluid resuscitation after traumatic shock. they evaluated three resuscitative strategies in a series of three in vivo hemorrhagic shock models with or without a simultaneous penetrating femur injury. after a 30-minute shock period, animals were randomized to receive no resuscitation, to receive 15 ml / kg hextend or to receive shed blood + 20 ml / kg lactated ringer 's solution. serial lactate levels and serial base deficit levels were used to identify the best response to resuscitation. of the three interventions, hextend had the best clearance of base deficit and lactate levels over the ensuing 150 minutes of observation. svo2 (derived from an invasive pa catheter) also identified colloid resuscitation with hextend as an effective resuscitative strategy within 30 minutes of resuscitation. similarly, hind limb sto2 (a non - invasive monitor) indicated enhanced resuscitation in response to hextend. taken together, these clinical and research data suggest that (derived from a non - invasive monitor) could provide sto2 information about the effectiveness of resuscitation equivalent to that of an invasive pa catheter or serial blood draws to measure base deficit or lactate levels. in the late 1980s, jim davis and his colleagues from san diego popularized early routine collection of the base deficit in trauma patients to evaluate for possible shock and to guide ensuing volume resuscitation. in subsequent publications they demonstrated that the admission base deficit in trauma patient predicts transfusion requirements, risk of complications and mortality. interestingly, sauaia and colleagues - who were interested in identifying early predictors of postinjury mof - identified the ed base deficit as the earliest independent predictor of mof. this observation was validated in a second study from the same group published in the late 1990s. these data indicated that the initial shock insult in a trauma patient is an important determinate of adverse outcomes, particularly the development of mof. with this in mind, cohn and colleagues decided to perform a study using sto2 monitoring in the emergency room to determine whether it could predict mof. they performed a prospective observational study involving seven us trauma centers over a 16-month time period, ending in 2006. they proposed that thenar sto2 and base deficit could equally predict mof and death in major torso trauma patients presenting in shock. entry criteria included major torso trauma (excluding severe head injury), ed shock (systolic blood pressure 10 units of packed red blood cells in 24 hours. this analysis identified that patients arrived in the icu with irreversible coagulopathy despite adherence to the existing mt protocol. the admission international normalized ratio was highly correlated with subsequent mortality, and the investigators concluded that fresh frozen plasma should be used earlier and more aggressively in the ed for patients that required a mt. they proposed that fresh frozen plasma (ffp) and packed red blood cells be used at a ratio of 1:1. john holcomb, who developed the original mt protocol at memorial hermann hospital, was also an advocate for the use of activated factor vii in damage - control patients, and subsequently further expanded this concept when he became the commander of the us army institute of surgical research in san antonio. he and his colleagues developed a new concept of damage control resuscitation, which emphasizes direct treatment of coagulopathy in trauma during initial resuscitative efforts. the primary goals of this strategy were early use of ffp, activated recombinant factor vii and fresh whole blood when available and to pursue hypotensive resuscitation until hemorrhage is definitively controlled. the data emerging from those conflicts suggest that this approach reduces mortality in patients sustaining serious torso and limb trauma. while these strategies are all very enticing, a central question emerged when faced with the clinical quandary of a patient with an unstable pelvic fracture who is in obvious shock : how much time does the clinician have to make these critical decisions ? when do you initiate aggressive ffp administration or administer factor vii ? after initial resuscitation, does the clinician triage the patient to the ct scanner, to the operating room or wait for the angiographers to come in to embolize potential pelvic bleeder ? to answer these questions, a retrospective analysis of the sto2 database was conducted. the specific aims of the analysis were : to define the current epidemiology of mt, which includes documenting early temporal events and confirming the association of mt with bad outcomes, including mof and death ; and, secondly, to determine feasibility of the early prediction of mt and the potential role of sto2 in these prediction models. in this 16-month observational study, there were 381 patients who met entry criteria ; 114 (30%) received mt (defined as > 10 units in the first 24 hours). the mt cohort versus the non - mt cohort had similar demographics, but the patients who received a mt had a notably higher iss (32 17 vs. 26 15). analysis of the data also showed that the mt patients arrived hypothermic, were in severe shock and were notably coagulopathic. their initial international normalized ratio was 1.7 1.4. comparing baseline characteristics between the two cohorts, all variables with the exception of temperature were significantly less deranged in the non - mt cohort. these data demonstrated that, upon arrival, mt patients are different from non - mt patients. they have higher iss, they have more severe shock and they are severely coagulopathic prior to aggressive inhospital resuscitation. most notably, mt is a very rapid process of care ; 40% of the patients met the threshold of 10 units within 2 hours and 80% met the threshold within 6 hours. by 6 hours looking at time to death in the first 24 hours, there were 26 early deaths in the mt cohort, of which two - thirds occurred within the first 6 hours. these data emphasize that critical decisions must be made in a limited amount of time. assessment of clinical outcomes such as icu - free days, vent - free days, hospital days, and percentages of death and mof showed that mt patients have longer icu stays and spend more time on mechanical ventilators. the mortality rate for mt patients was 33% ; the incidence of mof was 31%, and 50% of patients had the combined outcome of mof and death. when you compare these outcomes with the non - mt patients, it is clear that mt patients have much worse outcomes. we were also interested in identifying the early predictors of bad outcome (mof / death). using univariate analysis comparing good outcome versus bad outcome, a number of variables were identified. we then chose p 25 at 1 hour and 2 hours, and the sto2 value in the 1-hour, 2-hour and 3-hour models sto2 is therefore a good variable to help identify the patient who is going to die. given the amount of controversy that currently exists over the management of mt and the need for a clinical trial to address the potential role of aggressive early ffp analysis, we performed a second follow - up analysis of the sto2 database with a focused study on the " mt died early " cohort. we looked at the 114 patients who received a mt and divided them into three groups : g1 was the 27 early deaths, g2 included 31 patients who developed mof or died late, and g3 was the remaining 50 patients who had a mt but did not die. looking at the number of packed red blood cells over the first 6 hours for the various groups, the g1 mt died early group received 26.4 units of packed red blood cells but only 6.5 units of ffp. considering the bloody vicious cycle pathophysiology and comparing the three groups for temperature, acidosis and coagulopathy, the g1 group had more deranged physiology. interestingly, the coagulopathy in g1 was noted to worsen over the first 3 hours ; the mt died early patients arrived with a significantly higher international normalized ratio of 2.4, which increased to 3.8. consequently, approximately 70% of these patients bled to death during a time period corresponding to their worsening coagulopathy. from these data we conclude that this subgroup of patients arrived with coagulopathy, received an ordinary amount of packed red blood cells (that is, 26.4 units) but received only 6.5 units of ffp, and therefore their coagulopathy was not treated. when we look at the shock parameters (figure 4) of systolic blood pressure, base deficit and sto2, the major observation is that sto2 is very different in this cohort of patients who were dying early. we therefore concluded that, in the setting of mt, a dropping sto2 portends early death from exsanguination. g1, massive transfusion (mt) and dies in 24 hours ; g2, mt and dies in > 24 hours and/or multiple organ failure (mof) ; g3, mt and survived without mof. p - values reported for the group and hour from trauma center (tc) arrival factor and the interaction term. sbp, systolic blood pressure ; sto2, tissue hemoglobin oxygen saturation. reproduced with permission from in our ongoing experience, nirs or sto2 monitoring offers a continuous non - invasive monitor of hypoperfusion. in early clinical testing during active shock resuscitation, changes in skeletal muscle sto2 correlated well with changes in do2, base deficit and lactate levels. this clinical experience in active shock resuscitation has likewise been verified in laboratory studies. in a prospective clinical trial of sto2 monitoring obtained within the first hour after ed admission of major torso trauma patients who were presumed to be bleeding, the sto2 value predicted death and mof as well as or perhaps better than base deficit and lactate levels. additionally, in the setting of mt we observed that a drop in sto2 portends early death from exsanguination and may be helpful in making critical decisions. acs : abdominal compartment syndrome ; ct : computerized tomography ; do2 : oxygen delivery ; ed : emergency department ; ffp : fresh frozen plasma ; icu : intensive care unit ; iss : injury severity score ; mof : multiple organ failure ; mt : massive transfusion ; nirs : near - infrared spectroscopy ; pa : pulmonary artery ; sto2 : tissue hemoglobin oxygen saturation ; svo2 : mixed venous hemoglobin oxygen saturation. this article is part of critical care volume 13 supplement 5 : tissue oxygenation (sto2) in healthy volunteers and critically - ill patients. | introductionthe purpose of the present review is to review our experience with near - infrared spectroscopy (nirs) monitoring in shock resuscitation and predicting clinical outcomes.methodsthe management of critically ill patients with goal - oriented intensive care unit (icu) resuscitation continues to evolve as our understanding of the appropriate physiologic targets improves. it is now recognized that resuscitation to achieve supranormal indices is not beneficial in all patients and may precipitate abdominal compartment syndrome.resultsover the years, icu technology has provided physicians with specific physiologic parameters to guide shock resuscitation. throughout this time, the tissue hemoglobin oxygen saturation (sto2) monitor has emerged as a non - invasive means to obtain reliable physiologic parameters to guide clinicians ' resuscitative efforts. sto2 monitors have been shown to aid in early identification of nonresponders and to predict outcomes in hemorrhagic shock and icu resuscitation. these data have also been used to better understand and refine existing resuscitation protocols. more recently, use of nirs technology to guide resuscitation in septic shock has been shown to predict outcomes in high - risk patients.conclusionssto2 is an important tool in identifying high - risk patients in septic and hemorrhagic shock. it is a non - invasive means of obtaining vital information regarding outcome and adequacy of resuscitation. |
post - kala - azar dermal leishmaniasis (pkdl) is a dermal manifestation of leishmania donovani infection and often follows resolution of clinical visceral leishmaniasis (vl). however, it may also manifest without prior history of vl in a small minority of patients. pkdl is characterized by macular, papular, or nodular lesions or a mixture of them. it is quite common in sudan (occurring in > 50% of patients with vl), where it may occur concurrently or follows immediately after an episode of vl and heals spontaneously in majority of patients, whereas in the indian subcontinent it occurs in 220% of patients, 6 months to several years after an episode of vl. in a recent trial the prevalence of confirmed pkdl cases was 4.4 per 10 000 individuals and 7.8 if probable cases were also considered. several treatment regimens have been recommended for the treatment of pkdl in india, for example, 120 days of parenteral sodium stibogluconate (20 mg / kg body weight) or three courses of 20 daily infusions of amphotericin b with an interval of 20 days in between the courses. these inordinately long parenteral regimens invariably lead to either nonacceptance or poor compliance. in the last decade two new antileishmanial compounds, miltefosine and paromomycin, have been approved for the treatment of vl in india. there is a report about the efficacy of miltefosine at a higher dose 50 mg tid for 60 days (with a need to extend to 90 days if required) in small number of pkdl patients in bangladesh. of all the antileishmanial drugs, paromomycin is considerably cheaper and is produced in india. in this study the study was carried out at the kala - azar medical research center, muzaffarpur, the field site of the institute of medical sciences, banaras hindu university. patients with pkdl were enrolled between october 2007 and august 2009 and followed for one year after treatment. the study was approved by the institutional ethics committee, and written informed consent was taken from all enrolled patients and by the parents of patients of 3 times the upper limit of normal range, serum bilirubin > 2 times the upper limit of normal range, and serum creatinine or blood urea nitrogen (bun) above normal range were excluded from the study. patients with any noncompensated or uncontrolled condition, such as active tuberculosis, malignant disease, severe malaria, hiv, or other major infectious diseases, lactation, pregnancy, or inadequate contraception in females of childbearing potential for the treatment period, were also excluded from the study. once eligible for enrollment, patients were given daily paromomycin 11 mg / kg (base) intramuscularly for 45 days. clinical parameters like vital signs were assessed daily, and hematological and biochemical assessments were done on days 15, 30, and 45 of treatment. for monitoring the adverse events, except nephrotoxicity, common toxicity criteria of national cancer institute were used. if there was toxicity of grade 3 and above, the treatment was discontinued and the subject was removed from the study and offered rescue treatment. nephrotoxicity was defined as an increase in serum creatinine that was either double the baseline levels or more than 2.0 mg per deciliter [177 mol per liter ]. cure was defined as complete disappearance of skin lesion(s) after treatment, as reported by the patient and assessed by the trained physician at 12-month followup. those patients who failed treatment in the form of no response and increase in number and size of lesion at 1-year followup and those patients in whom treatment was stopped due to adverse events were offered rescue treatment with three 20-day courses of amphotericin b in doses of 1 mg / kg given 20 days apart. comparison of means was done by using paired student 's t - test for paired data. a p value less than 0.05 (< 0.05) was considered as statistically significant. thirty - one patients with pkdl were included in the study out of which three patients did not give a history of a prior episode of vl. the median duration of interval between treatment of vl and development of pkdl was two years. amongst the previously treated patients for vl, 22 patients were treated with sodium stibogluconate (ssg), 3 patients with amphotericin b and miltefosine each, and 1 patient with paromomycin. among the various forms of skin lesions patients one had nodular lesion on face / chin with hypopigmented macular lesion over trunk, arms, and lower limb, and the other had hypopigmented macular and nodular lesions over the whole body. one patient presented with erythematous lesion over face while one patient presented with nodular and erythematous lesion on face with hypopigmented macular lesion over the whole body. pm was well tolerated by all patients without any significant derangement in hematological and biochemical parameter. 14 patients (45.2%) complained of pain at injection site which was the most common side effect noted. posttreatment clinical, biochemical, and hematological parameters were similar to the baseline values except slight improvement in the platelet count (table 1). at one - year followup, out of 31 patients, 9 (37.5%) were cured with complete disappearance of lesion while 15 (62.5%) patients showed no improvement ; rather there was increase in lesion and appearance of nodular lesions in 11 patients. all the patients who improved were having macular lesions. among three patients who had nodular lesions, pm is very effective in the treatment of vl with a cure rate of ~95%. these results prompted us to use pm for the treatment of pkdl. since dose / duration of every antileishmanial drug in pkdl is 2 - 3 times that used for vl, we arbitrarily selected a regimen of pm for 45 days (~2 times of vl) for the treatment of pkdl. though 45 days of pm treatment did not result in any noticeable adverse event except the expected injection site pain, the cure rate with this relatively better tolerated regimen of pm was unfortunately far from satisfactory. since these patients were admitted in the hospital for the entire duration of treatment, and pm injection was administered by nursing staff ; compliance was ensured. in india cure rates are 6492% with ssg in doses of 20 mg / kg per day for 120 days. ssg, being a toxic drug with high volume (1012 ml) of intramuscular injections given in long regimen of 120 days, makes acceptance of this regimen very poor. similarly 60 injections of amphotericin b given over 100 days pose a real threat of nephrotoxicity. unfortunately, nearly every trial for pkdl consists of small number of patients with no randomization. clinical response may differ according to the type of lesions in pkdl ; nodules and papules disappear in 120 days, macules in 200 days. thus it becomes imperative to follow these patients for a sufficiently long duration to see the long term effects of treatment. though the cure rate was unacceptably low, it had noticeable activity and cured one - third of the patients. future studies using paromomycin with another antileishmanial drug like miltefosine could yield successful short duration regimen for the treatment of pkdl and should be tested. it is also important to know that the tolerance to the drug was excellent even though the drug was used for twice the duration of that used in vl. the unresponsiveness of pm in pkdl which is very effective for treatment of vl highlights that there is a lot to be still discovered about the mechanism and pattern of response of pm against different manifestations of leishmaniasis. | background. post - kala - azar dermal leishmaniasis (pkdl) plays an important role in maintaining endemicity of visceral leishmaniasis and its transmission. treatment regimens for pkdl are toxic and require 3 - 4 months of hospitalization. these long and arduous regimens result in extensive noncompliance. there is an urgent need to develop a safe, effective, and acceptable regimen for the treatment of pkdl. paromomycin (pm) has been recently approved in india for treatment of visceral leishmaniasis (vl) ; hence we tested its efficacy in patients with pkdl. methods. in this exploratory study, 31 patients with pkdl aged 10 years and above were administered pm 11 mg / kg daily intramuscularly for 45 days and followed up for one year. results. out of 31 patients, 7 patients were lost to followup at 1 year and 9 (37.5%) got cured with complete disappearance of lesion, while 15 (62.5%) showed no improvement by per protocol analysis. conclusion. cure rate with 45 intramuscular injections of pm was unacceptably low though there was no serious side effect of the drug. whether paromomycin can be used in multidrug therapy to shorten the duration of treatment should be the next logical step for investigation. |
alopecia areata (aa) is common cause of reversible hair loss afflicting approximately 1 to 2% of the general population. a wide range of clinical presentations can occur, from a single patch of hair loss to complete loss of hair on the scalp (alopecia totalis, at) or over the entire body (alopecia universalis, au). the cause of aa is unknown, although there is evidence to suggest that the link between lymphocytic infiltration of the follicle and the disruption of the hair follicle cycle in aa may be provided by a combination of factors, including cytokine release, cytotoxic t - cell activity, and apoptosis. it is also considered that a disequilibrium in the production of cytokines, with a relative excess of proinflammatory and th1 types, vs. anti - inflammatory cytokines may be involved in the persistence of aa lesions, as shown in human scalp biopsies. tumor necrosis factor - alpha (tnf-) is a multifunctional proinflammatory cytokine which has been implicated in the pathogenesis of several chronic inflammatory disorders with an autoimmune component. this cytokine is synthesized in epidermal keratinocytes along with several other cytokines and is known to be a very potent inhibitor of proliferation. the changes in serum tnf- levels were found in many diseases, such as psoriasis and systemic lupus erythematosus. in some of these diseases, serum tnf- concentration correlated with activity and intensity of the disease, and although it is well known that multiple cytokines simultaneously play role in aa, many authors have measured only one particular cytokine. our study has focused only on tnf- because there are only a few studies that have measured the serum levels of this cytokine with controversial results. therefore, the aim of our study was to evaluate serum levels of tnf- in aa patients and control subjects, and also to assess the difference between the localized and extensive forms of the disease such as at and au. the study included 60 patients with aa (36 females and 24 males ; median age, 35.6 years). forty - six patients had localized aa (laa) and 14 patients had at, au, or at / au. the patients who had received any treatment within previous 3 months were excluded from the study, as well as patients with any diseases based on the immune pathomechanism, which could influence serum concentrations of tnf-. control group consisted of 20 generally healthy subjects (11 females and 9 males ; median age, 32.6 years). serum levels of tnf- were measured by an enzyme - linked immunosorbent assay technique, using quantikine human tnf- immunoassay (r and d system, minneapolis, mn, usa). standards and samples are pipetted into the wells and any tnf- present is bound by the immobilized antibody. after washing away any unbound substances, an enzyme - linked polyclonal antibody specific for tnf- is added to the wells. following a wash to remove any unbound antibody - enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of tnf- bound in the initial step. the test distribution was done by kolmogorov - smirnov test, and comparisons were performed by t - test. the study group comprised of 60 (36 females and 24 males ; the mean age was 35.6 years, ranging from 5 to 69 years) patients with aa and 20 healthy controls (11 females and 9 males ; the mean age 32.6 years, ranging from 6 to 63 years). there were no significant difference in age and female / male ratio between the patients and controls (p > 0.05). the mean duration of aa was 14.5 25.4 (range, 1 - 119 months). in the total of patients with aa, 46 of them were laa and 14 were at, au, or at / au group. serum tnf- levels ranged from 8.8 to 17.0 pg / ml, with the highest values observed in the au patients. the mean serum tnf- in aa patients was 10.31 1.20 pg / ml (mean sd), whereas that of laa or extensive (at, au, or at / au) was 10.16 0.79 pg / ml or 10.40 1.03 pg / ml, respectively. patients with longer duration of the disease had higher concentration of tnf-, but not significantly [figure 1 ]. correlation between the duration of the aa and concentration of tnf-,r = 0.034 ; (rho) = 0.1142 ; 95% ci (-0,144 ; 0,358) ; p > 0.05;-n.s serum levels of tnf- in patients with aa were significantly higher than those in controls (p = 0.044). there was no significant difference in levels of tnf- between patients with laa and the extensive group (p=0.2272). serum concentrations (meansd) of tnf- in patients with aa, laa, at / au and in healthy controls recent progress in the understanding of aa has shown that the regulation of local and systemic cytokines plays an important role in its pathogenesis. hair loss may occur because proinflammatory cytokines interfere with the hair cycle, leading to premature arrest of hair cycling with cessation of hair growth. this concept may explain typical clinical features of aa such as a progression pattern in centrifugal waves and spontaneous hair regrowth in concentric rings, suggesting the presence of soluble mediators within affected areas of the scalp. tnf- is a multifunctional proinflammatory cytokine which has been implicated in the pathogenesis of many infections and inflammatory disorders. however, this cytokine not only acts as mediator of immunity and inflammation, but also affects not - immune responses within tissues such as cell proliferation and differentiation. in vitro studies have shown that tnf-, along with il-1 and il-, causes vacuolation of matrix cells, abnormal keratinization of the follicle bulb and inner root sheath, as well as disruption of follicular melanocytes and the presence of melanin granules within the dermal papilla. experiments in cultured human hair follicles by hoffmann. showed that tnf- completely abrogated hair growth. additionally, tnf- induced the formation of a club - like hair follicle, similar to catagen morphology of the hair bulb. a study by thein. examined cytokine profiles of infiltrating activated t - cells from the margin of involved aa lesions. it was found that t - cell clones from involved lesions inhibited the proliferation of neonatal keratinocytes. in examining the cytokine profiles and relating them to regulatory capacity, the authors found that t - cell clones that released high amounts of ifn- and/or tnf- inhibited keratinocyte growth. a limited number of studies in the literature have evaluated the serum levels of tnf- in patients with aa. the results presented in our study demonstrate that the mean serum levels of tnf- were significantly elevated in aa patients in comparison with healthy subjects. there was no significant difference in levels of tnf- between patients with laa and the extensive group. in contrast to our results, teraki. reported that serum levels of tnf- in patients with laa were significantly higher than those in patients with au. in the study of koubanova and gadjlgoroeva, serum levels of tnf- in patients with aa did not differ from that in controls. however, tnf- was lower in patients with severe form of aa than in patients with mild form. they hypothesized that similar levels of tnf- in patients with both forms of aa and controls may indirectly indicate the absence of systemic immunopathological reactions in patients with aa, and the lowering of tnf- level in the mild form may indicate the tendency to formation of immunodeficiency in patients with severe aa. in addition, lis. found that serum levels of stnf- receptor type i were significantly elevated in patients with aa in comparison with healthy subjects. as they conclude, these results indicate that immune mechanisms in aa are characterized by activation of t - cells and other cells, possibly keratinocytes. tnf- seems to be a useful indicator of the activity of aa and that it may play an important role in the development of this disease. further investigations are required to clarify the pathogenic role and clinical significance of tnf- and these findings may provide important clues to assist in the development of new therapeutic strategies for patients with aa. | background : alopecia areata (aa) is a common form of localized, nonscarring hair loss. it is characterized by the loss of hair in patches, total loss of scalp hair (alopecia totalis, at), or total loss of body hair (alopecia universalis, au). the cause of aa is unknown, although most evidence supports the hypothesis that aa is a t - cell - mediated autoimmune disease of the hair follicle and that cytokines play an important role.aims:the aim of the study was to compare the serum levels of tumor necrosis factor - alpha (tnf-) in patients with aa and the healthy subjects and also to investigate the difference between the localized form of the disease with the extensive forms like at and au.materials and methods : sixty patients with aa and 20 healthy controls were enrolled in the study. forty - six patients had localized aa (laa), and 14 patients had at, au, or at / au. the serum levels of tnf- were measured using enzyme - linked immunoassay techniques.results:serum levels of tnf- were significantly higher in aa patients than in controls (10.31 1.20 pg ml vs 9.59 0.75 pg / ml, respectively). there was no significant difference in serum levels of tnf- between patients with laa and those with extensive forms of the disease.conclusion:our findings support the evidence that elevation of serum tnf- is associated with aa. the exact role of serum tnf- in aa should be additionally investigated in future studies. |
the benguela upwelling system (bus) along the southwest african continental margin is one of the most productive upwelling regions in the world oceans (carr, 2001). decreasing water depth and increasing primary productivity result in (i) a > 100fold increase in sediment organic matter content from oligotrophic surface sediments near the south atlantic gyre (sag) to sediments underlying the hypoxic and seasonally anoxic water column of the bus (inthorn. lin., 2012), and (ii) a strong decrease in bottom water dissolved oxygen concentrations from lower shelf regions to the bus (lavik., the decrease in bottom water dissolved oxygen concentrations is reflected in a two order of magnitude decrease in benthic macrofaunal abundance (sanders, 1969). ammonia oxidation is carried out by chemolithotrophic ammoniaoxidizing archaea and bacteria (aoa and aob). as the first step towards the removal of fixed nitrogen via denitrification or anaerobic ammonium oxidation, it is a key process in the marine environment. as aoa and aob presumably take up the same ecological role in the environment, niche specialization and subsequently niche differentiation between aoa and aob have been suggested (erguder., 2009 ; schleper, 2010 ; pester., 2011 ; prosser and nicol, 2012). salinity (mosier and francis, 2008 ; santoro., 2008), ph (nicol., 2008 ; zhang., 2011) and oxygen (bouskill., 2012 ; pettridge., 2013) have been identified as important environmental variables for such niche differentiation. additionally, studies of terrestrial (di., 2009 ; jia and conrad, 2009 ; baolan., 2012 ; prosser and nicol, 2012) and marine environments (wuchter., 2006), and of aoa in culture (martenshabbena., 2009), suggest that aoa are better adapted to low ammonium concentrations, while aob outcompete aoa at high ammonium concentrations, and that therefore ammonium concentrations may be the driving force for niche partitioning between aoa and aob. in this study, we utilized the strong gradient in microbial activity and sediment organic matter content in the surface sediments along a 550 km transect from the rim of the sag into the bus on the inner continental shelf off namibia. sediments from seven stations were collected during the rv meteor cruise m761 in april 2008. the sediments ranged from highly oxidized, oligotrophic deep sea red clays in the sag to organicrich sites with oxygenrich bottom water on the slope and outer shelf, to extremely organicrich sediments underlying permanently hypoxic, seasonally anoxic bottom water on the inner shelf (fig. 1, table s1 for site characteristics ; lavik., 2009). we hypothesized that the drastic increase of ammonium concentrations from sag to bus surface sediments would drive a transition from aoadominated to aobdominated ammonia oxidizer communities along the transect. to this end, we analysed ammonium pore water concentrations as well as diversity and apparent abundance of benthic aoa and betaproteobacterial aob across the gradient. organicrich surface sediments (total organic carbon content higher than 10%) are indicated by shading (data from inthorn., 2006). oceanographically, the stations represent the abyssal plain (geob12815), the continental rise (geob12808, geob12811), the continental slope (geob12803, geob12802), the shelf break (geob12807) and the shelf (geob12806). sediment samples were transported on dry ice to aarhus university, denmark, where they were stored at 80c until processed further. at the abyssal plain (geob12815) and the lower rise (geob12808) stations, ammonium was below the detection limit (10 mol l) throughout the upper 30 cm below seafloor (cmbsf) (fig. the lower rise station geob12811 had an ammonium profile similar to the shelf break and slope stations, but here the ammonium flux (as predicted from transportreaction models) from below did not reach the sediment surface. at the shelf break and upper slope stations (geob12802, 12803 and 12807), ammonium concentrations were higher (1050 mol l), and the predicted ammonium flux from the subsurface reached the sediment water interface. station geob12806, the only station located in the intense upwelling zone, where the seabed consisted of an extremely organicrich diatom ooze (borchers., 2005), featured ammonium concentrations at least one order of magnitude higher (5721914 mol l) than all the other stations. the oxygen penetration depth predicted from in situ data from the region (wenzhfer and glud, 2002) decreased from 112 mm at the most oligotrophic station geob12815 to 15 mm at geob12803, and was below 10 mm at the upper slope, shelf break and shelf stations (fig. ammonium production in the oxic surface sediment, as estimated from carbon oxidation rates via the oxygen penetration depths (for details, see appendix s1, table s1), exceeded the ammonium diffusing into the oxic zone from below at all stations, most strongly at the offshore sites. since there was no accumulation of ammonium in the oxic zone, this locally produced ammonium must have been concurrently oxidized, and it constitutes the most significant source of ammonium for the aoa and aob. thus, the volumetric ammonium oxidation rate increased > 500fold from the sag to the bus. bioturbation was apparent at stations geob12802, geob12803, geob12811 and geob12815, but not geob12807 and geob12806 (zabel., 2008), indicating that actual sediment oxygenation may occasionally reach deeper than modelled (up to 100 mm) towards the sag but not at the bus. overall, the data documented that the organic matter gradient from the sag rim sediments towards the bus is reflected by an increase of ammonium concentration and a decrease of oxygen availability. sediment pore water concentrations of ammonium, estimated rates of organic matter remineralization, and abundance of prokaryotes and ammonia oxidizers. white data points represent ammonium concentrations below the detection limit (see appendix s1). smooth pore water profiles (grey lines), ammonium fluxes and volumetric rates of ammonium production were calculated by fitting transportreaction models to the measured pore water profiles according to berg and colleagues (1998). ammonium fluxes (mol m day) to the top 1 cm are shown in greyshaded boxes. the predicted o 2 penetration depth (table s1) is indicated by the hatching pattern. ammonium values were analysed onshore (for detailed methods, see goldhammer., 2011). b. sum of archaeal and bacterial 16s rrna gene copy numbers g of sediment. overlaid grey and black bars represent the relative abundance of bacterial (grey) and archaeal (black) 16s rrna gene copies (in %, not log scale). gene copy numbers were determined by qpcr on dna extracted after removal of extracellular dna (see appendix s1). primer sets were 806f (takai and horikoshi, 2000)/958r (delong, 1992) for archaea, and bac8f (reysenbach., 1994)/bac338rabc (daims., 1999) for bacteria. quantitative pcr was performed in technical triplicates, which were averaged. c. abundance of archaeal (black lines and symbols) and bacterial (grey lines and symbols) amoa gene copy numbers g of sediment. different symbols of the same colour represent results from replicate dna extractions, i.e. a black square and a black triangle at the same depth represent archaeal amoa gene copy numbers from two different dna extracts. the ratios between bacterial and archaeal amoa gene copy numbers are shown for each depth in greyshaded boxes. primers archamoaf and archamoar (francis., 2005) and amoa1f and amoa2r (rotthauwe., 1997) archaeal and bacterial 16s rrna gene copy numbers were determined by quantitative realtime pcr (qpcr) for several depths at the seven sampling stations (25 samples ; fig. total 16s rrna gene abundance within the uppermost sampled sediment layer (01 cmbsf) increased towards the coast, with abundance increasing from 1.15 10 copies g sediment at station geob12815 closest to the sag to 6.96 10 copies g sediment at station geob12806 beneath the upwelling cell. assuming average 16s rrna gene copy numbers of 1.64 for archaea and 3.98 for bacteria (averaged based on 165 archaeal and 2649 bacterial finished genomes available on 20 march 2014, on the integrated microbial genomes (img) webpage ; markowitz., 2006), the corresponding apparent cell numbers increased from 4.82 10 g sediment (geob12815) to 2.05 10 g sediment (geob12806) (table s2). this trend was even more pronounced in deeper sediments layers (> 10 cmbsf) where total 16s rrna gene abundance increased from 5.06 10 copies g sediment off shelf (2.34 10 cells g sediment ; 14 cmbsf, geob12815) to 2.01 10 copies g sediment near shore (6.49 10 cells g sediment ; 12.5 cmbsf, geob12806). the observed trend was probably caused by the low sedimentation rate in the gyre, and generally speaking older sediment with more recalcitrant organic matter below the surface at offshore sites compared with sites located closer to shore (inthorn. a subset of 19 samples used for bacterial and archaeal 16s rrna gene quantification, as well as four additional samples (geob12811 : 10 cm ; geob12807 : 24 cmbsf and 29 cmbsf ; geob12806 : 31 cmbsf), were screened for the presence of archaeal and bacterial amoa, which were detected across all stations and depths (fig. average archaeal amoa copy numbers ranged from 5.2 10 genes g sediment (14 cmbsf, geob12811) to 5.2 10 genes g sediment (14 cmbsf, geob12815). bacterial amoa copy numbers ranged from 1.45 10 genes g sediment (14 cmbsf, geob12808) to 6.6 10 genes g sediment (19 cmbsf, geob12803). these numbers are comparable to previous amoa surveys in marine sediments (e.g. dang., 2010 ;., 2012), logtransformed amoa copy numbers of aoa show a significant positive correlation with amoa copy numbers of aob across the analysed samples (pearson correlation ; r = 0.84, p = 3.2 10). this suggests that aoa and aob respond similarly to environmental parameters at the sampling sites. correlations were calculated between qpcr data and all available geochemical data along the transect, but none of them was significant (for details, see tables s5 and s6). amoa copy numbers of aoa and, to a lesser degree, also of aob showed a decreasing trend from the oligotrophic sediments near the sag towards the eutrophic upper slope and shelf sediments (fig. 3) was observed at the two shallow shelf stations geob12807 and geob12806, an area for which hypoxic and seasonally anoxic bottom water has been reported (brchert., 2009). gene copy numbers of bacterial (grey circles) and archaeal (black triangles) amoa (g dna) as determined by qpcr (for exact values, see table s3). data points for a given water depth and station represent the different sediment depths analysed at that station. the lines connect the average values across sediment depths for bacterial (grey) and archaeal (black) amoa copy numbers. most samples showed a dominance of amoa copy numbers of aob over those of aoa, with a trimean ratio of 7.4 (range : 0.2102 ; fig. a survey of published genomic information of aoa and aob (norton., 1996 ; norton., 2008 ; walker., 2010 ; blainey., 2011 ; and retrieved from img website : markowitz., 2006) showed that betaproteobacterial aob usually feature multiple amoa gene copies per genome compared with only one copy in aoa. specifically, aob of the genus nitrosospira, which formed the majority of aob detected in the present study (see below), contain three amoa copies per genome. taking this into account, apparent aoa and aob cell numbers were comparable (aob / aoa ratios of 0.9 and 1) in 5 of the 23 analysed samples (table s3). higher aob / aoa ratios (3.834) that can not readily be explained by varying copy numbers in the genome were detected in the upper, mostly oxic, surface sediment samples (01 cmbsf) of the five intermediate transect sites (all but stations geob12815 and geob12806), where ammonium concentrations were 21 mol l and active ammonium oxidation took place. this observation falsified our starting hypothesis of a shift from aoa to aob dominance from the ammoniumpoor gyre towards the ammoniumrich bus sediments, and suggests a more complex niche differentiation of ammonia oxidizers in this system. amoa gene copy numbers of aoa and aob generally increased by an order of magnitude from the surface layer (01 cmbsf) to the next deeper layer (5 cmbsf) (table s3), which indicates growth of aoa and aob in the top sediment layers. the proportion of archaeal relative to bacterial amoa increased with sediment depth at all sites except site geob12803 (fig. 2c, table s3), especially below the oxic zone, where ammonia oxidizers presumably are inactive. similar trends were previously observed in soil (leininger., 2006) and marine sediments (beman., 2012), and may be explained by the archaea 's suggested ability to better persist with little or no energy sources due to their lower basal power requirement (valentine, 2007 ; hoehler and jrgensen, 2013). archaeal and bacterial amoa diversities were analysed in surface sediments (01 cmbsf) of the abyssal plain site closest to the sag (geob12815), the upper rise site (geob12811) and the shelf break site (geob12807). with 13 operational taxonomic units (otus) of archaeal amoa (fig. s3), overall diversity was low, and neither diversity indices nor richness estimates showed a significant trend across the transect (table 1). archaeal amoa sequences affiliated with the nitrosopumilus cluster (npum), the nitrososphaera subcluster 1.1 (nsp) and five distinct sequence clusters (a e) without cultured representatives (npum and nsp nomenclature based on pester., clusters npum and nsp were rare at stations geob12815 and geob12811, but dominated the clone library at station geob12807. in contrast, clusters a and d dominated the clone libraries at stations geob12815 and geob12811, but were rare (cluster d) or absent (cluster a) at station geob12807. the remaining sequence clusters showed low abundance in clone libraries from all three stations. assuming that the observed amoa clone abundance represents aoa cell abundance, this distribution may indicate different adaptations of aoa in all clusters to ammonium, oxygen and organic carbon availability, with npum and nsp occupying the most eutrophic, oxygenlimited niche, and clusters a and d occupying more oligotrophic, oxygenrich niches. diversity, phylogenetic affiliation and relative abundance of aoa and aob clones from three sites along the bus transect based on 635 bp archaeal amoa gene fragment obtained with the primer set archamoaf and archamoar (francis., 2005). based on 491 bp bacterial amoa gene fragment obtained with the primer set amoa1f / amoa2r (rotthauwe., 1997). based on 510514 bp betaproteobacterial aob 16s rrna gene fragment obtained with primer set cto189fabc / cto654r (kowalchuk and stephen, 2001). diversity measures were calculated based on otu clustering using a 13% sequence divergence cutoff for archaeal amoa (pester., 2012), an 8% cutoff for bacterial amoa (this study ; fig. s1) and a 1% cutoff for 16s rrna genes (ebers and stackebrandt, 2006). phylogenetic affiliation of amoa and 16s rrna gene clones is based on consensus phylogeny of maximum likelihood (raxml version 7.4.2 ; stamatakis., 2008), maximum parsimony, and neighbour joining (both phylip 3.69 ; felsenstein, 2005) analyses (figs s2s4). all six bacterial amoa otus clustered with other marine amoa sequences in a sister group to the nitrosospira lineage (fig. 16s rrna gene analysis confirmed this affiliation, as all but one sequence affiliated with nitrosospira cluster 1 (stephen. the difference in the exact tree topologies can be explained by the different resolution of the amoa and 16s rrna genes (koops., 2006). nitrosospira cluster 1 contains currently no cultured representative and is the only nitrosospira cluster frequently found in marine environments (stephen., 1996 ; mccaig., 1999 ; koops., 2006). as all cultured nitrosospira species have low salt tolerance (koops., 2006), nitrosospira cluster 1 has been proposed as solely allochthonous inhabitants in marine settings without significant physiological activity (koops., 2006). on the other hand, their widespread distribution and even predominance in marine ammoniaoxidizing communities (bano and hollibaugh, 2000 ; freitag and prosser, 2003 ; freitag., 2006) suggest a more active role in the marine nitrogen cycle. our qpcr data showed (i) an increase of nitrosospira cluster 1 off coast towards the gyre, while one would expect the opposite trend if they were allochthonous input from land ; and (ii) an increase of apparent aob cell numbers from the top sediment layer (01 cmbsf) to the next deeper layer (see above), indicating active growth of nitrosospira cluster 1 at sampling sites geob12815, geob12803, geob12802 and geob1207. finally, at least at station geob12807 active ammonia oxidation by nitrosospira cluster 1 is implied, as aoa alone are unlikely to account for the estimated ammonia oxidation rate : published cellspecific rates for aoa are 0.20.5 fmol nh3 cell h (martenshabbena., 2009 ; tourna., 2011), while rates of > 2 fmol cell h would be needed if ammonia oxidation was solely performed by aoa (see appendix s1 for details of calculation). these combined observations indicate that nitrosospira cluster 1 actively grow and oxidize ammonia in these marine sediments. half saturation constants (km) for betaproteobacterial ammonia oxidation range from 10 to 3300 m (nh3 + nh4) (stehr., 1995 ; pommereningrser., 1996 ; jiang and bakken, 1999 ; schramm., 1999 ; bollmann., the presence and growth of nitrosospira cluster 1 in sediments with ammonium concentrations 500fold from the sag to the bus. bioturbation was apparent at stations geob12802, geob12803, geob12811 and geob12815, but not geob12807 and geob12806 (zabel., 2008), indicating that actual sediment oxygenation may occasionally reach deeper than modelled (up to 100 mm) towards the sag but not at the bus. overall, the data documented that the organic matter gradient from the sag rim sediments towards the bus is reflected by an increase of ammonium concentration and a decrease of oxygen availability. sediment pore water concentrations of ammonium, estimated rates of organic matter remineralization, and abundance of prokaryotes and ammonia oxidizers. white data points represent ammonium concentrations below the detection limit (see appendix s1). smooth pore water profiles (grey lines), ammonium fluxes and volumetric rates of ammonium production were calculated by fitting transportreaction models to the measured pore water profiles according to berg and colleagues (1998). ammonium fluxes (mol m day) to the top 1 cm are shown in greyshaded boxes. the predicted o 2 penetration depth (table s1) is indicated by the hatching pattern. ammonium values were analysed onshore (for detailed methods, see goldhammer., 2011). b. sum of archaeal and bacterial 16s rrna gene copy numbers g of sediment. overlaid grey and black bars represent the relative abundance of bacterial (grey) and archaeal (black) 16s rrna gene copies (in %, not log scale). gene copy numbers were determined by qpcr on dna extracted after removal of extracellular dna (see appendix s1). primer sets were 806f (takai and horikoshi, 2000)/958r (delong, 1992) for archaea, and bac8f (reysenbach., 1994)/bac338rabc (daims., 1999) for bacteria. quantitative pcr was performed in technical triplicates, which were averaged. c. abundance of archaeal (black lines and symbols) and bacterial (grey lines and symbols) amoa gene copy numbers g of sediment. different symbols of the same colour represent results from replicate dna extractions, i.e. a black square and a black triangle at the same depth represent archaeal amoa gene copy numbers from two different dna extracts. the ratios between bacterial and archaeal amoa gene copy numbers are shown for each depth in greyshaded boxes. primers archamoaf and archamoar (francis., 2005) and amoa1f and amoa2r (rotthauwe., 1997) archaeal and bacterial 16s rrna gene copy numbers were determined by quantitative realtime pcr (qpcr) for several depths at the seven sampling stations (25 samples ; fig. total 16s rrna gene abundance within the uppermost sampled sediment layer (01 cmbsf) increased towards the coast, with abundance increasing from 1.15 10 copies g sediment at station geob12815 closest to the sag to 6.96 10 copies g sediment at station geob12806 beneath the upwelling cell. assuming average 16s rrna gene copy numbers of 1.64 for archaea and 3.98 for bacteria (averaged based on 165 archaeal and 2649 bacterial finished genomes available on 20 march 2014, on the integrated microbial genomes (img) webpage ; markowitz., 2006), the corresponding apparent cell numbers increased from 4.82 10 g sediment (geob12815) to 2.05 10 g sediment (geob12806) (table s2). this trend was even more pronounced in deeper sediments layers (> 10 cmbsf) where total 16s rrna gene abundance increased from 5.06 10 copies g sediment off shelf (2.34 10 cells g sediment ; 14 cmbsf, geob12815) to 2.01 10 copies g sediment near shore (6.49 10 cells g sediment ; 12.5 cmbsf, geob12806). the observed trend was probably caused by the low sedimentation rate in the gyre, and generally speaking older sediment with more recalcitrant organic matter below the surface at offshore sites compared with sites located closer to shore (inthorn. a subset of 19 samples used for bacterial and archaeal 16s rrna gene quantification, as well as four additional samples (geob12811 : 10 cm ; geob12807 : 24 cmbsf and 29 cmbsf ; geob12806 : 31 cmbsf), were screened for the presence of archaeal and bacterial amoa, which were detected across all stations and depths (fig. average archaeal amoa copy numbers ranged from 5.2 10 genes g sediment (14 cmbsf, geob12811) to 5.2 10 genes g sediment (14 cmbsf, geob12815). bacterial amoa copy numbers ranged from 1.45 10 genes g sediment (14 cmbsf, geob12808) to 6.6 10 genes g sediment (19 cmbsf, geob12803). these numbers are comparable to previous amoa surveys in marine sediments (e.g. dang., 2010 ; cao., 2012 ; zheng., similar to marine sediments off the coast of california (beman., 2012), logtransformed amoa copy numbers of aoa show a significant positive correlation with amoa copy numbers of aob across the analysed samples (pearson correlation ; r = 0.84, p = 3.2 10). this suggests that aoa and aob respond similarly to environmental parameters at the sampling sites. correlations were calculated between qpcr data and all available geochemical data along the transect, but none of them was significant (for details, see tables s5 and s6). amoa copy numbers of aoa and, to a lesser degree, also of aob showed a decreasing trend from the oligotrophic sediments near the sag towards the eutrophic upper slope and shelf sediments (fig. 3) was observed at the two shallow shelf stations geob12807 and geob12806, an area for which hypoxic and seasonally anoxic bottom water has been reported (brchert., 2009) gene copy numbers of bacterial (grey circles) and archaeal (black triangles) amoa (g dna) as determined by qpcr (for exact values, see table s3). data points for a given water depth and station represent the different sediment depths analysed at that station. the lines connect the average values across sediment depths for bacterial (grey) and archaeal (black) amoa copy numbers. most samples showed a dominance of amoa copy numbers of aob over those of aoa, with a trimean ratio of 7.4 (range : 0.2102 ; fig. 2c). a survey of published genomic information of aoa and aob (norton., 1996 ; norton., 2008 ; walker., 2010 ; blainey., 2011 ; and retrieved from img website : markowitz., 2006) showed that betaproteobacterial aob usually feature multiple amoa gene copies per genome compared with only one copy in aoa. specifically, aob of the genus nitrosospira, which formed the majority of aob detected in the present study (see below), contain three amoa copies per genome. taking this into account, apparent aoa and aob cell numbers were comparable (aob / aoa ratios of 0.9 and 1) in 5 of the 23 analysed samples (table s3). higher aob / aoa ratios (3.834) that can not readily be explained by varying copy numbers in the genome were detected in the upper, mostly oxic, surface sediment samples (01 cmbsf) of the five intermediate transect sites (all but stations geob12815 and geob12806), where ammonium concentrations were 21 mol l and active ammonium oxidation took place. this observation falsified our starting hypothesis of a shift from aoa to aob dominance from the ammoniumpoor gyre towards the ammoniumrich bus sediments, and suggests a more complex niche differentiation of ammonia oxidizers in this system. amoa gene copy numbers of aoa and aob generally increased by an order of magnitude from the surface layer (01 cmbsf) to the next deeper layer (5 cmbsf) (table s3), which indicates growth of aoa and aob in the top sediment layers. the proportion of archaeal relative to bacterial amoa increased with sediment depth at all sites except site geob12803 (fig. 2c, table s3), especially below the oxic zone, where ammonia oxidizers presumably are inactive. similar trends were previously observed in soil (leininger., 2006) and marine sediments (beman., 2012), and may be explained by the archaea 's suggested ability to better persist with little or no energy sources due to their lower basal power requirement (valentine, 2007 ; hoehler and jrgensen, 2013). archaeal and bacterial amoa diversities were analysed in surface sediments (01 cmbsf) of the abyssal plain site closest to the sag (geob12815), the upper rise site (geob12811) and the shelf break site (geob12807). with 13 operational taxonomic units (otus) of archaeal amoa (fig. s3), overall diversity was low, and neither diversity indices nor richness estimates showed a significant trend across the transect (table 1). archaeal amoa sequences affiliated with the nitrosopumilus cluster (npum), the nitrososphaera subcluster 1.1 (nsp) and five distinct sequence clusters (a e) without cultured representatives (npum and nsp nomenclature based on pester., clusters npum and nsp were rare at stations geob12815 and geob12811, but dominated the clone library at station geob12807. in contrast, clusters a and d dominated the clone libraries at stations geob12815 and geob12811, but were rare (cluster d) or absent (cluster a) at station geob12807. the remaining sequence clusters showed low abundance in clone libraries from all three stations. assuming that the observed amoa clone abundance represents aoa cell abundance, this distribution may indicate different adaptations of aoa in all clusters to ammonium, oxygen and organic carbon availability, with npum and nsp occupying the most eutrophic, oxygenlimited niche, and clusters a and d occupying more oligotrophic, oxygenrich niches. diversity, phylogenetic affiliation and relative abundance of aoa and aob clones from three sites along the bus transect based on 635 bp archaeal amoa gene fragment obtained with the primer set archamoaf and archamoar (francis., 2005). based on 491 bp bacterial amoa gene fragment obtained with the primer set amoa1f / amoa2r (rotthauwe., 1997). based on 510514 bp betaproteobacterial aob 16s rrna gene fragment obtained with primer set cto189fabc / cto654r (kowalchuk and stephen, 2001). diversity measures were calculated based on otu clustering using a 13% sequence divergence cutoff for archaeal amoa (pester., 2012), an 8% cutoff for bacterial amoa (this study ; fig. s1) and a 1% cutoff for 16s rrna genes (ebers and stackebrandt, 2006). phylogenetic affiliation of amoa and 16s rrna gene clones is based on consensus phylogeny of maximum likelihood (raxml version 7.4.2 ; stamatakis., 2008), maximum parsimony, and neighbour joining (both phylip 3.69 ; felsenstein, 2005) analyses (figs s2s4). all six bacterial amoa otus clustered with other marine amoa sequences in a sister group to the nitrosospira lineage (fig. 16s rrna gene analysis confirmed this affiliation, as all but one sequence affiliated with nitrosospira cluster 1 (stephen. the difference in the exact tree topologies can be explained by the different resolution of the amoa and 16s rrna genes (koops., 2006). nitrosospira cluster 1 contains currently no cultured representative and is the only nitrosospira cluster frequently found in marine environments (stephen., 1996 ;, 1999 ; koops., 2006). as all cultured nitrosospira species have low salt tolerance (koops., 2006), nitrosospira cluster 1 has been proposed as solely allochthonous inhabitants in marine settings without significant physiological activity (koops., 2006). on the other hand, their widespread distribution and even predominance in marine ammoniaoxidizing communities (bano and hollibaugh, 2000 ; freitag and prosser, 2003 ; freitag., our qpcr data showed (i) an increase of nitrosospira cluster 1 off coast towards the gyre, while one would expect the opposite trend if they were allochthonous input from land ; and (ii) an increase of apparent aob cell numbers from the top sediment layer (01 cmbsf) to the next deeper layer (see above), indicating active growth of nitrosospira cluster 1 at sampling sites geob12815, geob12803, geob12802 and geob1207. finally, at least at station geob12807 active ammonia oxidation by nitrosospira cluster 1 is implied, as aoa alone are unlikely to account for the estimated ammonia oxidation rate : published cellspecific rates for aoa are 0.20.5 fmol nh3 cell h (martenshabbena., 2009 ; tourna., 2011), while rates of > 2 fmol cell h would be needed if ammonia oxidation was solely performed by aoa (see appendix s1 for details of calculation). these combined observations indicate that nitrosospira cluster 1 actively grow and oxidize ammonia in these marine sediments. half saturation constants (km) for betaproteobacterial ammonia oxidation range from 10 to 3300 m (nh3 + nh4) (stehr., 1995 ; pommereningrser., 1996 ; jiang and bakken, 1999 ; schramm. the presence and growth of nitrosospira cluster 1 in sediments with ammonium concentrations 90% is depicted with black pie piece colouring, while bootstrap support > 50% and 90% is depicted with black pie piece colouring, while bootstrap support > 50% and 90% is depicted with black pie piece colouring, while bootstrap support > 50% and < 90% are depicted with grey pie piece colouring. representative sequences for each otu and station are shown in bold, along with their embl nucleotide sequence database accession numbers. otu class and the number of clones for a given station are indicated in parenthesis. subgroup naming is based on stephen and colleagues (1996) and koops and colleagues (2006). transect stations with coordinates, water depth, representative bottom water temperature (data from mohrholz., 2008), oxygen and ammonium data. values in parentheses for the predicted o2 penetration depth indicate that those stations were within the mudbelt, and the predicted penetration depths are therefore maximum values. the predicted nh4 production rate in the oxic zone leads to an equal rate of nh4 oxidation. archaeal and bacterial 16s rrna gene copy numbers, estimated cell numbers per gram of wet sediment and bacteria / archaea ratios. cell numbers were estimated by using averages of 1.64 and 3.98 16s rrna gene copies per genome for archaea and bacteria (for details, see main text) respectively. archaeal and bacterial amoa gene copy numbers, estimated cell numbers and aob / aoa ratios. gene copy numbers are presented per gram of (wet) sediment and per gram of extracted dna. cell numbers were estimated by using averages of 1 and 3 amoa gene copies per genome for archaea and bacteria respectively. apparent relative abundance was calculated based on estimated aoa and aob cell numbers g sediment (this table) and total estimated cell numbers of archaea and bacteria g sediment as shown in table s2. samples that yielded a positive signal but concentrations below the quantification limit of the method are labelled with an asterisk (). pearson correlation coefficients (r) for comparisons between logtransformed qpcr data (gene copies g sediment) and geochemical data of sediment samples. (p < 0.01) is indicated with an asterisk (for detailed p values, see table s6). arc, archaea ; bac, bacteria ; bacamoa, bacterial amoa ; arcamoa, archaeal amoa ; dic, dissolved inorganic carbon. the used geochemical data of the samples can be retrieved from pangaea data publisher for earth & environmental science (http://www.pangaea.de). arc, archaea ; bac, bacteria ; bacamoa, bacterial amoa ; arcamoa, archaeal amoa ; dic, dissolved inorganic carbon. | summarysediments across the namibian continental margin feature a strong microbial activity gradient at their surface. this is reflected in ammonium concentrations of 700 m in upwelling areas near the coast. here we address changes in apparent abundance and structure of ammoniaoxidizing archaeal and bacterial communities (aoa and aob) along a transect of seven sediment stations across the namibian shelf by analysing their respective ammonia monooxygenase genes (amoa). the relative abundance of archaeal and bacterial amoa (g1 dna) decreased with increasing ammonium concentrations, and bacterial amoa frequently outnumbered archaeal amoa at the sediment water interface [01 cm below seafloor (cmbsf) ]. in contrast, aoa were apparently as abundant as aob or dominated in several deeper (> 10 cmbsf), anoxic sediment layers. phylogenetic analyses showed a change within the aoa community along the transect, from two clusters without cultured representatives at the gyre to n itrososphaera and n itrosopumilus clusters in the upwelling region. aob almost exclusively belonged to the n itrosospira cluster 1. our results suggest that this predominantly marine aob lineage without cultured representatives can thrive at low ammonium concentrations and is active in the marine nitrogen cycle. |
the use of nanoparticles for cellular imaging is among the most important clinical breakthrough of the past decade. in particular, superparamagnetic iron oxide (spio) nanoparticles enhance contrast in magnetic resonance imaging (mri), which allows clinicians to monitor anatomical, physiological, and molecular changes during the evolution of a disease or treatment. following intravenous injection, these nanoparticles accumulate in macrophages residing in the liver, bone marrow, and spleen, as well as tumors and sites of inflammation. accordingly, applications include the detection of inflammatory diseases, in vivo stem cell tracking,1 hyperthermia therapy,2 lymph node detection,3 and anticancer drug delivery.4 the current applications for spio nanoparticles are limited because these relatively large particles, with an average diameter of 80 nm, are rapidly internalized by the mononuclear phagocytic system of the liver and the spleen. this problem is currently addressed by the development of ultrasmall superparamagnetic iron oxide (uspio) nanoparticles (< 50 nm).5 in vivo studies recently showed that uspio nanoparticles of 35 nm in diameter have a longer half - life in the circulation system, allowing the labeling of macrophages migrating to remote areas.6,7 these studies opened the door to a number of new applications for molecular imaging because macrophages migrate and accumulate at sites of inflammation,8 autoimmune neuritis,9 renal ischemia,10 and solid organ transplant rejection.11 the use of uspio agents has allowed for the direct visualization of macrophage infiltration of carotid atheroma in clinical study.12,13 the surface coating of uspio nanoparticles also influences their stability and cellular uptake by macrophages.6 dextran - based coatings are preferred because of their low toxicity and they are biodegradable.14 for instance, dextran - coated spio nanoparticles and ferucarbotran are approved for liver mri. moreover, carboxydextran - coated spio nanoparticles have undergone clinical trials for mri evaluation of lymph node metastasis;15,16 however, the impact of uspio nanoparticles size and dextran coating composition on the uptake by macrophages has not been determined. the aim of the present study is to determine the impact of particle size and surface coating on the cellular uptake and relaxing time of spio nanoparticles in mouse macrophages by mri and microscopy. the uspio (28 nm) and spio (72 and 74 nm) nanoparticles were coated with alkali - treated dextran (atdm) or carboxymethyl dextran (cmdm). atdm (surface voltage potential : 15 mv) was tested with particle sizes of 28 and 74 nm. cmdm (surface voltage potential : 24 mv) was tested with particle sizes of 28 and 72 nm. all spio and uspio compounds were purchased from the meito sangyo company, ltd (aichi, japan).17 the mouse macrophage cell line raw264 was provided by the riken bioresource center (riken tsukuba institute, tsukuba, ibaraki, japan). raw264 cells (1 10) were cultured in minimum essential medium (sigma - aldrich, tokyo, japan), 10% fetal calf serum (nichirei corporation, tokyo, japan), and 0.1 mm nonessential amino acids (sigma - aldrich) in 93 mm 21 mm petri dishes (iwaki, tokyo, japan) with 18 mm square cover glasses (matusnami glass ind, ltd, osaka, japan). the contrast of proton mri images depends on the relaxation times of the nuclear magnetization (t1, longitudinal ; t2, transverse). iron oxide nanoparticles primarily affect t2 and work as negative contrast agents. a high intracellular concentration of spio and uspio nanoparticles results in the reduction of t2 relaxation time. prior to the mri experiment, the concentrations of uspio particles were adjusted to 0.005 mg fe / ml and prepared in five sample tubes with water samples serving as controls. mri experiments for the three times measurements of the t2 relaxation times were performed on an 11.7-t mri scanner (bruker biospin, ettlingen, germany) and used a volume rf coil with a 25 mm inner diameter for transmission and reception (m2 m imaging corp, cleveland, oh). t2 mapping was performed using a multislice, multiecho spin - echo sequence (repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6 25.6 mm ; matrix = 128 128 ; slice orientation = transaxial ; number of repetitions = 1) with echo times ranging from 10 to 100 ms in steps of 10 ms. t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point. the experiments were conducted to measure the t2 relaxation times of the macrophages labeled with spio or uspio nanoparticles. all uspio particles were dissolved in the cell growth medium at a concentration of 0.3 mg fe / ml, which is similar to the concentration used in another published report.18 cells (2 10) were incubated with medium containing uspio particles for 1, 2, or 4 h (same time points noted in a previous report)19 at 37c under 5% co2. after incubation, the medium was removed by two washes with phosphate - buffered saline (pbs) followed by fixation of the cells for 30 min with 7.5% formalin. the presence of iron oxides in the raw264 cells was detected by staining with berlin blue (kfe(3)fe(2)(cn)6) (wako pure chemical industries, ltd, osaka, japan)20 for 20 min. after washing away the berlin blue with pbs, the cell nuclei were counter - stained with nuclear fast red for 5 min. the nuclear fast red was removed by washing with pbs three times, and the cell specimens were fixed to the cover glass. for each experiment, the labeling efficiency of each type of uspio nanoparticle was assessed visually by three observers using a bz-9000 microscope (keyence corporation, osaka, japan). the labeling efficiencies (percentages of spio / uspio uptake) were qualitatively assessed from the microscope images in three independent experiments by estimating the number of iron - positive cells within five randomly selected 200 m fields. viable cells are not stained by trypan blue, while dead and lysed cells are stained. all uspio particles were dissolved in cell growth medium at a concentration of 0.3 mg fe / ml, which is a lower concentration when compared to a sample test because the concentrations of spio and uspio in macrophage cells were lower than sample spio and uspio concentrations. cells (2 10) were incubated in a medium containing uspio particles for 1, 2, or 4 h at 37c under 5% co2. after incubation, the medium was removed with two washes of pbs and the cells were fixed with formalin. cells were stained with trypan blue for 10 min and the viable and dead cells were counted under a microscope. the percentages of viable cells were qualitatively assessed by counting the number of trypan blue - positive cells within five randomly selected 200 m fields. all uspio nanoparticles were dissolved in cell growth medium at a concentration of 0.3 mg / ml. raw264 cells (2 10) were incubated with medium containing uspio particles for 30 min at 37c under 5% co2. after incubation, the medium was removed by two washes with pbs and the cells were fixed with formalin. the concentration of raw264 cells was adjusted to 4 10 cells / ml and the cells (0.5 ml) were dissolved in 0.1% agarose gel (0.5 ml) to give a final concentration of 2.0 10 cells / ml. the sample temperature was maintained at approximately 23c and the cells were imaged after settling into a pellet by gravity. for measurement of t2 relaxation time, t2 mapping was performed using a multislice multiecho spin - echo sequence (repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6 25.6 mm ; matrix = 128 128 ; slice orientation = transaxial ; number of repetitions = 1) with echo times ranging from 10 to 100 ms in steps of 10 ms. t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point. all statistical analysis was performed using prism (version 5 ; graphpad software inc, la jolla, ca). one - way analysis of variance with the bonferroni correction was applied in order to compare changes in percentages of spio and uspio uptake, cell viability, and t2 relaxation times for all spio / uspio nanoparticles and labeled cells. atdm (surface voltage potential : 15 mv) was tested with particle sizes of 28 and 74 nm. cmdm (surface voltage potential : 24 mv) was tested with particle sizes of 28 and 72 nm. all spio and uspio compounds were purchased from the meito sangyo company, ltd (aichi, japan).17 the mouse macrophage cell line raw264 was provided by the riken bioresource center (riken tsukuba institute, tsukuba, ibaraki, japan). raw264 cells (1 10) were cultured in minimum essential medium (sigma - aldrich, tokyo, japan), 10% fetal calf serum (nichirei corporation, tokyo, japan), and 0.1 mm nonessential amino acids (sigma - aldrich) in 93 mm 21 mm petri dishes (iwaki, tokyo, japan) with 18 mm square cover glasses (matusnami glass ind, ltd, osaka, japan). the contrast of proton mri images depends on the relaxation times of the nuclear magnetization (t1, longitudinal ; t2, transverse). iron oxide nanoparticles primarily affect t2 and work as negative contrast agents. a high intracellular concentration of spio and uspio nanoparticles results in the reduction of t2 relaxation time. prior to the mri experiment, the concentrations of uspio particles were adjusted to 0.005 mg fe / ml and prepared in five sample tubes with water samples serving as controls. mri experiments for the three times measurements of the t2 relaxation times were performed on an 11.7-t mri scanner (bruker biospin, ettlingen, germany) and used a volume rf coil with a 25 mm inner diameter for transmission and reception (m2 m imaging corp, cleveland, oh). t2 mapping was performed using a multislice, multiecho spin - echo sequence (repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6 25.6 mm ; matrix = 128 128 ; slice orientation = transaxial ; number of repetitions = 1) with echo times ranging from 10 to 100 ms in steps of 10 ms. t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point. the experiments were conducted to measure the t2 relaxation times of the macrophages labeled with spio or uspio nanoparticles. all uspio particles were dissolved in the cell growth medium at a concentration of 0.3 mg fe / ml, which is similar to the concentration used in another published report.18 cells (2 10) were incubated with medium containing uspio particles for 1, 2, or 4 h (same time points noted in a previous report)19 at 37c under 5% co2. after incubation, the medium was removed by two washes with phosphate - buffered saline (pbs) followed by fixation of the cells for 30 min with 7.5% formalin. the presence of iron oxides in the raw264 cells was detected by staining with berlin blue (kfe(3)fe(2)(cn)6) (wako pure chemical industries, ltd, osaka, japan)20 for 20 min. after washing away the berlin blue with pbs, the cell nuclei were counter - stained with nuclear fast red for 5 min. the nuclear fast red was removed by washing with pbs three times, and the cell specimens were fixed to the cover glass. for each experiment, the labeling efficiency of each type of uspio nanoparticle was assessed visually by three observers using a bz-9000 microscope (keyence corporation, osaka, japan). the labeling efficiencies (percentages of spio / uspio uptake) were qualitatively assessed from the microscope images in three independent experiments by estimating the number of iron - positive cells within five randomly selected 200 m fields. viable cells are not stained by trypan blue, while dead and lysed cells are stained. all uspio particles were dissolved in cell growth medium at a concentration of 0.3 mg fe / ml, which is a lower concentration when compared to a sample test because the concentrations of spio and uspio in macrophage cells were lower than sample spio and uspio concentrations. cells (2 10) were incubated in a medium containing uspio particles for 1, 2, or 4 h at 37c under 5% co2. after incubation, the medium was removed with two washes of pbs and the cells were fixed with formalin. cells were stained with trypan blue for 10 min and the viable and dead cells were counted under a microscope. the percentages of viable cells were qualitatively assessed by counting the number of trypan blue - positive cells within five randomly selected 200 m fields. all uspio nanoparticles were dissolved in cell growth medium at a concentration of 0.3 mg / ml. raw264 cells (2 10) were incubated with medium containing uspio particles for 30 min at 37c under 5% co2. after incubation, the medium was removed by two washes with pbs and the cells were fixed with formalin. the concentration of raw264 cells was adjusted to 4 10 cells / ml and the cells (0.5 ml) were dissolved in 0.1% agarose gel (0.5 ml) to give a final concentration of 2.0 10 cells / ml. the sample temperature was maintained at approximately 23c and the cells were imaged after settling into a pellet by gravity. for measurement of t2 relaxation time, t2 mapping was performed using a multislice multiecho spin - echo sequence (repetition time = 3000 ms ; slice thickness = 1 mm ; field of view = 25.6 25.6 mm ; matrix = 128 128 ; slice orientation = transaxial ; number of repetitions = 1) with echo times ranging from 10 to 100 ms in steps of 10 ms. t2 maps were calculated from a single exponential fitting of mri signal intensities at each echo time point. all statistical analysis was performed using prism (version 5 ; graphpad software inc, la jolla, ca). one - way analysis of variance with the bonferroni correction was applied in order to compare changes in percentages of spio and uspio uptake, cell viability, and t2 relaxation times for all spio / uspio nanoparticles and labeled cells. the mri contrast provided by each type of nanoparticle formulation was compared by measuring the t2 relaxation time. at 0.005 mg fe / ml, t2 relaxation time of the 74 nm atdm - coated nanoparticles was lower than for the other three formulations (figure 1a). since a low t2 relaxation time translates into a high mri negative contrast, these data suggest that atdm - coated spio nanoparticles would provide the best mri images among these formulations (figure 1b). microscopy analysis was used to compare the labeling efficiency of each nanoparticle formulation in terms of the percentage of macrophages labeled within 200-mm fields, and the rate of culture saturation over time. overall, the atdm - coated nanoparticles provided a more efficient labeling of the macrophages than the cmdm - coated nanoparticles (figure 2). quantitative analysis revealed that more than 60% of the macrophages were labeled by atdm - coated nanoparticles after 1 h, compared to < 10% with cmdm - coated nanoparticles, regardless of the particle size (figure 3a). this profile was maintained after 2 h. however, the efficiency of atdm - coated nanoparticle labeling had reached 100%, and cmdm - coated nanoparticles were detected only in 20%30% of macrophages. after 4 h, all nanoparticle formulations had a labeling efficiency superior to 95%, except for 20%30% for cmdm - coated 28 and 72 nm nanoparticles. figure 3b shows that none of the nanoparticle formulations affected the viability of the macrophages. altogether, these data suggest that the use of uspio nanoparticles (28 nm), instead of spio nanoparticles (72 and 74 nm), does not improve the labeling efficiency of macrophages. in contrast, atdm coating provides a more rapid and efficient labeling of most cells for at least 4 h, which is an asset for mri imaging. the t2 relaxation time of each nanoparticle formulation was measured following a 30-min labeling period of the macrophages to determine the impact of cellular uptake. figure 4a shows the different mri signal intensities obtained for the four types of nanoparticles. quantitative analysis revealed significantly higher t2 values for the cmdm - coated particles than the atdm - coated particles (figure 4b). among the atdm - coated particles, uspio particles generated an even brighter signal change than the spio particles. these data suggest that atdm - coated uspio particles would offer the best mri image contrast than all four formulations. finally, the impact of cell density on the contrast quality of mri images was tested by measuring the t2 relaxation time of macrophages labeled with atdm - coated 74 nm nanoparticles for 30 min. figure 5a shows that the t2 relaxation time decreases with increasing cell density. a linear relationship between relaxation time and cell density these data suggest that the ability of mri negative contrast agent is increased at high cell density. the mri contrast provided by each type of nanoparticle formulation was compared by measuring the t2 relaxation time. at 0.005 mg fe / ml, t2 relaxation time of the 74 nm atdm - coated nanoparticles was lower than for the other three formulations (figure 1a). since a low t2 relaxation time translates into a high mri negative contrast, these data suggest that atdm - coated spio nanoparticles would provide the best mri images among these formulations (figure 1b). microscopy analysis was used to compare the labeling efficiency of each nanoparticle formulation in terms of the percentage of macrophages labeled within 200-mm fields, and the rate of culture saturation over time. overall, the atdm - coated nanoparticles provided a more efficient labeling of the macrophages than the cmdm - coated nanoparticles (figure 2). quantitative analysis revealed that more than 60% of the macrophages were labeled by atdm - coated nanoparticles after 1 h, compared to < 10% with cmdm - coated nanoparticles, regardless of the particle size (figure 3a). this profile was maintained after 2 h. however, the efficiency of atdm - coated nanoparticle labeling had reached 100%, and cmdm - coated nanoparticles were detected only in 20%30% of macrophages. after 4 h, all nanoparticle formulations had a labeling efficiency superior to 95%, except for 20%30% for cmdm - coated 28 and 72 nm nanoparticles. figure 3b shows that none of the nanoparticle formulations affected the viability of the macrophages. altogether, these data suggest that the use of uspio nanoparticles (28 nm), instead of spio nanoparticles (72 and 74 nm), does not improve the labeling efficiency of macrophages. in contrast, atdm coating provides a more rapid and efficient labeling of most cells for at least 4 h, which is an asset for mri imaging. the t2 relaxation time of each nanoparticle formulation was measured following a 30-min labeling period of the macrophages to determine the impact of cellular uptake. figure 4a shows the different mri signal intensities obtained for the four types of nanoparticles. quantitative analysis revealed significantly higher t2 values for the cmdm - coated particles than the atdm - coated particles (figure 4b). among the atdm - coated particles, uspio particles generated an even brighter signal change than the spio particles. these data suggest that atdm - coated uspio particles would offer the best mri image contrast than all four formulations. finally, the impact of cell density on the contrast quality of mri images was tested by measuring the t2 relaxation time of macrophages labeled with atdm - coated 74 nm nanoparticles for 30 min. figure 5a shows that the t2 relaxation time decreases with increasing cell density. a linear relationship between relaxation time and cell density these data suggest that the ability of mri negative contrast agent is increased at high cell density. the present study suggests that spio / uspio labeling efficiency in cultured mouse macrophages was influenced by the particle size and surface coating. the labeling efficiencies of four spio / uspios in mouse macrophage cells could be visualized by microscopy and 11.7-t mri. oude engberink reported that spio particles with a diameter of 150 nm were more efficiently incorporated into mononuclear cells than uspio particles with a diameter of 30 nm.19 particles with larger diameters were more efficiently incorporated into macrophages.21,22 after 1 to 4 h of administration, there were no differences in labeling efficiencies between atdm at 28 nm and at 74 nm (figure 3a). in addition, there was no difference in labeling efficiencies by cmdm 1 h after administration. the diameter of spio used in previous studies was over 150 nm.21,22 the diameter of uspio used as a control was below 30 nm.23,24 the culture time varied from 30 min21 to 6 h.8 in our experiment, the diameters of uspio and spio particles that were compared were only about 30 and 70 nm and we show that particle size is not an important factor in the uptake of 28 and 74 nm iron nanoparticles irons. using an efficient labeling contrast agent is critical for cell imaging. the most commonly used cell - labeling method is to culture cells with a contrast agent. this method employs surface modification with cations,25 virus transfection,26 lipofectamine,27 and poly - l - lysine28 to increase the labeling efficiency. these positively - charged reagents are cocultured with negatively - charged uspio to alter the uspio into a positively - charged compound. such a charge alteration increases the affinity of uspio to negatively - charged cell membranes, thereby facilitating the intracellular uptake of the uspio contrast agent. the mechanism through which the positively - charged contrast agent passes through the cell membrane remains unclear, however, it may include cell wall destruction, adhesion, and internalization. the atdm - coated particles are more positively - charged compared to the cmdm - coated particles. our experiment demonstrated a more efficient uptake of iron particles during approximately 0.54 h culture for more positively - charged atdm (15 mv) than for cmdm (24 mv). furthermore, in vitro 11.7-t mri of labeled cells also demonstrated a shorter t2 relaxation time for atdm - labeled than for cmdm - labeled cells, although cmdm and atdm had the same diameter (figure 4b). the different charge and coating states of the surface potential may have influenced the uptake by cultured macrophages., it has been shown that aminosilane - and dextran - coated nanoparticles did not affect the cell viability of mouse macrophage cells.29 however, silane - coated spio nanoparticles negatively affected mouse macrophage cell viability in a dose - dependent manner.29 moreover, silica - coated spio nanoparticles, rather than dextran - coated nanoparticles, displayed dose - dependent cytotoxicity.30 in our study, none of the nanoparticle formulations affected the viability of the macrophages (figure 3b). our dextran - coated nanoparticles are safer than nanoparticles coated with aminosiline and silica.29,30 the different charge and coating states of the surface potential did not influence the cell viability of cultured macrophages in the four types of spio and uspio contrast agents that we used. in the in vivo imaging of labeled macrophages, a longer half - life of blood is critical for increasing accessibility to target organs and tissues.7,8,28,29 spio particles with a diameter of about 50 nm are rapidly incorporated into kupffer cells in the liver and reticuloendothelial system in the spleen. hence, the half - life of spio in the blood is as short as approximately 48 min.13,28 on the other hand, uspio particles with a diameter below 30 nm are barely recognized by the reticuloendothelial system. the half - life of uspio in blood is as long as approximately 2436 h in humans and a few hours in rats.6,7,31 the current study shows that cmdm - coated nanoparticles label macrophages at a significantly lower rate than atdm - coated nanoparticles, suggesting that they should remain soluble in blood circulation for a longer period of time. there are no differences in t2 relaxation time of labeled cells between atdm at 28 and 74 nm (figure 4b) ; however, the t2 relaxation time of contrast agent differs between atdm at 28 and 74 nm (figure 1). hence, the shortened t2 relaxation time of labeled cells can not simply be explained by the difference in the labeling efficiency of cells. also, no difference was noted in the t2 relaxation time of contrast agent between atdm at 28 and cmdm 28 nm (figure 1), even though the t2 relaxation times of atdm labeled cells were shorter than that of cmdm labeled cells (figure 4b). thus, atdm uspio with a smaller diameter was more efficiently incorporated into cells than cmdm uspio, thereby providing a more effective in vivo contrast agent for macrophages. we conclude that the best contrast agent would be atdm - coated 74 nm spio nanoparticles. despite recent interest in smaller particles (uspio), this study shows that coating plays a more important role than size to optimize mri contrast, while increasing the nanoparticle labeling time. | purposemagnetic resonance imaging (mri) using contrast agents like superparamagnetic iron oxide (spio) is an extremely versatile technique to diagnose diseases and to monitor treatment. this study tested the relative importance of particle size and surface coating for the optimization of mri contrast and labeling efficiency of macrophages migrating to remote inflammation sites.materials and methodswe tested four spio and ultrasmall superparamagnetic iron oxide (uspio), alkali - treated dextran magnetite (atdm) with particle sizes of 28 and 74 nm, and carboxymethyl dextran magnetite (cmdm) with particle sizes of 28 and 72 nm. mouse macrophage raw264 cells were incubated with spios and uspios, and the labeling efficiency of the cells was determined by the percentage of berlin blue - stained cells and by measuring t2 relaxation times with 11.7-t mri. we used trypan blue staining to measure cell viability.resultsanalysis of the properties of the nanoparticles revealed that atdm - coated 74 nm particles have a lower t2 relaxation time than the others, translating into a higher ability of mri negative contrast agent. among the other three candidates, cmdm - coated particles showed the highest t2 relaxation time once internalized by macrophages. regarding labeling efficiency, atdm coating resulted in a cellular uptake higher than cmdm coating, independent of nanoparticle size. none of these particle formulations affected macrophage viability.conclusionthis study suggests that coating is more critical than size to optimize the spio labeling of macrophages. among the formulations tested in this study, the best mri contrast and labeling efficiency are expected with atdm - coated 74 nm nanoparticles. |
leptin deficiency or dysfunction is one of the main causes for insulin resistance (ir) and lipid metabolism disorders [1, 2 ]. it has been found that the majority of type 2 diabetes patients have higher levels of body fat, but normal or increased leptin in the plasma [36 ], indicating leptin resistance (lr). certain levels of leptin effectively could stimulate amp - activated protein kinase (ampk) to phosphorylate acetyl - coa carboxylase (acc), which in turn reduces the acc activity, decreases fatty acid synthesis, and increases the oxidation of fatty acid (fa), consequently, maintaining the balance of lipid metabolism in the body. studies have shown that even one week of a high fat diet can cause leptin to increase rapidly, leading to fat accumulation in peripheral tissue ir. obese persons with high serum leptin levels tend to experience a downregulation of leptin receptor in hypothalamus, adipose tissue, and liver, which causes peripheral tissues to become lr and promotes lipid accumulation [1113 ]. excessive lipid deposition in nonfat tissue has been known to have a toxic effect on cells and to reduce sensitivity to insulin, eventually leading to diabetes and metabolic syndrome. moreover, type 2 diabetes and liver steatosis often coexist [1517 ], but the causal mechanism is unclear. the possible trigger for type 2 diabetes might be related to leptin resistance, which further inhibits the liver ampk - acc signaling pathway and causes liver and systemic metabolic disorders. exercise can reduce body fat by increasing energy consumption and improve the leptin resistance [18, 19 ]. previous studies have shown that exercise could upregulate the expression of leptin receptor and induce changes of the jak - stat3 signaling pathway in the hypothalamus and peripheral tissues in leptin - resistant rats [20, 21 ]. after exercise, normal rats experienced increased skeletal muscle growth, increased activity of the liver ampk and malonyl - coa decarboxylase (mcd) pathways, and decreased activity of acc, which further increased fatty acid oxidation and reduced glyceride synthesis. our previous study suggested that chronic and acute exercise could both reduce obesity and decrease blood sugar level in type 2 diabetic rats as well as improve the phosphorylation and gene expression related to improved skeletal muscle ; ampk1/2 and its subunits (ampk1, ampk2) reduced acc phosphorylation. it is unknown, however, whether exercise affects the liver leptin - ampk signaling pathway or whether it can improve liver lipid metabolism. in our present study, a type 2 diabetic rat model, given a high fat diet and a low dose of stz, was used to address this question. acute and chronic exercise variables were chosen to study whether exercise could affect protein expression and protein phosphorylation involved in the liver leptin - ampk - acc signaling pathway. the relationship between the liver leptin - ampk - acc signaling pathway and lipid metabolism, as well as the effect of acute and chronic exercise on that pathway, was investigated. 15-month - old male sd rats (450470 g) were provided by the animal center of the academy of military medical sciences of the chinese people 's liberation army (certification number scxk (army) 2007 - 004). animals were housed under standard conditions (22 2c, humidity 50 10%, cycles of 12 h light/12 h dark). experimental procedures were performed in accordance with the guidance suggestions for the care and use of laboratory animals, formulated by the ministry of science and technology of the people 's republic of china in 1998, and were approved by the animal ethics committee of china medical university. rats were randomly divided into a control diet group (con) and a high fat diet group (hfd). the control diet contained 57.3% carbohydrates, 18.1% protein, 18.8% cellulose, and 4.5% fat and the high fat diet consisted of 23% soy protein, 19.8% pork fat, 19.8% corn oil, 24.5% sucrose, and 5% cellulose and was supplemented with a 1.4% vitamin mixture, 6.7% mineral mixture, and 0.2% choline bitartrate. after 8 weeks, hfd rats were administered 30 mg / kg stz (citrated buffer, ph 4.4, sigma) by intraperitoneal injection, while con rats were injected with the same volume of citrate buffer (1 ml / kg). four weeks after injection with stz, all animals with fasting blood above 7.8 mmol / l and postprandial glucose above 11.1 mmol / l were considered to be diabetic [24, 25 ]. diabetic rats were then divided into a diabetic control group (dc), diabetic chronic exercise group (dce), and diabetic acute exercise (dae). rats were trained to swim 1020 min per session for more than 2 days to reduce water - induced stress. two or three rats per group were placed in a plastic cylindrical pool of 45 cm in diameter and 60 cm of deep, with a water temperature of 34 - 35c. after the initial training, the rats underwent chronic exercise for 1 hour / day, 5 days / week, for 8 weeks. the exercise program was conducted in accordance with the luciano e program with some modifications. acute exercise was conducted in two 90-minute sessions with a 45-minute interval between each session. the exercise program was performed in accordance with the chibalin av program with some modification. animal model preparation and exercise sessions were conducted in the shenyang institute of physical education laboratory. 2436 hours after the final session of chronic exercise or 816 hours after acute exercise, all rats were anesthetized by single dose intraperitoneal injection of amobarbital (15 mg / kg). blood samples were collected from tail veins and serum was separated by centrifugation at 1100 g for 10 min. serum glucose, triglyceride, total cholesterol, free fatty acid, hdl - c, ldl - c, and serum leptin levels were measured using an autoanalyzer (rt-1904c ; rayto, china). the rats fasted for 12 hours were anesthetized and given 1.5 iu / kg of synthetic insulin (sigma). blood samples were collected at 0, 5, 10, 15, 20, 25, and 30 min after injection, centrifuged at 1100 g for 15 min at 4c, and stored at 20c to determine glucose concentrations. the plasma glucose (t1/2) was calculated from the slope of the last square fit of the plasma glucose concentration during the linear phase of decline. after itt, the animals were sacrificed under anesthesia by intraperitoneal injection of sodium thiopental (200 mg / kg, following the recommendations of the us national institutes of health). the liver was isolated and placed in liquid nitrogen and then immediately transferred to 80c. western blotting analysis and the detection of liver glycogen, faa, and tg were performed later. the frozen livers were weighed, digested with 1 mol / l naoh (1 : 9 wt / vol) at 80c for 10 min, neutralized with 1 mol / l hcl, and mixed with 6 mol / l hcl to a final concentration of 2 mol / l hcl. the resulting solution was incubated at 85c for 2 hours and neutralized again with 5 mol / l naoh. a glucose hexokinase assay kit (sigma) was used to determine the concentration of hydrolyzed glucose and glucose content was determined as micromolar per gram of tissue. liver ffa was determined with fatty acid kit (sigma) following the manufacturer 's instructions. the liver tg was determined with triglyceride determination kit (sigma) following the manufacturer 's instructions. the frozen liver was thawed, weighed, roughly cut, placed in protein extraction solution (1% triton x-100, 100 mm tris, ph 7.4, containing 100 mm sodium pyrophosphate, 100 mm sodium fluoride, 10 mm ethylenediaminetetraacetic acid, 10 mm sodium vanadate, 2 mm phenylmethyl sulfonylfluoride, and 0.1 mg / ml aprotinin), and ultrasonicated at maximum speed at 4c for 30 s (jy92-iin ; scientz, ningbo, china). the homogenate was centrifuged at 9 000 g at 4c for 40 min (hc-3618r ; zonkia, hefei, china). then, 100 g of tissue extract was mixed with an equal volume of 3 sample buffer solution (6.86 m urea, 4.29% sds, 300 mm dtt, and 43 mm trishcl, ph 6.8) at room temperature for 30 min, subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page, 10% polyacrylamide gels), and transferred to a polyvinylidene difluoride membrane at 4c for 2 h. the membrane was blocked using trihydroxymethyl aminomethane buffer salt + tween-20 (tbst) containing 5% bovine serum albumin (sigma) and washed with tbst (ph 7.4). the used antibodies include leptin, leptin receptors, phosphorylated (p)-ampk1 (thr), ampk1, p - ampk2 (thr), ampk2, p - ampk1/2 (thr), ampk1/2, acetyl - coa carboxylase (acc), and p - acc (ser) (cell signaling technology, beverly, ma, usa ; 1 : 1 000 dilution). bands of interest were visualized by enhanced chemiluminescence and absorbance was determined using fluorchem v2.0 gel imaging analysis software (alpha innotech, san leandro, ca, usa). differences between groups were compared by one - way analysis of variance (anova). statistical analyses were performed using jmp software (sas institute, cary, nc). table 1 reports the body weights and biochemical parameters of different groups of rats. when comparing with the control group, the dc group shows a significant increase of the concentrations of blood glucose, leptin, triglyceride, total cholesterol, free fatty, acid and ldl - c. the dc group also shows the decreased insulin sensitivity and hdl - c level, respectively. on the other hand, the body weight, epididymal fat mass, and blood insulin concentration exhibit small variations. all these changes are the typical metabolism characteristics of type ii diabetes, proving that the animal model prepared using high fat diet plus stz - induced disorders is suitable for the current study. data in table 1 also illustrates the effect of chronic exercise on the dc rats. chronic exercise could significantly reduce the blood glucose, leptin, triglycerides, total cholesterol, free fatty acids, and ldl - c. in contrast, chronic exercise could also increase insulin sensitivity and hdl - c levels. the acute exercise reduces blood glucose but increases the blood glucose disappearance rate and there were no significant effects on dyslipidemia and plasma leptin concentrations. compared to the con group, liver glycogen of the dc group significantly decreased, but tg and ffa levels were significantly elevated (table 2). this indicates a disorder of the liver glycolipid reserves and faa. as shown in table 2, chronic exercise effectively increased glycogen content and reduced liver tg and faa, whereas acute exercise seemed to have no effect on the liver glucose and lipid reserves. to understand the underlying mechanisms of the different exercises on the type 2 diabetes rats, the protein expression and protein phosphorylation profiles were examined using western blotting (figure 2) and these image data were quantified with gel analysis software and presented in histograph (figure 1). the dc group had a significantly increased expression of leptin with respect to that of the control group. the leptin expression returned to the basal level after either chronic or acute exercise (figure 1(a)). however, the expression of leptin receptor showed an opposite change : a relatively low level of leptin receptor for the dc group and a restored level for the chronic exercise (dce) and acute exercise (dae) groups, respectively (figure 1(a)). interestingly, the acc expression exhibited a similar trend to that of leptin, and the phosphorylated acc exhibited a similar trend to that of leptin receptor (figure 1(e)). amp - activated protein kinase (ampk) is a kinase responsible for the downstream protein phosphorylation. previous studies have identified several phosphorylation positions on each subunit, and the phosphorylation could occur separately or simultaneously. as shown, protein expression and the phosphorylation levels of ampk1/2, ampk1, and ampk2 were greatly reduced in the dc group (figures 2(b), 2(c), and 2(d)). after chronic exercise, the protein expression and the protein phosphorylation of ampk1/2, ampk1, and ampk2 were elevated back to the normal level in both dce and dae groups. the acc phosphorylation was reduced in the dc group compared to the control group, which is consistent with the increased liver tg and ffa levels shown in table 2. it suggests that the disruption of the leptin - ampk - acc signaling pathway might be associated with liver lipid deposition. chronic exercise can effectively repair the damage of the liver leptin - ampk - acc signaling pathway. therefore, in the dce group, liver glycogen and the leptin and insulin sensitivity were significantly increased. moreover, tg and ffa decreased, which helped lowering both blood lipids and blood glucose. in the dae group, the leptin - ampk - acc signaling pathway was still active 816 hours after acute exercise, which increased insulin sensitivity, but had no significant effect on liver glycolipid storage. our results indicate that a long - term high fat diet plus low dose of stz could induce disorders of the whole system and of liver lipids in middle - aged rats, which are associated with both insulin and leptin resistance. these phenomena are similar to the onset, progression, and metabolic characteristics of type 2 diabetes in humans [36 ]. the interruption of the liver leptin - ampk - acc signaling pathway might be one of the glucose and lipid metabolism disorders found in type 2 diabetes. eight weeks of chronic exercise not only effectively improved the leptin - ampk - acc signaling pathway, but also alleviated the liver and whole system lipid disorders and partially reversed leptin and insulin resistance. acute exercise could activate the leptin - ampk - acc signaling pathway and reduce the blood glucose level for at least 816 hours but has no significant effect on hepatic glucose and lipid metabolism. it has been widely accepted that excessive fat accumulation is strongly correlated with the insulin resistance and leptin resistance in peripheral tissues (muscle and liver) [14, 3035 ]. excessive nutritional and lipid deposition could lead to an increase in the number of fat cells and further stimulates the leptin secretion. once leptin binds its receptor, triglyceride synthesis will be inhibited, which further promotes the oxidation of free fatty acids in order to avoid excessive lipid deposition and maintain lean body mass. some studies have suggested that skeletal muscle leptin can activate the ampk pathway in two ways. in the first activation, leptin acts directly with skeletal muscle to induce the rapid and transient activation of ampk, and in the second activation mechanism, one is the adrenal system activating ampk through the hypothalamus sympathetic skeletal muscle and inducing acc phosphorylation, which further reduces the synthesis of fatty acids. this activated malonyl - coa decarboxylase pathway reduces the level of malonyl - coa (ma), inhibits the synthesis of fat, eliminates the inhibition of carnitine palmitoyltransferase 1 (ctp1), promotes long - chain fatty acids in the mitochondrial inner membrane, and increases fat oxidation and decomposition [8, 39, 40 ]. lacking leptin or the leptin receptor in diabetic rats (fa / fa and zdf) reduces the ampk activity in skeletal muscle and in liver, and promotes fat storage. administration of leptin or ampk activators can effectively prevent the development of diabetes. however, the effects on the liver leptin - ampk - acc pathway of diabetic rats induced by high fat diet plus low dose of stz are unclear. our results indicate that in the dc group, expression of liver leptin significantly increased, but expression of leptin receptor decreased. moreover, expression and phosphorylation of ampk1/2, ampk1, and ampk2 were effectively inhibited. the acc phosphorylation was also inhibited, which was associated with a decline of glycogen and an increase of tg and ffa. this data suggests that the liver leptin - ampk - acc signaling pathway is related to hepatic glucose and lipid metabolic disorder and might explain the etiology of fatty liver incidence in patients with diabetes. changes in body weight may play a role in the prevention of hyperglycemia caused by regular physical activity, although it is not likely to be the only explanation. in our study, diabetic trained animals showed a slight increase in weight, which puts in doubt whether improvements in insulin sensitivity are consequences of weight gain effects. for this purpose, blood triglyceride, total cholesterol, and free fatty acid concentrations were also examined and found to be remarkably reduced by exercise training. this relative decrease in biochemical parameters in diabetic trained animals may have contributed, at least in part, to their improved insulin sensitivity. furthermore, diabetic animals were submitted to acute exercise, which had no effect on body weight. while the impact of chronic exercise on leptin was related to physical condition, chronic exercise does not have any effect on the blood leptin levels of athletes but reduces the leptin levels of nonathletes with normal weight [42, 43 ], obese individuals, and obese rats and also reduces body fat and accumulation of skeletal muscle lipids, which can prevent or mitigate leptin resistance. appropriate exercise can effectively reduce serum leptin of diabetic rats and humans, ease leptin resistance, and inhibit the development of diabetes. research indicates that chronic exercise could downregulate leptin receptor gene expression in the hypothalamus and improve the insulin resistance of aging rats. another study suggested that chronic exercise could reduce the expression of the liver leptin receptor gene and decrease plasma leptin levels in rats with a high fat diet. in our previous study, we found that chronic exercise could reduce body fat and blood leptin levels but elevate gene expression of the leptin receptor in the adipose tissue of obese rats as well as improve leptin resistance. in agreement with our previous study, decreased leptin expression and increased expression of the leptin receptor were also found in the high fat diet plus low dose stz - induced type 2 diabetic rats, 2436 hours after chronic exercise. in agreement with published data, endurance exercise could also increase gene expression of the hypothalamic leptin receptor and activation of the jak2-stat3 signaling pathway, reducing leptin and insulin levels. increased expression of the leptin receptor was found in hypertrophied triceps of professional tennis players. the impact of chronic exercise on the ampk pathway was mostly studied in skeletal muscle. chronic exercise can effectively reduce ceramide levels in the skeletal muscle of the high fat diet rats and restore insulin - stimulated glucose transport in skeletal muscle. moreover, leptin stimulated phosphorylation in the skeletal muscle ampk - acc pathway, and promoted the oxidative decomposition of the fa [44, 50 ]. in zucker and oletf rats, where both lines have metabolic abnormalities, chronic exercise induced increased phosphorylation of acc, and fat decomposition, as well as reduced fat synthesis in skeletal muscle. the impact of chronic exercise on the liver leptin - ampk - acc pathway has not been reported, although increase of liver acc phosphorylation and ampk 1-2-subunit mrna / protein expression has been found. in our present study, we found that not only was liver ampk1/2, ampk1, and ampk2 protein expression increased, but increased phosphorylation levels were also seen, which further induced acc phosphorylation, inhibited tg synthesis, promoted ffa oxidation, reduced lipid storage in the liver, and abolished insulin and leptin resistance. the impact of acute exercise on leptin could be influenced by many factors such as exercise stress and intensity, as well as the physical condition of the individual. bouassida reported that the effects of one - time exercise on leptin levels were related to energy consumption and exercise time. energy consumption less than 800 kcal or < 60 min of active movement does not change the level of serum leptin ; however, energy consumption greater than 800 kcal or 60 min could stimulate the lipolysis and reduce serum leptin level. 816 hours after acute exercise (90 min 2), leptin expression was significantly decreased and its receptor expression was significantly increased in the liver of diabetic rats. moreover, ampk1/2, ampk1, ampk2 and acc phosphorylation levels increase, but no significant effects are noticeable on liver lipid and glycogen storage. our data suggested that acute exercise could activate the liver leptin - ampk - acc signaling pathway, promote lipid mobilization, and inhibit lipid synthesis. this result has also been shown in other tissues. in agreement with our previous studies, ampkthr and the acc phosphorylation levels enhanced skeletal muscle ampk2 activity, ampk2, and acc phosphorylation levels were also found in similar human studies [52, 53 ]. moreover, koh. further confirmed that acute treadmill exercise reduces acc adipocyte activity in rats. impaired liver leptin - ampk - acc signaling pathways were closely related to glucose and lipid metabolism disorders in high fat diet plus low dose of stz - induced type 2 diabetic rats. we further confirmed that chronic exercise could indirectly repair the leptin - ampk - acc signaling pathway in these rats, alleviate liver and body lipid disorders, and improve the ir and lr. acute exercise could also indirectly activate the liver leptin - ampk - acc signaling pathway and increase insulin sensitivity, but it should be noted that irreversible liver lipid disorders are induced by a high fat diet. | aim. to investigate the effects of acute and chronic exercise on glucose and lipid metabolism in liver of rats with type 2 diabetes caused by a high fat diet and low dose streptozotocin (stz). methods. animals were classified into control (con), diabetes (dc), diabetic chronic exercise (dce), and diabetic acute exercise (dae) groups. results. compared to con, the leptin levels in serum and liver and acc phosphorylation were significantly higher in dc, but the levels of liver leptin receptor, ampk1/2, ampk1, and acc proteins expression and phosphorylation were significantly lower in dc. in addition, the levels of liver glycogen reduced significantly, and the levels of tg and ffa increased significantly in dc compared to con. compared to dc, the levels of liver ampk1/2, ampk2, ampk1, and acc phosphorylation significantly increased in dce and dae. however, significant increase of the level of liver leptin receptor and glycogen as well as significant decrease of the level of tg and ffa were observed only in dec. conclusion. our study demonstrated that both acute and chronic exercise indirectly activated the leptin - ampk - acc signaling pathway and increased insulin sensitivity in the liver of type 2 diabetic rats. however, only chronic and long - term exercise improved glucose and lipid metabolism of the liver. |
canine ehrlichiosis, a tick borne disease, was first recognized by donatien and lestoquar (1935) and has since been reported in dogs geographical widespread (bretischwerdt 1995). at the end of 1960 an epidemic outbreak of the disease with high mortality has been reported in american military dogs and south asia. this severe form was initially given the name canine tropical pancytopenia (william 1981). ehrlichia canis is a gram negative highly pleomorphic, obligate intracellular bacterium which is enveloped with a rippled thin outer membrane (marvomatis. it is considered to be the major causative agent of canine monocytic ehrlichiosis (cme) in dogs (huxsoll. rhipicephalus sanguineus, a brown - dog tick, kennel tick or pan - tropical dog tick belonging to ixodidae family is a ubiquitous tick responsible for transmitting e. canis, (jeremy. it is a one - host tick that feeds on dogs in all three stages of life cycle. ticks acquire e. canis by feeding on infected dogs and transmit infection for at least 155 days afterward to other dogs (groves. they can also act as vector of important pathogens of humans such as coxiella burnetii, rickettsia conorii, r. rickettsii and bartonella henselae being of zoonotic concern (wikswo. this tick species is known to be a vector of e. canis, babesia canis, b. gipsony, hepatozoon canis, and anaplasma platys in dog (gal. the acute phase is characterized by fever, anorexia, lymphadenomegaly, epistaxis and petechia (neer and harrus 2006). during the subclinical phase dogs appear healthy and have the potential to remain persistent carrier (waner. the disease can be diagnosed by the detection of e. canis morulae in monocyte in blood smears or serologically detection of specific antibodies by the use of ifa test, dot - elisa and western blot immunoassay or by the detection of e. canis in tissue and blood by means of pcr (matthewman. ifa is considered the gold standard serological diagnostic technique for e. canis (ristic. the objectives of this study were to determine the seroprevalance of canine ehrlichiosis and risk factors of this disease in companion dogs population of mashhad, north khorasan of iran. the study was performed on total 250 owned pet dogs (119 females and 131 males) between september 2009 until november 2010 referred to veterinary teaching hospital of ferdowsi university of mashhad for their annual vaccination, as well as with clinical illness. the following details were obtained for each dog : sex, breed, age, body temperature, location of dog s home, appetite status, examination of lymph node, crt, infestation by tick, epistaxis and reason for referred to the hospital. sera were separated by centrifuge and stored at 20 c until assayed. for each case blood smear was prepared and stained with giemsa and direct microscopic examination was performed to detect morula on white blood cells especially on monocytes and lymphocytes. anti- e. canis antibodies were detected by flu ehrlichia immune fluorescence kit (flu ehrlichia canis, megacor, austria) with following method : sera were added to the slides after dilution (1:40) in phosphate - buffered saline (pbs) ph 7.2. slides were placed in humid chamber and incubated for 30 minute at 37 c after that, those were washed twice in pbs. then we added one drop anti - dog fitc (conjugate) to each slides and those were returned to humid chamber and incubated for 30 minute at 37 c. after these steps, slides were washed as described before and were air - dried then 23 drops of mounting fluid were added to each slides and a cover slip was placed. the slides were analyzed at 400 magnification with ifa microscopy and were compared each wall with negative and positive control. a positive reaction appears as bright sharp regularly stained inclusion bodies in cytoplasm of infected cell. sera were positive at the 1:40 were prepared serial dilution 1:80 1:160 1:320 1:640 1:1360 and tested again with ifa. all data were collected and because of low seropositive cases for e. canis, statistical analysis was not performed. complete blood count showed 67 anemic (26.8%), 40 thrombocytopenic (16%) dogs. furthermore, 101 dogs in study population were diagnosed with abnormal leukogram findings including 20 leukopenia (8%), 24 leukocytosis (9.6%), 24 neutropenia (9.6%), 33 neutrophilia (13.2%). 7 dogs (2.8%) showed anemia and thrombocytopenia synchronously. 1 dog (0.4%) had morulae (fig. a morulae of ehrlichia canis (arrowed) in a blood smear from one of seropositive dogs (morulae in cytoplasm of lymphocyte) in physical examination, 12 dogs (4.8%) were infested with tick. 15 dogs (6%) had lymph node enlargement, 6 dogs (2.4%) had fever and 4 dogs (1.6%) had epistaxis (table 1). signalment and antibody titer in seropositive dogs two (0.8%) of the 250 dogs have been examined were found to be seropositive by the ifa. both of them were adult (above 1 year) and the number of platelets, leukocytes and neutrophils were normal. this dog showed inappetance and depression, had large submandibular lymph node and infested with r. sanguineus on physical examination. this study is the first investigation of the seroprevalence of e. canis antibodies among dogs in north khorasan of iran. prevalence of ehrlichiosis was also reported from other regions of iran : jafari. (1997) in shiraz (south west of iran) have examined 180 dogs. seventeen dogs (9.44%) were found positive for the presence of e. canis in their white blood cells. (2009) used ifa and ica to detect antibodies against e. canis in 123 apparently healthy dogs. seventeen (13.8%) dogs in ifa test and 13 dogs (10.6%) in ica were seropositive for cme. in blood smears from three infected dogs (16.6%) morulae were observed in monocytes. 2010) have reported seroprevalence of cme in 198 companion dogs of different ages by means of ifa and ica 9.6 % in ahvaz (west of iran). morulae of e. canis were observed in monocyte of four infected dogs (2.02%). blood samples from 980 dogs (510 domestic dogs and 475 wild dogs) in west azerbaijan and 820 dogs (520 domestic dogs and 300 wild dogs) in east azerbaijan of iran were obtained by asri and others in 2001 and tested by ifa for diagnosis of canine ehrlichiosis. sixty seven percent of wild dogs and 38 % of domestic dogs in west azerbaijan and for east azerbaijan 58 % and 39 % were serologically positive for ehrlichia. the main variants have been diagnosed were e. canis (75%), e. platys (20%) and e. equi (5%). in our study because of low seroprevalence of e. canis, we could not reach any correlation between age and cme but in many investigations the prevalence has been significantly differed among age groups. in shiraz 1997) the animals of all ages seemed equally susceptible to disease. in ahvaz (avize. the prevalence rate was 16.8 % in above 3 years old and 11.86 % in 13 years old compared with dogs less than 1 year old (1.41%). in kerman (akhtardanesh. 2009) high association was observed between age and seropositive dogs. possible explanations for more infection in older group include the immunologic status of the host or more exposure to the vector ticks (rodriguez - vivas ri. 2005). german shepherd dog has been reported to be more susceptible to cme (nyindo. 1980, harrus. 1997). in shiraz (jafari. no significant difference was proved between sex and various breeds with presence of e. canis antibodies (waner. the clinical signs of cme may vary among and within geographic locations (harrus. the probable reasons include e. canis strain pathogenicity, dose of infectious organism, breed of dog, immuno status of the host and co - infection with other tick - borne parasites (rodriguez - vivas. this change is found in all stage of disease but is more severe in chronic phase as a result of bone marrow hypoplasia (troy. 1980). death may occur as a consequence of hemorrhages and secondary infections (hendricks and bob 2004). in our study because of low prevalence of e. canis we could not show any relationship between seropositive dogs and hemathologic changes but platelet and leukocyte count in both seropositive dogs were normal. in ahvaz (avize. 2009) the prevalence of ehrlichiosis was higher in dogs with thrombocytopenic although the difference was not significant and correlation was not observed between seronegative and seropositive dogs for hemathologic changes. in kerman (akhtardanesh. 2009) thrombocytopenia, leukopenia and anemia were just observed in dogs with high ifa titer (> 1:320). (2005) have found that the presence of thrombocytopenia, platelet - related bleeding and a seropositive response to e. canis in a patient increase the index of possibility for infection. rhipicephalus sanguineus and possibly the american dog tick, dermacentor variabilis are the vector for e. canis (groves. rhipicephalus sanguineus is widely distributed in the world but it is mainly in tropical and sub - tropical regions and also well adapted to the indoor environment where owned dogs are kept (uspensku and ioffe - uspensky 2002, dantas - torres 2010). dogs may acquire ticks in the city areas in parks and housing estates (siuda 1993). infestation by r. sanguineus has significant risk factor for e. canis seropositivity in brazil (trapp. 2002). in this study, 4.8 % (12 dogs) were infested by r. sanguineus. r. sanguineus was also the most common species in north - east of iran (razmi. 2003). as said above the indirect immunofluorescence antibody (ifa) test is considered the serological gold standard for diagnosis of cme (ristic. serological cross - reactivity occurs with other members of ehrlichiae like e. equi (baneth g. 1996), e. ristici (ristici. 1999), e. ewingii (anderson. 1992), e. chaffeensis, neorickettsia helminthoeca (rikisha 1991). in this study, ifa test was used and seropositive titers were 1:80 and 1:160. ifa test is more susceptible than other test but supplementary test such as pcr and western immunoblotting is needed for detection of active infection and distinguished between infections with different type of species. possible explanations for low seroprevalence of e. canis in this study are : selected population : exposures to tick in domestic dogs are lower because of location and observance of health condition by owners. the life conditions of dogs affected the seroprevalence of e. canis (roqueplo. (2010) indicate that risk of exposure to vector - borne disease in rural hunting dogs can be quite high in korea. (2003) showed that dogs in non - urban areas (9.9%) or they have living in outdoor (12.7%) had a higher prevalence of e. canis. 2008).weather conditions : the prevalence of e. canis is largely dependent on the distribution of the vector, r. sanguineus, which occurs mainly in tropical and sub - tropical regions but it has worldwide distribution (jeremy. (2008) have determined the prevalence of canine ectoparasite infestation in pet dogs from the shiraz area of southern iran. overall, 160 dogs were examined for ectoparasites, and 142 r. sanguineus ticks were found on 13 dogs. a significant correlation was observed between increases in temperature and decreases in humidity and increased ectoparasite infestation. the number of dogs infested with ectoparasites in summer and spring was significantly higher than in winter (p= 0.007). morales - soto and cruz - vazquez (1998) found r. sanguineus along the year in cuernavaca, mexico but the peaks of tick were found in april, july and november and the lower prevalence were in january. so season of sampling can affect seroprevalance of e. canis.type of serological test : ifa detects antibodies as early as 7 days after initial infection but some dogs may be negative until 28 days after infection or in acute phase of disease and also in chronic phase because of injury to immune system (groves. 1975) when e. canis antibody titers results are negative, a follow up examination in 2 to 3 weeks or serotesting for other agents is recommended (neer and harrus 2006). selected population : exposures to tick in domestic dogs are lower because of location and observance of health condition by owners. the life conditions of dogs affected the seroprevalence of e. canis (roqueplo. (2010) indicate that risk of exposure to vector - borne disease in rural hunting dogs can be quite high in korea. (2003) showed that dogs in non - urban areas (9.9%) or they have living in outdoor (12.7%) had a higher prevalence of e. canis. weather conditions : the prevalence of e. canis is largely dependent on the distribution of the vector, r. sanguineus, which occurs mainly in tropical and sub - tropical regions but it has worldwide distribution (jeremy. (2008) have determined the prevalence of canine ectoparasite infestation in pet dogs from the shiraz area of southern iran. overall, 160 dogs were examined for ectoparasites, and 142 r. sanguineus ticks were found on 13 dogs. a significant correlation was observed between increases in temperature and decreases in humidity and increased ectoparasite infestation. the number of dogs infested with ectoparasites in summer and spring was significantly higher than in winter (p= 0.007). morales - soto and cruz - vazquez (1998) found r. sanguineus along the year in cuernavaca, mexico but the peaks of tick were found in april, july and november and the lower prevalence were in january. type of serological test : ifa detects antibodies as early as 7 days after initial infection but some dogs may be negative until 28 days after infection or in acute phase of disease and also in chronic phase because of injury to immune system (groves. 1975) when e. canis antibody titers results are negative, a follow up examination in 2 to 3 weeks or serotesting for other agents is recommended (neer and harrus 2006). the cme has a worldwide distribution and a significant seroprevalence in dogs from southeast asia, africa, europe, central and south america was reported (cardenas. 2007). antibodies against e. canis were detected in neighbors and close countries to iran as 44.4 % in saudi arabia (sacchini. 2007), 21 % in turkey (batmaz. 2001), and 33 % in egypt (botros. 1995). in our study, seroprevalence of e. canis was estimated less than 1 %. so cme is not endemic in mashhad, but in kerman, ahvaz and azerbaijan is considered endemic (asri. besides, e. canis is a human health hazard and causes clinical signs of disease (perez. human ehrlichiosis is caused by e. chaffeensis, a. phagocytophilum and e. ewingii (dumler. 2007). co - infection of e. canis and a. phagocytophilum is possible (amusategui. a. phagocytophilum was reported in ixodes ricinus in north of iran (bashirbod. it is possible that more tick - transmitted pathogens can infect dogs, including e. canis, a. phagocytophilum, b. canis, hepatozoon canis, bartonella spp. so in dogs with clinical signs of thrombocytopenia, leukopenia, fever and epistaxis if they have negative result for e. canis, consider the possibility of infectious with other organisms. so in dogs that have one of these diseases, e. canis infectious should be considered (matthewman. ehrlichia canis infection in owned pet dogs from north of khorasan was not endemic from 2009 to 2010. | background : the aims of this study were to determine the seroprevalence of canine ehrlichiosis and risk factors of this disease in companion dogs population of mashhad, north east of iran. canine monocytic ehrlichiosis (cme) is a zoonotic disease transmitted by ticks, rhipicephalus sanguineus, and caused by an obligate intracellular bacterium, ehrlichia canis.methods:during september 2009 until november 2010, 250 companion dogs from mashhad, north - east of iran, were examined for serum antibody detection against e. canis by means of immunofluorescence assay test (ifat) and factors associated with a positive antibody response.results:there was a very low prevalence of anti - e. canis antibodies (0.8%, 2/250) among studied dogs. the antibody titers for two seropositive dogs were 1:80 and 1:160, respectively. one (0.4%) of seropositive dogs was infested with, r. sanguineus. in blood smears from one of infested dogs (0.4%), typical morulae of e. canis was observed in lymphocytes. the results confirm that the lowest occurrence of reactive dogs indoors probably related to low tick infestion.conclusion:this is the first report that describes serological evidences of canine monocytic ehrlichiosis in north - east of iran. results suggested that e. canis infection in owned pet dogs from north of khorasan was not endemic from 2009 to 2010. additional molecular studies are necessary to confirm e. canis infection and to identify the local strains of the organism. |
zhigancao, glycyrrhizae radix et rhizoma praeparata cum melle, originates from the processed dried roots or rhizomes of glycyrrhriza uralensis fisch. it is most frequently used in traditional chinese medical formulary to harmonize all drugs and detoxify the adverse effects of herbs. clinically, it treats disorders such as shortness of breath, fatigue, epigastria and abdominal pain, musculoskeletal and smooth muscle cramp and pain, and diarrhea. due to the numerous bioactive compounds in it such as terpenoids, saponins, polysaccharides, and flavonoids, zhigancao has been reported to possess anti - inflammatory activities, antioxidative [3, 4 ], neuroprotective, antiallergic, anticonvulsant activities, and so forth. two chalcone derivatives in figure 1, isoliquiritin apioside (ila) and isoliquiritin (il), which are important ingredients in zhigancao, are selected to research their pharmacokinetics. il has been reported to have shown various pharmacological activities such as antiangiogenic effect dependent upon antitube formation, antidepressant - like effects in mice induced by forced swimming and tail suspension, and inhibitory effects on tyrosinase. the bioavailability of drugs is the cornerstone for extending their in vitro biological functions to humans in vivo. the flavonoid glycosides show low oral bioavailability possibly because of the extensive conjugation of the free hydroxyl groups [1214 ] or / and glycosides hydrolysis to the aglycones in the intestinal lumen [1416 ]. the pharmacokinetic study of ila and il is essential for us to comprehend the bioavailability of two analytes after an oral administration of zhigancao extrat. to my knowledge, ila and il quantification method (lc - ms / ms) has been performed to study pharmacokinetics of multiple licorice flavonoids after an oral dose of xiaochaihu - tang to rats, but lc - ms / ms method is simply not available in most laboratories. in this study, we established the hplc method which was highly selective and sensitive for simultaneous quantification of ila and il in rat plasma. the method was used to explore the possible pharmacokinetics of ila and il after an oral administration of zhigancao extract. isoliquiritin apioside (ila) and isoliquiritin (il) were purchased from xi'an xiaocao botanical development co., ltd. wogonoside (internal standard, is) was obtained from national institute for food and drug control (beijing, china). zhigancao which was produced under the guidance of the theory of traditional chinese medicine science was provided by chifeng xinzhou traditional chinese medicine co., ltd. the material was authenticated as the dried roots of g. uralensis by professor ting - guo kang from liaoning university of traditional chinese medicine. hplc grade acetonitrile and analytical grade chloroform were purchased from shandong yuwang chemical industry co., ltd. the water in the study was purified with a milli - q water purification system from millipore (bedford, usa). powdered herb materials were extracted twice under reflux condition with 70% ethanol (1 : 10, w / v) for 1 h under 100c. the extract was filtered and evaporated. finally, the residue was dried in vacuum drying oven at 60c to obtain a powder state of zhigancao extract. the contents of ila and il in the extract were detected by hplc, 33.72 and 28.17 mg / g, respectively. the liquid chromatography system employed was agilent 1100 with a variable wave length uv detector (g1314a vwd)., 5 m, dikma technologies, china) analytical column with a endcapped c18 ods guard column. the mobile phase composed of water (containing phosphoric acid 0.1%, v / v) and acetonitrile (72 : 28, v / v) was filtered through 0.22 m millipore membrane filter. the flow rate was 1.0 ml / min. the detection wavelength was set at 360 nm (09 min) and 276 nm (912 min). stock solutions of is, ila and il with concentrations of 440, 384, and 480 g / ml, respectively, were prepared in methanol, and stored at 4c. the working solution for is was diluted with methanol to get a final concentration of 8.80 g / ml. stock solutions were diluted with methanol to serial standard working solutions at concentrations of 0.60, 1.20, 2.40, 4.80, 9.60, 19.2, and 38.4 g / ml for ila, 0.75, 1.50, 3.00, 6.00, 12.0, 24.0, 36.0, and 48.0 g / ml for il. these solutions were then added to blank plasma (1 : 10) to make standards of 0.0603.84 g / ml for ila and 0.0754.80 g / ml for il. the quality control (qc) samples which were used for the intra- and inter - day accuracy, and precision, extraction recovery and stability study, were prepared in the same way at 0.12, 0.48 and 3.07 g / ml for ila, and 0.15, 0.60 and 3.84 g / ml for il. the 200 l of rat plasma was mixed with 20 l is working solution. after protein was precipitated with 500 l of acetonitrile in a 1.5 ml polypropylene tube by vortexing for 3 min, the sample was centrifuged at 6,677 g for 5 min. the supernatant was transferred into a 2.0 ml tube and was added with 1,000 l of chloroform. after vortexing and centrifugation, 20 l of water phase was injected for analysis. the analysis method was completely validated using spiked rat plasma samples, including selectivity, linearity, intra- and inter - day precision, accuracy and stability, according to the fda guideline for method validation of bioanalytical assays. the selectivity of the method was demonstrated by comparing chromatograms of blank plasma samples (without is) obtained from rats, plasma samples spiked with the analytes and is, and plasma samples after an oral dose. all blank plasma samples were prepared and analyzed to ensure the absence of interfering peaks. the linearity of the method was assessed by plotting calibration curves in plasma at seven concentration levels in triplicate on three consecutive days. the lower limit of quantification (lloq) was defined as the lowest concentration of the calibration curve, that was measured with accuracy and precision by analyzing samples in six replicates at the concentration of 0.060 g / ml for ila and 0.075 g / ml for il. precision and accuracy were evaluated by determining qc samples at three concentrations in six replicates on the same day (intra - day accuracy and precision) and three consecutive days (inter - day accuracy and precision). accuracy was measured by relative error (re) and precision was evaluated by intra- and inter - day relative standard deviation (rsd). the extraction recovery was evaluated by comparing the peak areas of the extracted qc samples at three concentrations in six replicates with those of unextracted standards that represented 100% recovery. similarly, the recovery of is was evaluated at a single concentration of 0.88 g / ml in the same way. the stability of ila and il in rat plasma was evaluated by comparing the mean of back - calculated concentration of the stored qc samples at three concentrations in triplicate with the spiked concentration of analytes. the qc samples treated as sample preparation were kept at room temperature for 12 h and then the stability was determined. the freeze - thaw stability was determined afterthree freeze (20c, 24 h) and thaw (room temperature) cycles. long - term stability was assessed by keeping qc samples at 20c for 15 days. female sprague - dawley rats (200 20 g) were kept in environmental controlled breeding room for 7 days until the experiment. the rats were fasted for 12 h but allowed water ad libitum before the zhigancao extract was orally administered at a dose of 1.21 g / kg. orbital venous blood samples (0.5 ml) were collected before dosing, and at 0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h after an administration. after centrifuging at 6,677 g for 5 min, the plasma samples were obtained and frozen at 20c until analysis. hplc analysis procedure was applied to analyze plasma concentration - time profiles of ila and il. data was processed by noncompartmental method using drug and statistics (das) 2.0 software package (chinese pharmacological society, shanghai, china). the selectivity was evaluated by analyzing blank samples, spiked samples at lloq, and middle qc levels, and actual samples obtained from rats after an oral administration of zhigancao extract. for all blank samples, ila, il, and is retention windows were free from endogenous interfering peaks. the calibration curves were linear over the concentration range of 0.0603.84 g / ml for ila and 0.0754.80 g / ml for il by weighted (1/x) linear least - squares regression method. the res of the back - calculated values of the standards from their nominal values were constantly within 15% for all values, including the lloq. the lloq measurement showed the respective averages 0.060 g / ml with rsd 14.5% for ila and 0.077 g / ml with rsd 12.3% for il. typical regression equations were calculated as follows : ila, y = 1.024x 0.004 (r = 0.9954) ; il, y = 1.004x + 0.007 (r = 0.9968). the precision and accuracy data was shown in table 1. the intra- and inter - day rsd values were lower than 10%, and the re values were within 5%. the extraction recoveries of ila and il from rat plasma were 79.5 4.2%, 82.5 4.4%, 84.7 4.0% at 1.2, 4.8, and 3.07 g / ml and 81.3 4.9%, 82.3 6.6%, 86.4 3.4% at 1.5, 6.0, and 3.84 g / ml, respectively. these results indicated that the extraction method was suitable to extract ila, il, and is from plasma. as shown in table 2, stability of analytes showed no significant sample loss over 12 h at room temperature, three freeze - thaw cycles, and 15 days storage condition. the developed method was applied in pharmacokinetic study of ila and il in rat plasma after an oral administration of zhigancao extract (at a dose containing 40.8 mg / kg ila and 34.1 mg / kg il, resp.). the mean plasma concentration - time profiles (n = 6) were shown in figure 3. the assay was sensitive enough for the determination of ila and il in rat plasma after an oral administration of zhigancao extract. in this study, we established an hplc method to simultaneously quantify ila and il in rat plasma. due to the stronger polarity of ila and il, the recovery with methanol was comparable to acetonitrile, during both processes the analyte concentration in the plasma was diluted when 2.5-fold organic solvent volume was added, so that ila and il couldnot be detected at a low concentration. yet evaporating supernatant to dryness and then dissolving it in a small amount of solvent to increase the concentration would be time - consuming and would cause the loss of analyte. so removing the organic solvent from the supernatant was employed. when the supernatant was added with chloroform, a clear water phase could be obtained by precipitating the protein with acetonitrile, but no stratification could be observed when methanol was used instead of acetonitrile therefore, plasma samples were processed by precipitating protein with acetonitrile which wogonoside was selected as the is because of its appropriate retention and extraction recovery. some other compounds such as rutin, baicalin and hesperidin were also tested with the selected condition above, but without ideal results. due to its satisfactory separation from the analytes, wogonoside was selected as the is. because wogonoside couldnot be detected at 360 nm at the concentration 0.88 g / ml, the wavelength was switched to 276 nm at 9 min. the extrapolated fraction of the auc0 accounted for only 5 - 6%, which indicated a suitability of the analytical method for ila and il pharmacokinetic study. these two analytes exhibited consistent tendencies in plasma concentration - time profiles and similar tmax, t1/2z and mrt values after an oral administration of zhigancao extract. the fact that these two compounds had the similar pharmacokinetic behavior could be tentatively attributed to their having similar structures. comparing the pharmacokinetic data of ila with that of il, cmax and auc of ila were lower than that of il although there was more content of the former in zhigancao extract. it was possible that ila could be hydrolysis to the il in the intestinal tract or transformed into il after being absorbed. we have developed and validated a selective and sensitive hplc method for simultaneous quantification of ila and il in rat plasma. the method was successfully applied to the pharmacokinetic study of ila and il in rat plasma after an oral administration of zhigancao extract. | a sensitive hplc method was developed for the quantitative determination of isoliquiritin apioside (ila) and isoliquiritin (il) in rat plasma. after protein precipitation with acetonitrile, chloroform was used to separate lipid - soluble impurities from the plasma samples and remove acetonitrile. a chromatography was carried out on diamonsil c18 (150 4.6 mm ; 5 m) analytical column, using a mobile phase consisting of water (containing phosphoric acid 0.1%, v / v) ; acetonitrile (72 : 28, v / v) at a flow rate of 1.0 ml / min. the wavelength - switching technology was performed to determine ila and il at 360 nm and wogonoside (internal standard, is) at 276 nm. the calibration curves of ila and il were fairly linear over the concentration ranges of 0.0603.84 g / ml (r = 0.9954) and 0.0754.80 g / ml (r = 0.9968), respectively. the average extract recoveries of ila, il, and is were all over 80%. the precision and accuracy for all concentrations of quality controls and standards were within 15%. the lower limit of quantification (lloq) was 0.060 g / ml for ila and 0.075 g / ml for il. the method was used in pharmacokinetic study after an oral administration of zhigancao extract to rats. |
consumption of green tea is inversely associated with risk for cardiovascular diseases and stroke,1 as well as with lower cardiovascular and total mortality.2 improvement of endothelial function is thought to be one major mechanism for these beneficial effects. green tea improved flow - mediated dilation in chronic smokers3 and in healthy individuals.4 a meta - analysis indicates that green and black tea ingestion increases endothelial - dependent vasodilation.5 because endothelial dysfunction is characterized by reduced availability of nitric oxide (no), interventions able to stimulate vascular no production represent a promising tool in the prevention of cardiovascular diseases. the catechin epigallocatechin-3-gallate (egcg) represents the most potent physiologically active tea compound in vitro. egcg has been shown to induce vasodilatory effects in isolated blood vessels in various studies.612 besides an no - dependent mode of action, other no - independent mechanisms for egcg - induced vasodilation were also proposed. to elucidate conclusively whether egcg - induced vasodilation is dependent on no, we studied effects of egcg in isolated aortic rings of endothelial no knockout (enos) mice compared with wild type controls. eight- to 10-week - old male enos mice (strain b6.129p2-nos3/j) from jackson laboratory were used for the experiments. the background strain c57bl/6j with functional enos expression (wild type) served as control. extraction of organs from genetically modified animals was approved by the local authority (landesamt fr gesundheit und soziales, berlin) under the permit number t0026/05. mice were anesthesized by intraperitoneal injection of thiopental (0.3 g / kg body weight). thoracic aortae from anesthesized mice were rapidly excised, cleaned of connective tissue, and cut into rings of 1 mm in length for organ - chamber experiments. rings were then mounted on platinum hooks in 10-ml jacketed organ baths containing modified krebs henseleit solution (composition, in mmol / l : nacl 144, kcl 5.9, cacl2 1.6, mgso4 1.2, kh2po4 1.2, nahco3 25, and d - glucose 11.1) and 1 mol / l of diclofenac. the solution in the bath was maintained at 38c with a gas mixture of 5% co2 and 95% o2. after equilibration, the reactivity of rings was tested with kcl (40 mm). after repeated washouts, the tension of rings was again adjusted to 1 g. after equilibration, the rings were precontracted with the 1-receptor agonist phenylephrine (200 nm). to obtain cumulative concentration response curves, egcg (sigma aldrich, dissolved in water) at concentrations of 5, 10, and 15 m was added at 30-minute intervals to the rings. for the relaxation experiments with tea, 1.2 g of darjeeling green tea (provided by king 's teagarden, berlin, germany) was brewed with 100 ml of boiled water for 3 minutes. relaxations to cumulative doses (10 nm5 m) of the endothelium - dependent vasodilator acetylcholine were also performed. maintenance of smooth muscle integrity was confirmed by evaluation of endothelium - independent vasodilation to sodium nitroprusside (snp, 0.1100 nm). statistical calculations were carried out with student t tests for independent samples for pair - wise comparisons of mean values, and with mann whitney rank sum test when comparing medians. eight- to 10-week - old male enos mice (strain b6.129p2-nos3/j) from jackson laboratory were used for the experiments. the background strain c57bl/6j with functional enos expression (wild type) served as control. extraction of organs from genetically modified animals was approved by the local authority (landesamt fr gesundheit und soziales, berlin) under the permit number t0026/05. mice were anesthesized by intraperitoneal injection of thiopental (0.3 g / kg body weight). thoracic aortae from anesthesized mice were rapidly excised, cleaned of connective tissue, and cut into rings of 1 mm in length for organ - chamber experiments. rings were then mounted on platinum hooks in 10-ml jacketed organ baths containing modified krebs henseleit solution (composition, in mmol / l : nacl 144, kcl 5.9, cacl2 1.6, mgso4 1.2, kh2po4 1.2, nahco3 25, and d - glucose 11.1) and 1 mol / l of diclofenac. the solution in the bath was maintained at 38c with a gas mixture of 5% co2 and 95% o2. after equilibration, the reactivity of rings was tested with kcl (40 mm). after repeated washouts, the tension of rings was again adjusted to 1 g. after equilibration, the rings were precontracted with the 1-receptor agonist phenylephrine (200 nm). to obtain cumulative concentration response curves, egcg (sigma aldrich, dissolved in water) at concentrations of 5, 10, and 15 m was added at 30-minute intervals to the rings. for the relaxation experiments with tea, 1.2 g of darjeeling green tea (provided by king 's teagarden, berlin, germany) was brewed with 100 ml of boiled water for 3 minutes. relaxations to cumulative doses (10 nm5 m) of the endothelium - dependent vasodilator acetylcholine were also performed. maintenance of smooth muscle integrity was confirmed by evaluation of endothelium - independent vasodilation to sodium nitroprusside (snp, 0.1100 nm). statistical calculations were carried out with student t tests for independent samples for pair - wise comparisons of mean values, and with mann whitney rank sum test when comparing medians. vasodilation to the no donor snp was not impaired in enos mice compared with wild type mice. moreover, we observed a slight but significantly enhanced relaxation to snp in enos mice (fig. p < 0.05 aortic rings of wt versus enos mice at equal concentrations of ach. b, cumulative doses of the endothelium - independent vasodilator snp (0.1100 nm) were given to the rings to illustrate maintenance of smooth muscle integrity. p < 0.05 aortic rings of wt versus enos mice at equal concentrations of snp. next, we exposed phenylephrine - precontracted aortic rings to cumulative concentrations of egcg in a time interval of 30 minutes between applications. low doses of egcg (515 m) induced concentration - dependent vasorelaxations in wild type mice. in aortic rings of enos mice, however, egcg had no effect on vascular tone. an original recording of a representative experiment is shown in figure 2a. in accordance with our previous results obtained in rats,7 treatment of aortic rings from wild type mice with in contrast, neither initial contractions nor any relaxation in response to egcg could be detected in enos mice (fig. egcg of 5 m led to a relaxation of 82.3 (4.2) % of precontraction, and doses of 10 m and 15 m resulted in 56.9 (5.0) % and 41.3 (5.4) % vasodilation, respectively. however, egcg completely failed to induce vasodilation in rings of enos mice (fig. 2). to extend our findings to the whole beverage, green tea was added to aortic rings of wild type and enos mice. green tea in lower concentrations caused vasodilation only in wild type aortic rings, whereas a slight vasorelaxation was also obtained in enos mice at higher concentrations (fig. original recording of a single experiment of egcg - induced vasorelaxation in phenylephrine - precontracted aortic rings of enos and wt mice (a). the graphs show an initial transient contraction after each egcg dose in wt aortic rings that is followed by sustained relaxation. b, cumulative concentrations of egcg were added to phenylephrine - precontracted aortic rings of enos and wt mice. shown p < 0.05 aortic rings of wt versus enos mice at equal concentrations of egcg cumulative doses of green tea were added to phenylephrine - precontracted aortic rings of enos and wt mice. p < 0.05 aortic rings of wt versus enos mice stimulated with equal amounts of green tea. n.s., not significant ; wt, wild type. predominantly, a no - dependent mechanism for egcg - induced vasorelaxation has been described, as demonstrated by pharmacological inhibition of endothelial no production.7,1012 however, alternative modes of action are discussed. the egcg - induced inhibition of ca influx in smooth muscle cells as a mechanistic factor for vessel relaxation was proposed.6 inibition of various phosphodiesterase (pde) isoforms was also suggested as an underlying mechanism for vasodilating effects of egcg. egcg reduced the enzymatic activities of isolated pde1-pde5 isoenzymes, with strongest inhibitory effects on pde1 and pde2. consequently, the contraction - induced decrease in cyclic nucleotide levels (camp and cgmp) in aortic smooth muscle cells was reversed by egcg.8 in addition, an increase in endothelial prostacyclin production by egcg was reported at the cellular level.13 we demonstrate in our study that the vasodilating effects of egcg strongly rely on the presence of enos and thus no production in endothelial cells. however, several mechanisms can result in stimulation of cellular no production, for example, the generation of reactive oxygen species.10,11 besides vasodilation, a number of studies describe vasoconstrictive effects of egcg. does egcg induce rather vasorelaxation or vasoconstriction ? a transient ca - dependent contractile response to egcg was observed in resting rat aortae (without precontraction).14 egcg - induced contractions involved the formation of h2o2, which results in ca influx in smooth muscle cells through nonvoltage - dependent ca channels.15 the apparent contradiction between these opposite effects (vasoconstriction vs. relaxation) could be attributed to the time - course in egcg - induced changes in vessel tone. egcg results in vasoconstriction7,8,12,14,15 that is transient in resting rings8,14,15 or followed by vasodilation in precontracted vessels.7,8,12 an initial activation of ca influx into smooth muscle cells through voltage - operated ca channels followed by inhibition of voltage - operated ca channels was suggested as potential explanation for biphasic actions of egcg on vessel tone.9 interestingly, this biphasic mode of action was completely absent in enos mice in our study. the lack of egcg - induced contractions was also obtained after pharmacological inhibition of enos and after removal of the endothelium in rat aortae.7,12 the reason for this endothelial - dependent and no - dependent transient vasoconstriction induced by egcg is unknown at present. this would not be surprising because loss of endothelial no likely increases sensitivity to exogenous no. in summary, irrespective of potential upstream signaling molecules and pathways involved, our study clearly demonstrates that the egcg - induced changes in vessel tone (both initial contractions and subsequent relaxations) are exclusively dependent on the presence of enos and endothelial no production. | abstract : the underlying mechanisms for the vasodilating effects of the tea catechin epigallocatechin-3-gallate (egcg) are still not fully understood. besides nitric oxide (no)-dependent effects, other modes of action are discussed. to elucidate whether the no pathway is a prerequisite in mediating vasodilating effects, we investigated egcg - induced vasorelaxation in isolated aortic rings of endothelial nitric oxide knockout (enos/) mice. vasodilation to acetylcholine was fully prevented in aortic rings of enos/ mice, confirming lack of vascular no production. vasodilation to the exogenous no donor sodium nitroprusside was preserved in enos/ mice aortic rings. low concentrations of egcg (515 m) resulted in strong vasorelaxation in aortic rings of wild type mice, whereas it was completely absent in enos/ mice. in corroboration, relaxation in response to green tea was significantly inhibited in aortic rings of enos/ mice. these results demonstrate that egcg - induced vasodilation strongly relies on functional no synthase in endothelial cells and subsequent stimulation of no production in vessels. |
indeed, the ten leading companies newly marketed compounds increased their revenues by only ~10 %, and the average innovation deficit was ~1.31.8 new chemical entities per year (drews 2003). as the time from drug discovery to launch is currently ~12 years and costs ~$750 million / drug, the pharmaceutical industry is determined to reduce both the cost and time scale of this process ; it is, therefore, understandable that the strategies adopted by these companies are those which provide information in advance of costly clinical trials. a significant obstacle to this is determining the properties of a drug that facilitate its delivery to, and uptake by, target tissues and/or cells to avoid unsuccessful but nonetheless expensive clinical trials. the bioavailability of drugs and hence their ability to interact with their targets can be summarized by four notions grouped under the acronym adme, which stands for absorption, distribution, metabolism, and excretion. each of these notions involves a particular aspect of the physiological interactions between body tissues and drugs, which explain drug bioavailability. to circumvent the inherent difficulty linked to adme - related problems, lipinski and collaborators produced a set of rules to identify the optimum physicochemical properties required for an oral compound to achieve maximum bioavailability, i.e. to cross all biological barriers before reaching its target. they studied all marketed drugs and deduced similarities or common important properties of all the different active compounds. in this context the first rule is based on the lipophilic index of drugs (octanol water partitioning : log p 0 $ $ \end{document } is the magnitude of the interaction). in this case, each negatively charged lipid can be in two states, occupied (i.e. interacting with hydrogen ion) or non - occupied (i.e. free of hydrogen ion). it follows that the partition function of a negatively charged lipid is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \zeta = 1 + e^{{\left ({ e_{0 } + \mu } \right)/k_{b } t } } $ $ \end{document } (\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu \sim k_{b } t\ln \left ({ c_{{h^ { + } } } \times v_{0 } /v_{{h^ { + } } } } \right) $ $ \end{document } is the chemical potential of hydrogen ion in solution and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c_{{h^ { + } } } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{{h^ { + } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v_{0 } $ $ \end{document } are, respectively, the volume concentration of hydrogen, the volume of an hydrogen ion, and the typical volume of ions in the cytosol). using statistical physics, the probability that a lipid is free from hydrogen is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p_{0 } = 1/\zeta $ $ \end{document}. this last relationship in conjunction with eq. 9 provides the relationship between the free surface area per lipid and the volume concentration of ions in solution. | with a predicted 382.4 per 100,000 people expected to suffer from some form of malignant neoplasm by 2015, and a current death toll of 1 out of 8 deaths worldwide, improving treatment and/or drug design is an essential focus of cancer research. multi - drug resistance is the leading cause of chemotherapeutic failure, and delivery of anticancer drugs to the inside of cancerous cells is another major challenge. fifteen years ago, in a completely different field in which improving drug delivery is the objective, the bioavailability of oral compounds, christopher lipinski formulated some rules that are still used by the pharmaceutical industry as rules of thumb to improve drug delivery to their target. although lipinski s rules were not formulated to improve delivery of antineoplastic drugs to the inside of cancer cells, it is interesting to note that the problems are similar. on the basis of the strong similarity between the fields, we discuss how they can be connected and how new drug targets can be defined in cancer. |
the anterior cruciate ligament (acl) is one of major knee ligaments and is critical to knee stability. injury to the acl can be a debilitating musculoskeletal injury seen most often in athletes. the incidence of acl injuries is currently estimated at approximately 200,000 in usa annually, with 100,000 acl reconstructions performed each year. in general, the incidence of acl injury is higher in people who participate in high - risk sports, such as basketball, football, skiing, and soccer. the goal of the acl reconstruction surgery is to prevent instability and restore the function of the torn ligament, creating a stable knee so that the young can go back to the sporting activities. acl reconstruction is uasually performed using either the patellar bone tendon and semitendinosis and gracilis tendons, and both are not free from complications. the most common is the graft failure and stretching of the graft due to delay in the tendon - bone healing and tendon - bone incorporation of a tendon - graft within the bone tunnel. improvement of graft healing to bone is crucial to facilitate early and aggressive rehabilitation and a rapid return to full activity. to counteract this bone morphogenetic factors have been used with good results to improve the bone in growth in the tendon. (2012) reported a potential role of growth factors and bio - scaffolds for improving healing and mechanical integrity of the acl injury that is reconstructed with a tendon - graft. it was shown that the use of a collagen - platelet - rich plasma scaffold stimulated healing of a defect in the canine acl. many other growth factors have been used in the early and better bone ingrowth at the site of acl reconstruction with bone tendon - graft and tendon - graft. sadat - habdan msenchymal stimulating peptide (shmsp) was discovered at university of dammam, dammam and king fahd hospital of the university, alkhobar and was patented in 2008 (united states patency and trade office, us 7,399,826, b1 given on july 15, 2008). shmp is a 13 amino acids with a molecular weight of 1460 kd, which is now available in the synthesized form. a recent study showed that when topically applied there was early and better healing in diabetic animals. the objective of this study is to assess the efficacy of a bone growth factor (shmsp) in the rate of healing of bone tendon interface and osteo - integration of the tendon at the tunnel. rabbits were procured and were left in the animal house for 2 weeks for acclimatization to the surrounding. under ketamine 50 ml / kg weight and xylazine 35 ml / kg weight animals were anesthetized. a 3 cm long lateral half of tendoachilles tendon of the left side was harvested and a bone tunnel was made in the region of acl and a 1/0 ethilon suture was passed through the end of the harvested tendon [figure 1 ]. the tendon was place in the amorphous powder of the shmsp as a growth factor at a dose of 5 ml / kg body weight a 2.5 mm drill hole was made at the acl going between the tibia and the femur. the tendon was passed through the bony tunnel made and secured to each end of the tunnel with a 2/0 dexon [figures 2 and 3 ]. in the control group, the procedure was repeated without the addition of the shmsp. both groups of animals were kept in the similar circumstances and monitored on a regular basis. after 4 weeks, 5 animals from each group were euthanized and 8 weeks the rest of the animals were euthanized. the lower limb was disarticulated at the hip joint and stored in 2% formalin at a temperature of 4c and before histopathological analysis was done. harvesting of the tendon harvesting and drilling of the tendon positioning of the tendon the two groups were compared specifically for the bone in growth in the drill hole made at the tibial end through, which the tendon - graft was passed. the study was approved by the institutional review board of the university of dammam and funded by the deanship of scientific research of university of dammam, saudi arabia. figures 14 shows harvesting of the tendon, drilling, and position of the graft. in all the animals of the study group at 4 weeks showed, newly formed osteoid was observed at places early of the bone formation encroaching the tunnel, where in the control group tunnel was filled with the granulation tissue [figures 5 and 6 ]. photomicrograph of control group at 4 weeks showing fibro - collagenous tissue (fibrosis) ; (h and e, 40) photomicrograph of study group at 4 weeks showing early bony exostosis in the tunnel ; (h and e, 40) by 8 weeks in the study group, the canal was totally obliterated with increased mineralization of the new bone and at places seen extending onto the periosteal surface. in the control, there was minimal change in the formation of the new bone formation but there was more granulation tissue leading to form the connective tissue [figures 7 and 8 ]. photomicrograph of control group at 8 weeks showing the whole tunnel is filled with granulation tissue with no signs of any new bone formation ; (h and e, 40) photomicrograph of study group at 8 weeks showing abundant new bony formation and little granulation tissue in the tunnel ; (h and e, 40) our study showed that in animals in which shmsp was used to augment healing of the tendon - graft in the osseous tunnel there was exuberant bone formation, which was higher and more organized when compared to the control group of animals. the changes were subtle initially but 8 weeks the difference was more pronounced and appreciable. the histological specimens showed increased trabecular bone close to the grafted tendons as early as 4 weeks after implantation. different methods and growth factors were used to improve healing of the bone tendon interface. found that improved bone formation around a tendon - graft using recombinant human bone morphogenetic protein-2 (bmp-2) in an extra - articular bone tunnel in a dog model. similarly, nicklin. showed that exogenous osteogenic protein-1 results in improved bone formation at the tendon - bone interface in a sheep model. injected fibrin sealant (ifs) combined with bmp after acl reconstruction showed that the rate of new bone formation of ifs - bmp composite was significantly and achieved a more prolonged osteogenic effect. coated mesenchymal stem cells to the tendon - graft thereby enhancing the of tendon - graft osteo - integration. in our study, we coated the tendon with the shmsp to stimulate the osteo - integration in the tunnel with satisfactory results. in this study, we used shmsp a small polypeptide, which was reported as an angiogenesis factor and showed results comparable to other growth factors. the healing pattern between the tendon and the drill hole in the bone through which the tendon passes is not clearly understood but one this is certain that it takes many months probably to heal and incorporate and till that time certain activities are to be curtailed postsurgery. for a young athlete to be away from sporting activities is quite difficult and early activity to jeopardize the repair causing up to 10,000 revision yearly of acl reconstruction in the usa alone. if a osteo - integration of the bone there is still no clinical evidence regarding the use of growth factors, due to the fact that dosage of these factors still remain undeterminable as most of the half - life of growth factors is too short to stimulate the healing for weeks. with regard to shmsp which was used on a daily basis and we believe that our study has some limitations and the one which stands out that we did not perform bio - mechanical tests to assess the strength of the healing which was so convincing histologically and secondly a small sample of 10 animals on each side of the study arm. our study shows that local application of shmsp on the tendon - graft and instilling the growth factor in the tunnel itself enhanced the oseto - integration of the tendon into the bony tunnel created. we believe there is opportunity to convert this animal - based study into a clinical study when the safety of shmsp is established. | background : reconstruction of the anterior cruciate ligament (acl) involves use of semintendinosis and gracilis tendons graft that is transplanted into bone tunnels at the femoral and tibial insertion sites and the sites and the bone tendon interface is a weak link in the early healing period due to slow rate of healing. we hypothesized that an addition of bone growth factor like sadat - habdan mesenchymal stimulating peptide (shmsp) could enhance bone tendon healing rate so that re - rupture of the tendon does not take place.methodology:twenty skeletally mature rabbits underwent acl reconstruction of the right knee. in 10 of the rabbits at the site of the tendon - graft 5 mg / kg body weight of shmsp was put in the bone tunnel. in 10 other animals, nothing was added. at eight and 12 weeks 5 animals from each group were sacrificed. the tendon - graft site was harvested and sent for histopathological examination to assess the healing at the tendon - bone graft to the tibial tunnel.results:there were no deaths in both the groups. one rabbit of the control group developed an infection. in all the animals of the study group from 4 weeks onward showed bone formation, wherein the control group only granulation tissue was observed. by 8 weeks in the study group, the canal was totally obliterated with the new bone formation which extended onto the periosteal area. in the control, there was minimal change in the formation of the new bone formation.conclusion:addition of a growth factor like shmsp would enhance the osteo - integration of the tendon - graft in the bony tunnel after acl reconstruction in vivo. |
over the past year, multiple noteworthy trials with the potential to evolve current clinical practice and guidelines have been presented at international meetings including the american college of cardiology (acc, san diego, usa, march 2015), european association for percutaneous cardiovascular interventions (europcr, paris, france, may 2015), american diabetes association (ada, ma, usa, june 2015), european society of cardiology (esc, london, uk, august 2015), transcatheter cardiovascular therapeutics (tct, san francisco, usa, october 2015), heart rhythm congress (hrc, birmingham, uk, october 2015), and the american heart association scientific sessions (aha, florida, the authors describe new clinical data placed in the context of acute coronary syndrome (acs), interventional cardiology, heart failure, atrial fibrillation, electrophysiology, and coronary prevention. the results of major clinical trials in cardiology presented at major conferences in 2015 were reviewed by the authors. search terms included acute coronary syndrome, atrial fibrillation, coronary prevention, electrophysiology, heart failure, and interventional cardiology. trials were selected based on relevance to the cardiovascular (cv) community, the potential to change clinical practice, and the potential to guide further phase 3 research. this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors. advances in heart failure (hf) have featured prominently in many 2015 conferences with particular focus on addressing the side effects of currently used drug classes. mineralocorticoid antagonists (mras) have a class 1a recommendation for reduction in mortality and hospitalization in patients with heart failure with reduced ejection fraction (ef). however, this drug class is typically under - prescribed due to concerns regarding hyperkalemia and renal impairment in high - risk patients. finerenone is a novel non - steroidal mra with greater receptor selectivity than spironolactone and better receptor affinity than eplerenone in vitro [1, 2 ], which may have the potential to reduce side effects of hyperkalemia and renal impairment. the arts - hf (mineralocorticoid receptor antagonist tolerability study - heart failure) double - blind controlled phase 2b trial (clinicaltrials.gov # nct01807221) randomized 1060 patients with heart failure (hf), type 2 diabetes mellitus (t2 dm) chronic kidney disease (ckd) to finerenone (titrated from one of five doses from 2.5 to 20 mg) or eplerenone (titrating from 25 to 50 mg) [1, 2 ]. although the proportion of patients with a relative decrease of 30% in 90-day nt - probnp (primary endpoint) was similar in all groups, the measurement of nt - probnp to predict clinical outcome is not well established, and the subgroup of patients receiving finerenone 1020 mg showed a 44% reduction the secondary clinical composite endpoint of 90-day all - cause death, cv death, hospitalization, or emergency presentation for worsening chronic hf [1, 2 ]. finerenone 1020 mg appeared better tolerated than eplerenone with a smaller increase in potassium (0.119 vs. 0.262 mmol / l ; p = 0.016) and lower incidence of estimated glomerular filtration rate (egfr) decrease > 40% (4.2% vs. 9.4%) [1, 2 ]. hyperkalemia is a common concern in hf patients using renin - angiotensin - aldosterone (raas) inhibitors. in a previous trial, opal - hk (clinicaltrials.gov # nct01810939), investigators showed a short - term sustained decrease in potassium in patients with hf taking the potassium binder patiromer with raas inhibitors. the phase 2 amethyst dn trial (clinicaltrials.gov # nct01371747) assigned 304 patients with ckd, hypertension (htn), t2 dm, serum potassium above 5 meq / l and who were taking raas inhibitors to patiromer (starting dose stratified by baseline potassium). meq / l in patients with hf (n = 105) or without hf (n = 199) for up to 1 year (p 50% being treated with anthracycline trastuzumab and radiation to a protective strategy of candesartan or metoprolol vs. placebo. patients receiving candesartan had a significantly smaller decline in ef from baseline vs. placebo (0.8% vs. 2.6% ; p = 0.026). although small in size, this study suggests potential benefit from candesartan therapy in this patient group. previous studies have yielded conflicting conclusions regarding the mortality effect of digoxin among patients with congestive heart failure (chf) [11, 12 ]. thus conducted an observational study using a danish nationwide registry of patients discharged from hospital with both hf and atrial fibrillation (af), excluding those receiving antiarrhythmic therapy (e.g., amiodarone), those who died within 30 days of discharge, and those not taking a vitamin - k antagonist, then matching the 8078 digoxin - treated patients with 8078 control patients, based on age, sex, comorbidities, cha2ds2vasc score, medication, and heart - failure severity. the risk of death was reduced [hazard ratio (hr) 0.92 ; (ci 95% 0.860.98) and risk of readmission increased hr 1.07 ; 95% ci 1.011.12 ] with digoxin users. when death was considered as a competing risk, the collective incidences of readmission were similar at 64.9% in digoxin users and 64.4% in non - users. the authors concluded that in digoxin - naive patients with af, digoxin use was associated with a slightly reduced risk of death. a novel heart failure strategy may be attenuation of myocyte sodium overload by beta 3 agonists. in the beta 3 agonists treatment in hf (beat - hf) trial (clinicaltrials.gov # nct01876433) [14, 15 ], 70 patients with hf were randomized to the beta 3 agonist mirabegron or placebo for 6 months [14, 15 ]. while mirabegron was not associated with improvement in patients with a baseline ef > 40% (mean difference 0.4% ; 95% ci 3.5 to 3.8 ; p = 0.82) an exploratory analysis of patients with a baseline ef 40% noted a 5.5% increase in ef (95% ci 0.610.4 ; p 20%) were randomized to intracoronary injection of 4 ml bcm (n = 201) vs. saline control (n = 102) 25 days after their primary pci. injection of bcm was not associated with any difference vs. saline in the primary endpoint (change in lv end diastolic volume index (lvedvi) from baseline to 6 months), or in secondary endpoints including 6-min walk test, nyha grade, cv hospitalization, or death. bcm injection was associated with five temporary angiographic occlusions vs. one with saline. the disappointing discrepancy between pre - clinical data and preservation i findings may have been related to microvascular obstruction and edema preventing entry of bcm into the myocardium. hf may be complicated by sleep - disordered breathing and early treatment may improve outcomes. seventy patients with acute hf diagnosed with sleep apnea by overnight polysomnography within 4 weeks of discharge were treated with continuous positive airway pressure (cpap) treatment. of these, 37 (53%) met the definition of good compliance with the cpap device. patients with good cpap compliance showed a significant decrease in 6-month readmission rates after therapy compared with the 6-month readmission rates pre - therapy (mean se : 1.5 0.2 events ; p 50% calls completed) had lower 180-day mortality compared to non - adherent patients (8.3% vs. 26% ; p 0.001) [25, 26 ]. patients with good remote monitoring adherence (> 50% days) had lower 180-day mortality (6.6% vs. 21.4% ; p 0.001) and lower 180-day readmission rates (41.3% vs. 61.1% ; p 0.001) [25, 26 ]. while good adherence to a telephone and telemonitoring program appears to be of clinical benefit, as this was a post hoc observation, further prospective investigation is needed. until recently, the absence of a specific reversal agent for non - vitamin k antagonist oral anticoagulants (noacs) has been seen as a limitation. in late 2015, the first specific reversal agent idarucizumab was approved for reversal of the direct thrombin inhibitor dabigatran. approval was based on interim results of reverse - ad (clinicaltrials.gov # nct02104947) [28, 29 ] in 90 patients51 with serious bleeding (group a) and 39 requiring an urgent procedure (group b). the anticoagulant effects of dabigatran were rapidly (within minutes) and completely reversed in 88 to 98% of the patients who had had elevated clotting times at baseline. unbound dabigatran concentration at 24 h remained below 20 ng / ml in 79% of patients. one thrombotic event occurred within 72 h of idarucizumab in a patient who had not received anticoagulant re - initiation. factor xa oral anticoagulants do not yet have an approved reversal agent but a recombinant engineered version of human factor xa with the ability to bind factor xa inhibitors has been tested in healthy older (aged 5075) volunteers receiving apixaban 5 mg twice daily or rivaroxaban 20 mg once daily. in apixaban participants, reduced anti - xa activity by 94% (vs. 21% receiving placebo ; p 1.8 mmol / l despite maximum tolerated statin were randomized in 2:1 ratio to alirocumab (150 mg) or placebo subcutaneous injection every 2 weeks for 78 weeks. at week 24, ldl - c in the alirocumab group had dropped 62% more from baseline than with placebo (p 140 mmhg, eligible for diuretic use and with 1 other component of metabolic syndrome to therapy with amiloride alone, to combination amiloride plus hydrochlorothiazide, or to hydrochlorothiazide alone. two - hour glucose concentrations after an oral glucose tolerance test, averaged at 12 and 24 weeks (the primary endpoint), were significantly lower with amiloride alone than hydrochlorothiazide alone (0.55 mmol / l, 95% ci 0.96 to 0.14 ; p = 0.0093) and in the combination group compared to hydrochlorothiazide alone [0.42 mmol / l (0.84 to 0.004) ; p = 0048 ]. mean home systolic blood pressure was reduced similarly with single treatments, but combination therapy led to an additional 3.4 mmhg reduction compared with hydrochlorothiazide (95% ci 0.95.8 ; p = 0.007). hyperkalemia occurred in seven (4.8%) amiloride patients compared to three (2.3%) in the combination group. in summary, combination therapy appeared to be a favorable treatment option being neutral for glucose and potassium but with greater effects on blood pressure. lower fasting glucose and hba1c levels with ranolazine have been suggested by previous trials in the setting of coronary disease [45, 46 ]. a prospective randomized study presented at the american diabetes association (ada) 2015 scientific sessions (clinicaltrials.gov # nct01472185) evaluated the effect of ranolazine on type 2 diabetics with a hba1c of 710% (5386 mmol / mol) compared with placebo. ranolazine compared with placebo was associated with a greater reduction in the primary end point of fall in hba1c from baseline at 24 weeks [mean difference 0.56% (6.1 mmol / mol) ; p 24 h to 30 days) device thrombosis (0.9% vs. 0.1% ; rr 6.26 ; 95% ci 0.8248.04 ; p = 0.04) although the relative difference was less at 1 year (1.5% vs. 0.7% ; rr 2.08 ; 95% ci 0.785.51 ; p = 0.13). intravascular imaging is usually recommended to guide optimal b vs. deployment but a surprisingly low use was noted in absorb iii. the first - generation absorb b vs. has relatively thick struts and device thrombosis was less prevalent in vessels > 2.25 mm. nevertheless, a meta - analysis of six trials including absorb japan and absorb iii did confirm a small but significant increase in device thrombosis. thus, appropriate patient / lesion selection, careful lesion preparation, optical coherence tomography to guide sizing post dilatation and extended dapt (to 2 years) is recommended minimize risk of device thrombosis. reducing bleeding complications following pci is an important goal given the association of bleeding with adverse clinical outcomes. the matrix (minimizing adverse haemorrhagic events by transradial access site and systemic implementation of angiox) (clinicaltrials.gov # nct01433627) study randomized 8404 patients with acs, undergoing urgent angiography (and 80% with follow - on pci) to radial or femoral access [62, 63 ]. radial access was associated with a significant reduction in non - cabg major bleeding (1.6% vs. 2.3%, rr 0.67 ; 95% ci 0.490.92 ; p = 0.013), a 14.6% trend to reduction in the primary endpoint of mace (death, mi or stroke ; 8.8% vs. 10.3% ; 95% ci 0.740.99 ; p = 0.0307 although not - significant at an of 0.025) and significant reductions in secondary endpoints of all - cause mortality (1.6% vs. 2.2%, rr 0.72 ; 95% ci 0.530.99 ; p = 0.045) and nace (net composite of death, mi, stroke, or major bleeding ; 9.8% vs. 11.7% ; hr 0.83 ; 95% ci 0.730.96 ; p = 0.0092). such results strongly support radial access being preferred as the default access site. a separate randomization within the matrix study to bivalirudin vs. unfractionated heparin bail out glycoprotein iib / iiia inhibition did not show any difference in mace or nace. in some patients with poor radial access, the ulnar artery route, although technically more challenging, may be a useful alternative to avoid femoral cannulation. the single - center ajmer ulnar artery working group study (ajular) presented at acc2015 randomized 2533 patients undergoing pci by experienced operators (> 50 ulnar procedures) to ulnar or radial access. there was no significant difference in the primary endpoint (composite mace, site cross - over, major vascular events during hospital stay ; 14.6% tua vs. 14.4% tra 95% ci ; p = 0.92), or individual components of the composite endpoint. requirement for creation of a subcutaneous pacemaker pocket and insertion of a transvenous lead in conventional pacing systems can be associated with important complications such as pocket hematoma or infection, pneumothorax, or hemothorax. the micra transcatheter pacing system study (clinicaltrials.gov # nct02004873) evaluated use of a leadless pacemaker (fig. 6) implanted in the right ventricular apex via femoral vein implantation and secured by small tines in 725 patients (719 successfully implanted). the primary safety endpoint (freedom from system or procedure related major complications) was achieved in 96% and the primary efficacy endpoint (percentage of patients with low and stable pacing capture thresholds at 6 months) in 98.3%both of which were higher than the minimum performance goals from conventional pacing historical control data. major complications, which were less common than in the historical control dataset, included cardiac perforation or effusion / tamponade (1.6%), groin site complication (0.7%), and pacing issues (0.3%) but no (0%) dislodgments.fig. reproduced with permission of medtronic, inc traditional pacemaker vs. micra. reproduced with permission of medtronic, inc another leadless pacemaker the nanostim lp (st. jude medical)was evaluated among 300 patients in the leadless ii study (clinicaltrials.gov # nct02030418). the primary safety endpoint (6-month freedom from device - related serious adverse events) was met in 93.3% and primary efficacy endpoint (acceptable 6-month pacing threshold and sensing amplitude) was met in 90%both also exceeding minimum performance goals from conventional pacing historical control data. in total, 6.7% had serious adverse events (1.7% device dislodgement with percutaneous retrieval, 1.3% cardiac perforation and 1.3% pacing - threshold elevation requiring percutaneous retrieval and device replacement). as each of the leadless pacemaker studies are observational, larger randomized studies with longer term data including safety and retrieval data is required. current guidelines suggest it is reasonable to perform af ablation in selected patients undergoing cardiac surgery but acknowledge that supporting data are limited. thus, in a cardiothoracic surgical trials network (ctsn) study (clinicaltrials.gov # nct00903370) 260 patients with persistent including longstanding persistent af requiring mitral valve surgery were randomized to ablation or no - ablation groups. patients assigned to ablation were further randomized to pulmonary vein isolation (pvi) or the more complex biatrial maze. those receiving concomitant af ablation were over twice as likely to be free from af on 72-h holter at both 6 and 12 months (63.2% vs. 29.4% ; p < 0.001) although there no difference whether performed by pvi or biatrial maze (61% vs. 66% ; p = 0.6). ablation was associated with increased need for permanent pacemaker implantation (21.5 vs. 8.1 per 100 patient - years, p = 0.01). there was no difference in major cardiac or cerebrovascular adverse events at 12 months but the study was not powered to show benefit in this respect. although the ctsn study did not find an advantage for the more complex biatrial maze over pvi, with a transcatheter approach, persistent af may benefit more extensive ablation. the belief trial (clinicaltrials.gov # nct01362738) randomized 173 patients with long - standing persistent af undergoing pulmonary vein antrum and non - pulmonary vein trigger ablation to additional left atrial appendage (laa) electrical isolation (n = 85) or no laa isolation (n = 88). those undergoing laa electrical isolation had significantly higher freedom from af at 1-year (56% vs. 28% ; hr, 1.92 ; p = 0.001). there was no significant difference in secondary endpoints including mortality, stroke, or re - hospitalization. anticoagulation strategy was assessed in the randomized active - controlled multi - center study with blind - adjudication designed to evaluate the safety of uninterrupted rivaroxaban and uninterrupted vitamin k antagonists [vka ] in subjects undergoing catheter ablation for non - valvular atrial fibrillation (venture - af ; clinicaltrials.gov # nct01729871, n = 248 patients). no adverse safety endpoints were reported for rivaroxaban and only rarely for vka (major bleeding 0.4% ; composite of stroke / systemic embolism / mi / vascular death / bleeding 0.8%). while the trial was too small to assess superiority, it suggests rivaroxaban is a reasonable alternative to vitamin k antagonists in patients undergoing catheter ablation in the setting of uninterrupted anticoagulation. the legacy study (long - term effect of goal directed weight management on af cohort : a 5-year follow - up study) (actrn12614001123639) evaluated the long - term impact of weight loss and weight fluctuation on rhythm control in obese individuals with symptomatic paroxysmal or persistent af. all patients received weight loss counseling with optional participation in a weight management clinic or self - managed program. those achieving 10% vs. < 10% weight loss had reduced af burden and symptom severity (p < 0.001) and a sixfold greater likelihood of arrhythmia - free survival (p < multivariate analyses found weight loss and weight fluctuation to be independent predictors of outcomes (p < 0.001). the trial of continuous or interrupted chest compressions during cpr conducted by the resuscitation outcomes consortium) (clinicaltrials.gov # nct01372748) assessed whether outcomes with non - trauma related cardiac arrest after continuous compressions with positive - pressure ventilation (intervention group) differed from those after compressions that were interrupted for ventilations at a ratio of 30 compressions to two ventilations (control group). however, there was no significant difference in survival to hospital discharge (8.9% vs. 9.7%, 95% ci 1.5 to 0.1 ; p = 0.07) or recovery of favorable neurologic function at discharge (7.0% vs. 7.7%, 95% ci 1.4 to 0.1 ; p = 0.09). the prospective multicenter imaging study for evaluation of chest pain (promise) study (clinicaltrials.gov # nct01174550) randomized 10,003 symptomatic patients to a strategy of initial anatomical testing with the use of coronary computed tomographic angiography (cta) or to functional testing (nuclear stress testing 68%, stress echocardiography 22% or exercise electrocardiography 10%). median cumulative radiation exposure per patient was lower with cta (driven by significantly higher exposure with nuclear imaging vs. cta). those assigned to cta vs. functional testing were more likely to undergo cardiac catheterization (12.2% vs. 8.1%) but of these, most patients in the cta group (72.1%) had obstructive coronary disease, whereas less than half in the functional group (47.5%) had obstructive coronary disease). revascularization < 90 days was undertaken in 6.2% of the cta group vs. 3.2% in the functional group (p < 0.001), including 72 patients and 38 patients, respectively, who underwent cabg. over a median follow - up of 25 months there was no difference in the incidence of the primary end - point (death, mi, hospitalization for unstable angina, or major procedural complication ; 3.3% vs. 3.0% ; adjusted hr 1.04 ; 95% ci 0.831.29 ; p = 0.75). thus, cta appears to identify coronary disease more accurately than functional testing although the clinical impact in promise was unclear. a second large multicenter cta study (the scot - heart trial) scot - heart randomized 9849 patients with an initial clinical diagnosis of coronary heart disease (chd) in 47% and angina due to chd in 36% to usual care plus cta at ~6 weeks or to usual care alone (clinicaltrials.gov # nct01149590). use of cta reclassified diagnosis of chd in 27% and angina in 23% (vs. 1% and 1%, respectively, with usual care p < 0.0001) and led to a change in planned investigation in 15% (cancellation of 121 functional tests, cancellation of 29 invasive angiograms, but scheduling of an additional 94 invasive angiograms, 88% of which showed obstructive disease requiring revascularization). at a median follow - up of 1.7 years, there was a 38% trend to reduction in fatal and non - fatal mi (26 vs. 42, hr 0.62, 95% ci 0.381.01 ; p = 0.0527). scot - heart thus confirmed that cta helps clarify diagnosis, enables more appropriate use of invasive angiography / intervention and may reduce the future risk of mi. in this paper, we have highlighted and summarized numerous important studies in the field of cardiology that have been presented to major international cardiology meetings throughout 2015. many of these studies will contribute to updating of current practice guidelines and others will play an integral role in the advancement and development of new therapeutic strategies. ian menown has received grants to institution, honoraria and/or conference sponsorship from : boston scientific, sanofi aventis, biosensors, meril life, orbus neich, menarini, astra zeneca, bayer, boehringer ingelheim, daichii sankyo, lilly, bristol myers squibb, and pfizer. this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors. this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. | introductionmultiple significant, potentially practice changing clinical trials in cardiology have been conducted and subsequently presented throughout the past year.methodsin this paper, the authors have reviewed and contextualized significant cardiovascular clinical trials presented at major international conferences of 2015 including american college of cardiology, european association for percutaneous cardiovascular interventions, american diabetes association, european society of cardiology, transcatheter cardiovascular therapeutics, heart rhythm congress, and the american heart association scientific sessions.resultsthe authors describe new trial data for heart failure (including eplerenone, finerenone, patiromer, sacubitril / valsartan, the beta 3 agonist mirabegron, sitagliptin, empagliflozin, alginate - hydrogel lv epicardial implant), anticoagulation (idarucizumab and reversal agents, adherence programmes, practice in ablation), transcatheter aortic valve replacement (long - term data, valve - in - valve use, the triguard embolic deflecting device), patent foramen ovale closure, cardiovascular prevention (pcsk9 inhibitors, hypertension treatment) and antiplatelets strategies (extended duration therapy with clopidogrel or ticagrelor). trial data are also described for contemporary technologies including the biofreedom polymer - free drug coated stent, bioabsorbable stents, pci strategies, left main treatment, atrial fibrillation ablation techniques, leadless pacemakers and the role of coronary computed tomographic angiography.conclusionsthis paper summarizes and contextualizes multiple pertinent 2015 clinical trials and will be of interest to both clinicians and cardiology researchers. |
non - alcoholic fatty liver disease (nafld) is a major cause of chronic liver disease globally, and is characterized by a spectrum of steatosis, inflammation and fibrosis. non - alcoholic steatohepatitis (nash) is a more severe form of nafld, and is characterized by lobular inflammation and hepatocyte ballooning;1,2 furthermore, nash is presumed to lead to cirrhosis in a quarter of the afflicted patients. the growth of nafld as an epidemic is related to increasing prevalence of obesity and sedentary life - style. the traditional risk factors for nafld include age, sex, central obesity leading to insulin resistance, and development of metabolic syndrome. furthermore, genetic predisposition is also presumed to have a role, given the differences noted amongst different ethnic groups.3,4 in the united states, the prevalence of nafld is gradually increasing and is currently estimated to affect one - third of adults.15 moreover, nash is predicted to be the leading cause of liver transplant in the coming years. despite the growing prevalence, however, the treatment options remain extremely limited. until recently, nafld was believed to be a disease of the industrialized world, primarily related to sedentary life - style. studies have suggested a wide amount of diversity in prevalence based on country of interest. the average prevalence in europe is 2030% and in china appears to be 524%.1,5 in india, the prevalence is estimated to be between 1632%.5 this is believed to be due to the increasing industrialization of these nations, along with changes in life - style and diet. furthermore, recent studies have also suggested that nash / nafld can also affect seemingly non - obese asians.6,7 this has come to be known as the asian paradoxas a disease that is associated with high bmi in the western world may not predict accurately the risk of developing nafld in the asian world.68 in this review, we look specifically at south asians afflicted with nafld. south asia is a growing epicenter for industrialization and changing life - styles, and also increasing prevalence of nafld. the south asian region includes the countries of india, nepal, bhutan, bangladesh, sri lanka, maldives, pakistan and afghanistan. the importance of gaining a better and complete understanding nafld in this region is essential in view of its growing population and immigration to western nations. in this review, we describe the prevalence of nafld in south asians and further describe the nafld phenotype with respect to lean nafld as well as the burden of hepatocellular carcinoma (hcc) in south asia. first, we searched the pubmed database using the terms nash and south asia, nafld and south asia, non - alcoholic liver disease and south asia, non - alcoholic steatohepatitis and south asia, and fatty liver and south asia. furthermore, we followed up this wide search for articles with a subsequent search using the terms nafld and individual countries comprising south asia (i.e. afghanistan, bangladesh, bhutan, india, nepal, maldives, pakistan and sri lanka). additionally, we performed an embase database search for the period from 1980 to 2016 with the search terms nash and south asia, nafld and south asia, non - alcoholic liver disease and south asia, nonalcoholic steatohepatitis and south asia, along with fatty liver and asia. the last terms used for this comprehensive search were nafld and (afghanistan, bangladesh, bhutan, india, nepal, maldives, pakistan or sri lanka). the studies that were included in the review were those with diagnosis of nafld either by aid of ultrasound (us) or biopsy. we did not exclude studies based on study participants age, degree of obesity, insulin resistance or diabetes (type 1 diabetes or type ii diabetes). we also did not exclude studies based on type of center or geographical location (rural vs. cosmopolitan center, or tertiary referral center vs. community hospital). several small population - based studies were reviewed, which provided estimates of nafld prevalence across south asian nations (fig. most of the studies, though, were from india, with a few studies from the other nations. a caveat to consider is that the true prevalence of nash is unclear, as diagnosis via biopsy is limited, with the majority of the data provided by us detection. also, the studies did not always differentiate clearly between tertiary and non - tertiary centers, which can confound the data due to differences of availability of diagnostic testing. of the few studies that utilized liver biopsy, there was estimation of prevalence in patients undergoing bariatric surgery at a tertiary care center9 (table 1). not surprisingly, the prevalence of nafld was 65.7% in this population, with 33.6% having nash and 14.1% having advanced fibrosis.9 in table 1, we detail all the studies with the prevalence estimates. some of the key findings from the articles show that nafld is a disease not only in more developed cities but also noted in rural areas. a study in the indian state of haryana, showed prevalence of nafld to be 30% in patients from rural villages reporting for us.10 similarly, another study also conducted in rural india last year showed that the prevalence of nafld in patients undergoing routine us was 28.1%.11 also of note, nafld prevalence, even in non - diabetic patients, was found to be high, with an estimate of 24.7% in one study for an urban population.12 not surprisingly, the prevalence of nafld in diabetics was higher, with reported estimates of 57.287%.13,14 the study from prasanth.15 utilized review of histologic data and noted a prevalence of 62.6% for steatohepatitis, with 37% having fibrosis. the overall data, though, still remains variable, with another study conducted in a suburban population from two cohorts in established railway colonies showing a prevalence of 16.6%.16 in bangladesh, a single published study conducted in a tertiary care liver clinic estimated a prevalence of 42.4% of biopsy - proven nash cases.17 in comparison to this, a rural study in bangladesh showed overall prevalence of nafld to be 18.4%.18 this either may highlight an underestimation in the rural setting (likely due to the methodology of detection) or an overestimation in tertiary clinic (likely due to referral bias). it is worth noting that a study in london amongst the immigrant bangladeshi population appeared to show an increased susceptibility to developing nafld as compared to caucasians.19 nafld was also noted to be more common in diabetic bangladeshis, with a high prevalence of 59.4%.17 further future studies are needed to understand underlying potential genetic risk in this population for developing nafld. data from an urban tertiary care center in pakistan for type 2 diabetic patients assessed by us showed an overall prevalence of nafld of 72.4%.20 another study, though conducted in an urban setting in pakistan, noted a prevalence of 51% nafld in type ii diabetics.21 in a study carried out in rural sri lanka, nafld was noted to be prevalent, at 18%.22 again, as noted in other studies, a different study in an urban setting also showed a higher prevalence of 32.6%.23 we did not find any studies in our search with regard to nafld prevalence in the other south asian nations. nonetheless, it appears that nafld is a global epidemic with increasing prevalence in south asia. the risk factors for nafld in south asia appear to be similar to those in the western world, with some subtle differences. age, obesity, insulin resistance, and overall development of metabolic syndrome are the major risk factors.13 multiple population - based studies from south asia have revealed an association between htn, waist circumference, bmi and insulin resistance.7,9,24 in 2015, a study specifically looked at risk factors for nafld in india and suggested multiple risk factors, including bmi, homeostatic model assessment of insulin resistance (homa - ir), waist - hip ratio, diabetes, htn, family history of metabolic syndrome and sleep apnea.14,15,25 furthermore, certain dietary habits were also associated with nafld in india, which included non - vegetarian diet, fried food, spicy food and tea.15 a similar study revealed higher prevalence of all components of metabolic syndrome in nafld patients.10 finally, there are also non - modifiable risk factors associated with south asian nafld that are related to genetic and epigenetic variations, such as in single nucleotide polymorphisms (snps).26 interestingly, multiple studies from south asia also reveal the early - onset of nafld, with average age in the 40s, and a male predominance in this region.24,2630 the south asian phenotype of nafld appears to differ from that which is presumed in developed nations. south asian patients that present with nafld appear to have lower bmi and obesity rates.7 despite this, the prevalence of nafld is significant on this sub - continent. asian paradox. this phenomenon is hypothesized to be related to ethnic disparities in visceral fat distribution.27,28 in fact, computed tomography imaging studies evaluating distribution of fat in south asians has found both subcutaneous and total adipose tissue to correlate with pathological severity of nafld.3133 this has also led to concerns regarding the use of nomenclature broadly to define obesity, which may not be accurate in the south asian population. in fact, the average bmi in south asian patients is only 26 and does not reflect the true risk of developing nafld (table 2).2428 there is further implication, as even non - obese nafld has been shown to be an independent risk factor for coronary artery disease in this population.3436 hence, it is important for clinicians to be aware that asians with lower bmi are also at increased risk of cardiovascular and metabolic diseases. additionally, these patients develop insulin resistance despite having lower bmi as compared to western patients and subsequently are still high risk of developing nafld.37 hcc remains one of the most common causes of cancer - related deaths in the south asian sub - continent, with nafld / nash presumed to be the cause of a major portion of the inciting disease.3842 data from globocan 2012 showed that 782,000 new cases of hcc were reported that year, with 83% of those being in the developing world.43 unfortunately, epidemiological studies on incidence of hcc in the asia - pacific remain limited. by 2030, it is projected that china and india will have the highest burden of diabetes, with 4 of the 10 countries with the highest prevalence of diabetes also being in asia (namely indonesia, pakistan, bangladesh, and the philippines).44 since diabetes has a known association with nafld and nash, the increasing rates of this disease will also have implications on development of hcc. longitudinal studies on the natural history of nafld in south asian countries are lacking, but studies from the western countries clearly point to fatty liver progressing to cirrhosis and hcc.45,46 it is then worrisome that a similar phenomenon may take place in asia. true incidence of nash leading to hcc is further difficult to ascertain given the relatively long duration from diagnosis of nafld to development of nash.46 from longitudinal studies performed in the west, it has been shown that rates of hcc from nash are similar to those of hcc from infection with the hepatitis c virus (hcv).46 as of yet, the only longitudinal study that has been published on an asian population is out of japan. yatsuji.47 showed that the 5-year occurrence rate of hcc in patients with nash - related cirrhosis and patients with hcv - related cirrhosis was not statistically different. amongst available data from the south asian nations, india has some of the highest rates of nafld, and it is possible to roughly estimate rates of nash - associated hcc from numbers from similar studies. india is estimated to have 120 million people with nafld,42 31% of those patients with demonstrated nash on histology48 (i.e. 37 million individuals with nash). from studies in western nations, it has been proposed that 10% of nash patients39,49 will go on to develop cirrhosis (i.e. 3.7 million individuals). of them, 25% of nash cirrhotics may go on to develop nash hcc,49 representing almost 1 million patients in india with nash - related hcc.33 mahady.48 estimated the prevalence of hcc in another unique way, by utilizing data on the prevalence of diabetes to estimate prevalence of nash and hcc. sixty - three million indians are proposed to have type 2 diabetes, with 70% having nafld (44 million).5052 nearly a third are proposed to have nash (i.e. 13 million). it is projected that 5% of those with nash (i.e. 650,000) may develop hcc.48 the studies on prognosis for nash - related hcc in south asia are also limited, and to date only studies from japan exist. the 5-year recurrence rate of nash hcc as compared to hcv - related hcc were not statistically different.53 this differed from a recent usa - based study, where it was found that patients with nash hcc had better survival rates as compared to those with hcv hcc.54 visceral, rather than overall, body fat has been seen to correlate with occurrence of hcc.55 visceral body fat is related to the degree of inflammation and fibrosis in nash. the pro - inflammatory environment can impact the liver and other tissues48 and patients with more visceral fat had higher rates of hcc recurrence.56 this is particularly important in south asia, given the higher rates of visceral body fat as related to their western counterparts.38 to conclude, nafld is a growing epidemic and is not limited to the western world. emerging data notes a high prevalence of nafld in the south asian population. this is likely related to changes in dietary modification from industrialization and development of metabolic risk factors for this disease. notably, the prevalence of nafld is increasing in the south asian population and there appears to be a male predominance. this population also appears to be predisposed to developing this disease earlier, compared to the western population. interestingly, bmi does not fully appreciate the risk of developing this condition in the south asian population, due to variation in ethnic distribution of adiposity. with the increasing prevalence of nafld in this subcontinent, it is becoming clear that it will contribute to increasing incidence of hcc as well, and further population - based studies are needed to understand the risks that may serve to inform future public health initiatives. | abstractnon - alcoholic fatty liver disease (nafld) and non - alcoholic steatohepatitis (nash) are national and global epidemics. the disease is characterized by a spectrum of liver steatosis (fat deposition), inflammation (in nash) and fibrosis. nafld and specifically nash can lead to cirrhosis, which carry risks of progression to portal hypertension and hepatocellular carcinoma (hcc). nash is also associated with higher mortality from cardiovascular causes. most of the data for nafld has been obtained from the perspective of developed nations, although the disease is increasing and threatening to reach epidemic proportions across the world. emerging data is notable for high prevalence of nafld in south asian populations, presumably resulting from a combination of underlying genetic polymorphisms and changes in socio - economic status. it is also notable that an asian paradox has been defined for nafld based upon the observation of lower than pre - defined body mass index (bmi), otherwise termed as lean nafld, among this population. yet, data remains limited in regards to the characteristics of nafld / nash in this population. in this article, we present a review of the literature and discuss the prevalence, associated risk factors and burden of hcc in south asians with nafld. |
type 2 diabetes (t2d) is the most common form of diabetes and an increasingly prevalent metabolic disease. it is associated with microvascular and macrovascular complications and is considered one of the major causes of morbidity and mortality. according to the international diabetes federation (idf), there were approximately 382 million people worldwide with diabetes in 2013, and this number is expected to reach more than 592 million by 2035. in recent decades, several studies have shown the role of chronic low - grade inflammation and activation of the immune system in the pathogenesis of t2d and its complications [37 ]. however, the mechanisms by which chronic inflammation is involved in t2d are not completely clear. it has been reported that synthesis and release of pro- and anti - inflammatory cytokines such as tumor necrosis factor- (tnf-), interleukin- (il-) 10, and il-6 and growth factors such as transforming growth factor-1 (tgf-1) [813 ] may be involved in pathogenesis of t2d and its complications. single nucleotide polymorphisms (snps) in the cytokine genes are usually located in their regulatory regions and affect the levels of their expression [14, 15 ]. various studies have associated cytokine gene expression alterations with obesity, changes in insulin sensitivity, and risk of t2d [6, 7, 16 ]. some works have investigated the association between the tnf- -308g / a polymorphism (rs1800629) and diabetic retinopathy (drp), nephropathy (dnp), and neuropathy (dnr) in different populations [1724 ]. only a recent brazilian study found an association between proliferative drp and these tnf- polymorphism. dnp was associated with the polymorphisms il-10 -592c / a (rs1800872) and il-10 -819t / c (rs1800871). in addition, paine. associated the polymorphism il-10 -1082g / a (rs1800896) with drp and kolla. observed an association with dnr the human tgf-1 gene presents two hotspot focuses : codon 10t / c (rs1800470) and codon 25c / g (rs1800471). both polymorphisms were associated with drp by bernek., and el - sherbini (2014) associated the tt genotype of tgf-1 codon 10t / c polymorphism with dnp risk. il-6 gene polymorphism -174g / c (rs1800795) was associated with dnp in study conducted by muammer. the few studies that evaluated the association of the polymorphism ifn- + 874t / a (rs2430561) with diabetes complications found association between this polymorphism and drp and with dnr. in spite of the existence of several reports examining the association of polymorphisms in various cytokine genes, much controversy remains about their role in diabetes complications. no studies to date have examined in a single population a large number of polymorphisms (tnf- -308g / a, il-10 -1082g / a, il-10 -819t / c, il-10 -592c / a, tgf-1 codon 10t / c, tgf-1 codon 25c / g, il-6 -174g / c, and ifn- + 874t / a) and their association with the t2d complications and comorbidities. in this study, we investigated if these polymorphisms are associated with drp, dnp, and dnr and with comorbidities (hypertension, dyslipidemia, and obesity) in a group of brazilian t2d patients. this study was conducted with 102 brazilian individuals, including 19 men and 83 women with clinical and laboratory diagnosis of t2d, aged from 32 to 70 years (54.99 8.97 years), recruited from the clinic of endocrinology, santa casa hospital (belo horizonte, minas gerais, brazil) in the period from june 2012 to september 2013. drp was diagnosed by ophthalmoscopic examination through fundoscopic examination and slit lamp microscopic examination with present lens. dnp was defined as albumin excretion rate (aer) > 30 mg/24 h and without coexisting renal diseases from causes other than diabetes ; and no dnp was defined at an aer 0.05) (table 1). t2d patients with dnp presented significantly increased fasting and postprandial glucose levels and hba1c%. as seen in the drp group, patients with nephropathy were older and the bmi was lower than the group without this complication (p 10 years were the most frequent group presenting dnp (p = 0.001). no significant differences were observed for gender, hypertension, and dyslipidemia frequencies between dnp positive and negative patients, similarly to the results for the drp group reported above (table 2). regarding dnr, we observed a significant difference concerning age and gender between the groups presenting this complication or not (p = 0.012 and p = 0.003, resp.). the other variables were not significantly different between dnr positive and negative patients (p > 0.05), although a trend toward more dyslipidemic individuals was observed in the dnr group (p = 0.079) (table 3). next, we performed an analysis of genotype and allele frequencies involving a large number of polymorphisms for cytokine genes including tnf-, il-10, tgf-1, il-6, and inf- for all complication groups. we found that all polymorphisms were under hardy - weinberg equilibrium (p > 0.05 for all). we observed that drp is associated with the gg genotype and g allele in tgf-1 codon 25c / g polymorphism (p = 0.004, or 5.865, and ci 95% 1.68520.414 and p = 0.018, or 3.387, and ci 95% 1.06911.064). the drp patients presented the following genotype and allele frequencies : gg 92.42%, gc 6.06%, cc 1.52%, g 95.45%, and c 4.55%, whereas patients without drp showed the following frequencies : gg 72.22%, gc 27.78%, cc 0%, g 86.11%, and c 13.89%. in addition, dnp is associated with lower frequency of gg genotype in il-10 -1082g / a polymorphism (p = 0.049, or 2.357, and ci 95% 1.6129.076). the frequencies observed for patients with dnp, were gg 8.75%, ga 50.00%, and aa 41.25%, and patients without dnp, gg 25.00%, ga 25.00%, and aa 50.00%. no other polymorphism was associated with drp, dnp, or dnr (see table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/605965). since il-10 -1082g / a polymorphism showed association with dnp, an analysis of haplotype frequencies of il-10 polymorphisms (-1082g / a, -819t / c, and -512c / a) was performed in the patients grouped according to the t2d complications. however, no haplotype was associated with drp, dnp, or dnr (table 4). interestingly, individuals who presented the aa genotype in ifn- + 874t / a polymorphism presented lower fasting glucose levels when compared to the other genotypes : tt [median (iqr) = 146.0 (106.0) mg / dl ], ta [134.0 (65.0) mg / dl ], and aa [114.0 (53.5) mg / dl ] ; [tt ta (p = 0.783) ; tt aa (p = 0.045) ; ta aa (p = 0.015) ]. the patients also were evaluated according to the presence of comorbidities such as hypertension, dyslipidemia, and bmi categories concerning the presence of the same polymorphisms. the analysis of the genotype frequencies revealed the association of hypertension with the cc genotype and c allele for il-10 -592c / a polymorphism (p = 0.013, or 9.400, and ci 95% 1.50958.568 and p = 0.009, or 3.830, and ci 95% 1.20612.546, resp.). the frequencies observed were patients with hypertension (cc 50.00%, ca 39.36%, aa 10.54%, c 69.68%, and a 30.32%) and patients without hypertension (cc 25.00%, ca 25.00%, aa 50.00%, c 37.50%, and a 62.50%). a higher frequency of t allele for the tgf-1 codon 10t / c (p = 0.047, or 2.810, and ci 95% 1.8909.148) and c allele for the il-10 819t / c (p = 0.033, or 0.338, and ci 95% 1.1071.154) was also observed in patients with hypertension. moreover, a significant association was found between the polymorphism tgf-1 codon 10t / c and bmi groups (p = 0.026, or 1.963 and ci 95% 1.0073.826 and or 1.546 and ci 95% 1.8582.786). the cc genotype was more frequent in the group with bmi < 25 kg / m, while the tc genotype was more frequent in the group with bmi 30 the frequencies observed were bmi < 25 kg / m (tt 26.32%, tc 42.10%, and cc 31.58%), bmi 2530 kg / m (tt 48.00%, tc 40.00%, and cc 12.00%), and bmi 30 kg / m (tt 31.04%, tc 62.07%, and cc 6.89%). we also found a trend for association of the polymorphism il-10 -592c / a and the bmi groups (p = 0.058). in this case, the aa genotype was less frequent in the bmi 30 kg / m group. no other polymorphism was associated with hypertension, dyslipidemia, or bmi categories (table 2 in supplementary material). since il-10 -592c / a polymorphism showed an association and a tendency for association with hypertension and bmi groups, respectively, an analysis of haplotype frequencies for il-10 polymorphisms (-1082g / a, -819t / c, and -512c / a) was performed in the patients grouped according to t2d comorbidities. for this analysis, bmi was categorized into two groups : bmi < 30 kg / m and bmi 30 kg / m. however, no haplotype was associated with hypertension, dyslipidemia, and bmi categories (table 4). this study investigated the association of cytokine gene polymorphisms in t2d patients including t2d complications drp, dnp, and dnr, as well as comorbidities commonly observed in these patients : hypertension, dyslipidemia, and obesity. when the patients were grouped according to t2d complications, the clinical and laboratory characteristics revealed, as expected, that drp, dnp, and dnr are more common in older individuals with longer diagnostic t2d, because of their association with disease progression. significant difference in the levels of postprandial glucose was observed in the drp and dnp cases when compared to the t2d group without complications. these results suggest a relationship between the hyperglycemic status and the development of microvascular complications of t2d. although higher levels of fasting glucose, postprandial glucose, and hba1c were observed in patients with dnr, these levels were not significantly different from those found in t2d individuals without this complication. the small sample of individuals presenting dnr may explain the lack of significance concerning these variables in this group. patients with drp and dnp showed lower bmi when compared to individuals without these complications. these findings seem contradictory, because obesity is considered a main risk factor for t2d and its complications. this inconsistency may be explained by the fact that individuals with higher bmi were also those with shorter t2d onset and therefore presented fewer complications (data not shown). curiously, a higher frequency of women was observed in the dnr group, although the reason underlying this observation is not clear. studies involving a much larger sample may be necessary to confirm this finding and explore the possible causes for this observation. drp is the most frequent cause of newly detected cases of blindness in adults. in the present study, drp was associated with the gg genotype and g allele of tgf-1 codon 25c / g polymorphism. this polymorphism is located in the region of the gene encoding the signal peptide and causes a change in the amino acid sequence (g / arg c / pro). tgf-1 modulates ocular cell migration and proliferation by inducing fibroblast growth factor - like and platelet - derived growth factor, which accelerate the process of retinal neovascularization. tgf-1 is also involved in extracellular matrix deposition (an essential step in new vessel formation) and stimulates angiogenesis in patients with ischemia and proliferative drp. another case - control study with t2d patients with diabetic proliferative retinopathy found an association of the polymorphisms tgf-1 codon 25c / g (g allele) and tgf-1 codon 10t / c (t allele) with proliferative diabetic retinopathy. on the contrary, a recent meta - analysis found an association between tgf-1 codon 10t / c polymorphism and drp. although the present study did not find significant association with other polymorphisms, the studies published by paine. [18, 31 ] showed the association of the polymorphisms il-10 -1082g / a and ifn- + 874t / a with proliferative diabetic retinopathy. this discrepancy may be due to the genetic background of the populations studied since paine. dnp is a common cause of end - stage renal disease and the major cause of morbidity and premature mortality in patients with t2d. structurally, dnp is characterized by renal hypertrophy, mesangial matrix expansion, glomerulosclerosis, and tubulointerstitial fibrosis. recently, it has become evident that chronic inflammatory mechanisms contribute to the development and progression of dnp, such as infiltration of renal compartments by lymphocytes and monocytes (or macrophages) as well as local production of cytokines and chemokines in the kidney [41, 42 ]. studies have observed that acute phase markers of inflammation (c reactive protein, fibrinogen, and il-6) were correlated to proteinuria in t2d patients [43, 44 ] and showed that increased tnf- levels were linked with dnp progression. in our study, dnp was associated with lower frequency of gg genotype polymorphism il-10 -1082g / a, which is related with higher expression of this cytokine. in fact, il-10 is an anti - inflammatory cytokine and downregulates proinflammatory production of tnf-, il-6, and mcp-1. thereby, lower production of il-10 may be associated with a high production of proinflammatory cytokines and an exacerbated inflammatory response with subsequent renal injury in t2d patients. ezzidi., in a case - control study with t2d patients from tunisia, found a higher frequency of t allele (il-10 -819t / c) in the group with dnp.. found higher frequency of the genotypes aa and cc for the il-10 -592c / a polymorphism in individuals with the dnp in a taiwanese population. in contrast to the current results, no association was found between the il-10 -1082g / a polymorphism and dnp in patients from turkey. the polymorphism tgf-1 codon 10t / c was associated with dnp risk in a meta - analysis conducted by zhou.. thus, it is possible that the different polymorphisms may reflect the genetic background of the population studied. ifn- is a pivotal proinflammatory cytokine implicated in the induction of immune mediated inflammatory response. studies suggest that ifn- participates in the pathogenesis of diabetes mellitus by upregulating the expression of mhc i / mhc ii antigens and adhesion molecules on pancreatic cells [4951 ]. herein we report an association of the aa genotype polymorphism ifn- + 874t / a with lower glucose levels than those presented by patients carrying other genotypes. it is known that the allele + 874 t is associated with high ifn- levels, whereas the allele + 874a is associated with low production of this cytokine. thus, decreased expression of ifn- may contribute to the downregulation inflammatory response in t2d patients and consequently to allowing better glycemic control. it is known that obesity and visceral fat contribute to the development of hypertension, insulin resistance, and diabetes mellitus. typically, hypertension is a clinical condition commonly present in the patient at diagnosis of t2d, and the elevation of blood pressure often occurs before the onset of microalbuminuria. in our study we found an association between the cc genotype and c allele for il-10 -592c / a polymorphism and hypertension ; furthermore we found a trend for association of the same polymorphism and bmi groups, since the -592aa genotype was less frequent in the obese t2d patients. in addition, we observed an association of the c allele for the il-10 -819t / c polymorphism and patients with hypertension. for both il-10 polymorphisms, reported that increased il-10 levels were associated with improved systemic endothelial vasoreactivity in patients with elevated serum crp levels, a condition commonly observed in t2d patients. furthermore, zeyda. demonstrated that human adipose tissue macrophages (atms) produce high levels of il-10. consequently, increased levels of il-10 could be associated with an exacerbated inflammatory profile in which the balance between pro- and anti - inflammatory cytokines contributes to chronic low - grade inflammation observed in obesity and t2d, as well as being involved in increased blood pressure. the cc genotype was more frequent in the group with bmi < 25 kg / m, while the tc genotype was more frequent in the group with bmi 30 kg / m. this polymorphism consists of a t c transition at nucleotide 29 in the region encoding the signal peptide sequence, which results in a leu pro substitution at amino acid 10. studies have shown that the c allele increases the production of the tgf-1 protein [55, 56 ]. the tgf-1 is a multifunctional cytokine and shows anti - inflammatory action such as suppressing generation of free radicals, as well as vasculoprotective properties. tgf-1 can inhibit the adhesion and transmigration of neutrophils and t cells to the endothelium, inhibit production of adhesion molecules by the endothelial cells, and inhibit macrophage foam cell formation [5861 ]. the higher frequency of the cc genotype found in patients with a bmi < 25 kg / m suggests that the increased expression of tgf-1 and its potential anti - inflammatory effect can facilitate the control of adiposity, since obese individuals have higher frequency tc genotype. a higher frequency of t allele for the tgf-1 codon although some studies have demonstrated that tgf-1 is associated with increased risk of essential hypertension through the stimulation of endothelin-1 expression in the vascular endothelium, release of renin from the juxtaglomerular cells in the kidney, and regulation of angiotensin ii expression [6264 ], no study has evaluated the impact of this polymorphism on hypertension in patients with diabetes. thus, our results suggest that lower expression of tgf-1 could predispose to diabetic hypertension due to lack of anti - inflammatory and protective tgf-1 effects in vascular endothelium. in conclusion, this is the first study to evaluate the association of a large panel of polymorphisms of cytokine genes with complications and comorbidities in t2d patients. however, the small sample of this study is considered a limitation and further studies including clinical classifications concerning nonproliferative / proliferative drp and autonomic / chronic sensorimotor dnr may improve our current understanding about the link between the cytokine polymorphisms, their expression levels, and the development or progression of these complications. although the results are not conclusive regarding the association of polymorphisms of cytokine genes with microvascular complications in t2d and comorbidities, taken together, our results may be relevant for future molecular studies aiming to predict possible t2d complications. | aims. the polymorphisms of pro- and anti - inflammatory cytokines may be involved in type 2 diabetes (t2d) pathogenesis and its complications. methods. we investigated in 102 t2d patients the association of the cytokine polymorphisms in the tnf-, il-10, il-6, tgf-1, and ifn- genes with the t2d microvascular complications and comorbidities (hypertension, dyslipidemia, and obesity). cytokine genotypes were determined by pcr using cytokine genotyping tray kit. results. diabetic retinopathy was associated with gg genotype and g allele in tgf-1 codon 25c / g polymorphism (p = 0.004 and p = 0.018) and the nephropathy was associated the lower frequency of gg genotype in il-10 -1082g / a polymorphism (p = 0.049). hypertension was associated with the cc genotype and c allele for il-10 -592c / a polymorphism (p = 0.013 and p = 0.009) and higher frequencies of t (p = 0.047) and c (p = 0.033) alleles of the tgf-1 codon 10t / c and il-10 -819t / c polymorphisms, respectively. the tgf-1 codon 10t / c polymorphism was associated with the bmi groups (p = 0.026) : the cc genotype was more frequent in the group with bmi < 25 kg / m2, while the tc genotype was more frequent in the group with bmi = 30 kg / m2. conclusions. our findings suggest that tgf-1 and il-10 polymorphisms are involved in complications and comorbidities in t2d patients. |
down syndrome (ds) is one of the most frequent congenital disorders in humans. many of them develop symptoms of alzheimer s disease (ad) at a relatively young age, and a high proportion of ds individuals develop dementia at a later stage. ds is caused by a trisomy of chromosome 21 as a consequence of nondisjunction during meiotic cell division. although the mechanisms leading from trisomy 21 to the clinical disease pattern are not yet understood, the 30 genes located in the so - called down syndrome critical region (dscr ; 21q22.122.3) of chromosome 21 are highly likely to be associated with the disease. there is mounting evidence that overexpression of dyrk1a (dual - specificity tyrosine phosphorylation - regulated kinase 1a), a protein kinase encoded by a gene located within dscr, contributes to mental retardation in ds. since dyrk1a is also connected to neurological disorders such as ad, the early onset of ad in ds individuals has been related to dyrk1a overexpression. two histopathological features are found in the brains of ad patients : extracellular -amyloid plaques and intracellular tangles consisting of hyperphosphorylated tau protein. hyperphosphorylation of tau by dyrk1a diminishes its microtubule - stabilizing effects and increases aggregation. the neurological impairment of mice with modified dyrk1a expression has also been attributed to deregulated splicing, leading to an imbalance of 3r - tau and 4r - tau isoforms. the increased 3r - tau concentration (by a factor of up to 4) observed in ds has been associated with changes of the neuronal cytoskeleton and neurofibrillary degeneration. dyrk1a - overexpressing mice also exhibit increased production of a peptide, which has been attributed to phosphorylation of the amyloid precursor protein (app) and presenilin 1 by dyrk1a. dyrk1a is a member of the dyrk protein kinases (dyrk1a, dyrk1b, dyrk2, dyrk3, dyrk4), which are part of the cmgc superfamily and share structural similarity of the catalytic domain and a small sequence nearby, the so - called dyrk homology box (dh - box). although dyrks are serine / threonine kinases, autophosphorylation occurs at a conserved tyrosine residue of the activation loop. this autophosphorylation is constitutive and seems to be not related to regulation. in the light of its involvement in ds and other neurodegenerative disorders, the inhibition of dyrk1a with small chemical inhibitors has been suggested as a therapeutic strategy. selective dyrk1a inhibitors would also be valuable tools for the investigation of the role of dyrk1a in physiological and pathobiochemical processes. to date, most protein kinase inhibitors exert their activity by competing with atp in the binding pocket of the kinase. since atp binding sites of protein kinases share a common structure with subtle differences, selectivity with regard to closely related kinases is not easily achieved. when selective dyrk1a inhibitors are to be developed, special attention should therefore be devoted to other members of the cmgc superfamily, consisting of cdks, mapkinases (such as erks), gsk-3, and cdc - like kinases. the structures and properties of dyrk1a inhibitors have been summarized in recent reviews. a well established dyrk1a inhibitor is the -carboline alkaloid harmine (1, chart 1), which however also shows high affinity for serotonine and tryptamine receptor binding sites, acts as monoamine oxidase a (mao a) inhibitor, and also inhibits clks and therefore is inappropriate for use as a cellular chemical probe. epigallocatechin 3-gallate (egcg) is a polyphenolic constituent of green tea reported to inhibit dyrk1a in a non - atp - competitive manner, but it is chemically reactive and also interacts with numerous intracellular signaling pathways by other mechanisms. the benzothiazole derivative indy showed potent inhibition of dyrk1a and related kinases (dyrk1b, dyrk2, dyrk3, clk1, clk4, ck1, and pim1). a prodrug of this compound, proindy (2), has been shown to protect xenopus tadpoles that overexpress dyrk1a against head malformation during development. leucettine l41 (3), derived from the marine natural product leucettamine b, is an atp - competitive inhibitor of dyrks and clks that also interacts with gsk-3 and ck2 to a lower extent. it modulates pre - mrna splicing, protects ht22 hippocampal cells from glutamate - induced cell death, induces autophagy, and inhibits phosphorylation of tau on thr212. similar to the agents mentioned before, the recently reported chemical dyrk1a inhibitors comprising meridianines, indirubin 5-carboxylates, thiazolo[5,4-f]quinazolines, pyrido[2,3-d]pyrimidines, 3,5-diaryl-7-azaindoles (dandys), kh - cb19, 2,4-bisubstituted thiophenes, and hydroxybenzothiophenes either show limited selectivity against structurally closely related dyrk and clk kinase isoforms or were not tested on these enzymes. in this regard, a dyrk1a inhibitor with high selectivity over other cmgc kinases would be useful for biochemical and cellular studies and as lead motif for the development of new pharmaceuticals targeting neurodegenerative diseases. to identify novel hit matter, we tested a small diverse in - house compound library against dyrk1a and the following cmgc protein kinases : cdk1/cyclin b, cdk2/cylin a, cdk5/p25, ck1, gsk-3, and erk2. the only compound showing a moderate selective dyrk1a inhibition (ic50 = 2.6 m) was the 11h - indolo[3,2-c]quinoline-6-carboxylic acid 5a (table 1). experimental dyrk1a inhibitors described so far have suffered from poor selectivity toward other closely related protein kinases, namely, other dyrks and members of the clk family. we have developed two 10-iodo-11h - indolo[3,2-c]quinoline-6-carboxylic acids 5j and 5o as the first selective inhibitors of dyrk1a. docking studies and x - ray structure analyses revealed a nonclassical binding mode in which inhibitors were oriented via the 10-halogen substituent toward the hinge region of the kinase. although the 10-substituent obviously was of central importance for the hinge inhibitor attraction, the nature of this interaction is not yet clear. the title compounds demonstrate that within the cmgc family of protein kinases selective inhibition of dyrk1a is possible. with the aim of developing compounds that are better suited for cellular assays or animal disease models, further studies in this compound class will be directed to increase solubility and cell permeability. starting materials were purchased from acros organics (geel, belgium) or sigma - aldrich (steinheim, germany). solvents were used as commercially available grades for synthesis, with the exceptions of toluene, thf, ch2cl2, and diethyl ether, which were dried and purified by published methods.5b, 5c, and 5f were prepared as reported previously. synthetic procedures and structure characterization data for the following compounds are available in the supporting information : 5a, 5d e, 5g n, 5p w, 6, 7, 10n, s, t. melting points (mp) were determined on an electric variable heater (electrothermal ia 9100, barnstedt international, southend - on - sea, u.k.) in open glass capillaries. ir spectra were recorded as kbr discs on a thermo nicolet ft - ir 200. h nmr spectra and c nmr spectra were recorded on the following instruments : bruker avance drx-400, bruker avance iii-400, and bruker avance ii-600 (bruker, billerica, ma, usa) ; internal standard tetramethylsilane ; signals in ppm (scale). signals in c spectra were assigned based on the results of c dept135 experiments (nmr laboratories of the chemical institutes of the technische universitt braunschweig). elemental analyses were determined on a ce instruments flashea 1112 elemental analyzer (thermo quest). mass spectra were recorded on a finnigan - mat 95 (thermo finnigan mat, bremen, germany). accurate measurements were conducted according to the peak match method using perfluorokerosene (pfk) as an internal mass reference. (ei) ms : ionization energy 70 ev (department of mass spectrometry of the chemical institutes of the technische universitt braunschweig). tlc : polygram sil g / uv254, macherey - nagel, 40 mm 80 mm, visualization by uv illumination (254 and 366 nm). isocratic elution : elite lachrom (merck / hitachi), pump l-2130, autosampler l-2200, diode array detector l-2450, organizer box l-2000 ; column, merck lichrocart 1254, lichrosphere 100, rp 18, 5 m ; flow rate 1.000 ml / min ; volume of injection, 10 l ; detection (dad) at 254 and 280 nm ; auc, % method ; time of detection 15 min, net retention time (tn), dead time (tm) related to dmso. gradient elution : elite lachrom (merck / hitachi), pump l-2130, autosampler l-2200, uv detector l-2400, organizer box l-2000 ; column, merck lichrocart 125 - 4, lichrosphere 100, rp 18, 5 m ; flow rate 1.000 ml / min ; volume of injection, 10 l ; detection at 254 nm ; auc, % method ; net retention time (tn), dead time (tm) related to dmso. for all gradient runs, mixtures of acn and water or aqueous formic or trifluoroacetic acid were used as specified for particular compounds. preparation of h2o + (et3nh)2so4 buffer (ph 2.6) for isocratic hplc : triethylamine (20.0 ml) and sodium hydroxide (242 mg) are dissolved in water to 1 l. the solution is adjusted to ph 2.6 by addition of sulfuric acid. all compounds that were biologically tested were of > 95% purity with the exception of 5h (hplc, 93.6% at 254 nm and 94.8% at 280 nm detection wavelength) and 5w (hplc, 95.1% at 254 nm and 94.2% at 280 nm detection wavelength). absorption maxima (max) were extracted from the spectra recorded by the dad in the hplc peak maxima in isocratic runs (software, ez chrom elite client / server, version 3.1.3.). an appropriate substituted 3,4-dihydro-1h-1-benzazepine-2,5-dione (1 equiv) and an appropriately substituted phenylhydrazine hydrochloride (1.21.5 equiv) and sodium acetate (1.21.5 equiv) are stirred in acetic acid (10 ml) at 70 c for the indicated time. sulfuric acid (0.1 ml) is added, and stirring at 70 c is continued for the indicated time. after cooling to room temperature, the mixture is poured into 5% aqueous sodium acetate solution (20 ml) and kept at 8 c for 2 h. a precipitate is formed, which is filtered off with suction, washed with water and petrol ether. the appropriate paullone 4 (1 equiv), nhpi (2 equiv), and cobalt(ii) acetate tetrahydrate (0.25 equiv) are dissolved in the given volume of dmf. oxygen is bubbled through the stirred mixture for the given time at the indicated temperature. if no precipitate appears, water (5 ml) is added and the resulting precipitate is separated and purified by centrifugation, decanting, and washing with water. the resulting solid is washed with a small amount of acetone. the predried product is finally dried at 130 c in vacuo. ethanol (100 ml) is added to a solution of an appropriate 11h - indolo[3,2-c]quinoline-6-carboxylic acid 5 in dmso (5 ml). hydrogen chloride gas is bubbled through the solution for 30 min at room temperature. after removal of the ethanol by evaporation, water (100 ml) is added and the mixture is kept at 8 c for 1 h. the resulting precipitate is filtered off, washed with water, and dissolved in acetone (5 ml). after addition of silica gel (2 g), the mixture is evaporated. the product is subsequently eluted from the silica gel by flash chromatography using the indicated eluent. synthesis was according to general procedure a from 3,4-dihydro-1h-1-benzazepine-2,5-dione (88 mg, 0.50 mmol), 2-iodophenylhydrazine hydrochloride (203 mg, 0.750 mmol), and sodium acetate (62 mg, 0.75 mmol). reaction times were 1 h before and 4 h after addition of sulfuric acid, respectively. formic acid 5:5:1) yielded a dark yellow powder (51 mg, 28%) ; mp (dec) starting at 288 c. ir (kbr) : 3194 cm (nh), 1666 cm (c = o). h nmr (dmso - d6, 400.4 mhz) : (ppm) = 3.47 (s, 2h), 6.90 (t, 1h, j = 7.6 hz), 7.227.31 (m, 2h), 7.40 (ddd, 1h, j = 7.9/7.4/1.6 hz), 7.59 (dd, 1h, j = 7.5/0.9 hz), 7.71 (d, 1h, j = 7.9 hz), 7.91 (dd, 1h, j = 7.8/1.5 hz), 10.11 (s, 1h, lactam nh), 11.29 (s, 1h, indole nh). c nmr (dmso - d6, 100.7 mhz) : (ppm) = 31.6 (ch2) ; 118.0, 121.1, 122.1, 123.3, 128.3, 128.5, 131.5 (ch) ; 76.7, 109.4, 122.4, 127.1, 133.6, 135.6, 139.4 (c) ; 171.7 (c = o). c16h11in2o (374.18). ms (ei) : m / z (%) = 374 [m ] (100), 373 [m h ] (35), 345 [m cho ] (49). hrms (ei) : m / z [m ] calcd 373.991 06, found 373.990 84. hplc (isocr) : 96.1% at 254 nm and 92.9% at 280 nm, tn = 4.7 min, tm = 1.0 min (acn / h2o 50:50) ; max : 230 and 313 nm. synthesis was according to general procedure a from 8-methoxy-3,4-dihydro-1h-1-benzazepine-2,5-dione (500 mg, 2.44 mmol), 2-iodophenylhydrazine hydrochloride (993 mg, 3.67 mmol), and sodium acetate (301 mg, 3.67 mmol). reaction times were 3 h before and 16 h after addition of sulfuric acid, respectively. purification by column chromatography (toluene ethyl acetate formic acid 5:5:1) yielded a dark yellow powder (340 mg, 34%) ; mp (dec) starting at 290 c. ir (kbr) : 3205 cm (nh), 1680 cm (c = o). h nmr (dmso - d6, 600.1 mhz) : (ppm) = 3.47 (s, 2h), 3.81 (s, 3h), 6.82 (d, 1h, j = 2.6 hz,), 6.88 (t, 1h, j = 7.7 hz), 6.91 (dd, 1h, j = 8.7/2.6 hz), 7.55 (dd, 1h, j = 7.5/0.9 hz), 7.67 (d, 1h, j = 7.8 hz), 7.85 (d, 1h, j = 8.7 hz), 10.04 (s, 1h, lactam nh), 11.19 (s, 1h, indole nh). c nmr (dmso - d6, 150.9 mhz) : (ppm) = 55.2 (ch3), 31.6 (ch2), 106.6, 109.9, 117.6, 121.0, 129.6, 130.9 (ch), 76.5 (c(11)), 107.6, 115.3, 127.1, 133.8, 136.9, 139.0, 159.2 (c). c 50.51, h 3.24, n 6.93, found c 50.38, h 3.29, n 6.72. ms (ei) : m / z (%) = 404 [m ] (100), 403 [m h ] (36), 375 [m cho ] (42). hrms (ei) : m / z [m ] calcd 404.001 62, found 404.001 61. hplc (isocr) : 96.0% at 254 nm and 95.2% at 280 nm, tn = 5.2 min, tm = 1.0 min (acn / h2o 50:50). synthesis was according to general procedure b1 from 11-iodo-7,12-dihydroindolo[3,2-d]benzazepin-6(5h)-one (4j, 43 mg, 0.11 mmol), nhpi (36 mg, 0.22 mmol), and cobalt(ii) acetate tetrahydrate (7 mg, 0.03 mmol) in dmf (2 ml). oxygen was bubbled through the reaction mixture at room temperature for 4 h. a yellow powder was formed, which was purified by washing with acetone (34 mg, 82%) ; mp (dec) 358359 c. ir (kbr) : 3224 cm (nh), 1659 cm (c = o). h nmr (dmso - d6, 400.4 mhz) : (ppm) = 7.10 (t, 1h, j = 7.7 hz), 7.357.42 (m, 1h), 7.567.62 (m, 2h), 7.82 (dd, 1h, j = 7.7/1.0 hz), 8.30 (dd, 1h, j = 8.0/1.0 hz), 8.38 (d, 1h, j = 8.0 hz), 11.54 (s, 1h, nh), 11.95 (s, 1h, cooh). c nmr (dmso - d6, 100.7 mhz) : (ppm) = 121.2 (2c), 123.4, 124.4, 129.2, 131.1, 134.8 (ch) ; 76.7 (c(10)), 115.5, 117.2, 127.3, 135.2, 139.3, 142.5, 161.8 (c) ; 176.3 (c = o). ms (ei) : m / z (%) = 388 [m ] (37), 360 [m co ] (100). hrms (ei) : m / z [m ] calcd 387.970 32, found 387.968 99. hplc (isocr) : 96.7% at 254 nm and 98.6% at 280 nm, tn = 2.8 min, tm = 1.0 min (acn / h2o + (et3n)2so4 50:50). hplc (grad) : 97.4% at 254 nm, tn = 10.0 min, tm = 1.1 min (acn / h2o ; 0 min, 10/90 13 min, 90/10 (linear) ; 20 min, 90/10). synthesis was according to general procedure b1 from 11-iodo-3-methoxy-7,12-dihydroindolo[3,2-d]benzazepin-6(5h)-one (4o, 40 mg, 0.10 mmol), nhpi (33 mg, 0.20 mmol), and cobalt(ii) acetate tetrahydrate (7 mg, 0.03 mmol) in dmf (2 ml). oxygen was bubbled through the reaction mixture for 1 h. for the purification by column chromatography, impurities were removed initially by elution with toluene ethyl acetate diethylamine (1:1:1) prior to elution by toluene ethyl acetate yellow powder (20 mg, 48%) ; mp (dec) 346348 c. ir (kbr) : 3200 cm (nh), 1669 cm (c = o). h nmr (dmso - d6, 600.1 mhz) : (ppm) = 3.86 (s, 3h), 7.01 (dd, 1h, j = 9.0/2.6 hz), 7.07 (t, 1h, j = 7.7 hz), 7.17 (d, 1h, j = 2.5 hz), 7.78 (dd, 1h, j = 7.5/0.9 hz), 8.31 (dd, 1h, j = 7.9/0.7 hz), 8.36 (d, 1h, j = 9.0 hz), 11.44 (s, 1h, nh), 11.76 (s, 1h, cooh). c nmr (dmso - d6, 150.9 mhz) : (ppm) = 55.5 (ch3) ; 104.9, 110.6, 121.1, 124.2, 130.8, 134.4 (ch2) ; 76.3, 110.3, 114.2, 127.5, 137.0, 139.1, 143.0, 161.1, 161.5 (c) ; 175.5 (c = o). ms (ei) : m / z (%) = 418 [m ] (47), 390 [m co ] (100), 375 [m 43 ] (24), 347 [m 71 ] (22). hrms (ei) : m / z [m ] calcd 417.980 89, found 417.981 26. hplc (isocr) : 96.3% at 254 nm and 97.7% at 280 nm, tn = 3.2 min, tm = 1.0 min (acn / h2o + (et3n)2so4 50:50). hplc (grad) : 99.4% at 254 nm, tn = 10.8 min, tm = 1.2 min (gradient, acn / h2o ; 0 min, 10/90 13 min, 90/10 (linear) ; 20 min, 90/10). synthesis was according to general procedure c from 10-iodo-3-methoxy-11h - indolo[3,2-c]quinoline-6-carboxylic acid (5o, 120 mg, 0.300 mmol). diethylamine 2:2:1) yielded pale yellow crystals (100 mg, 75%) ; mp 214217 c. ir (kbr) : 3384 (nh), 1726 cm (c = o). h nmr (dmso - d6, 400.4 mhz) : (ppm) = 1.45 (t, 3h, j = 7.1 hz), 3.99 (s, 3h), 4.59 (q, 2h, j = 7.1 hz), 7.14 (t, 1h, j = 7.8 hz), 7.46 (dd, 1h, j = 9.1/2.6 hz), 7.63 (d, 1h, j = 2.6 hz), 7.93 (dd, 1h, j = 7.6/1.0 hz), 8.33 (dd, 1h, j = 8.1/0.9 hz), 9.05 (d, 1h, j = 9.1 hz), 12.34 (s, 1h, nh). c nmr (dmso - d6, 100.7 mhz) : (ppm) = 14.2 (ch2ch3) ; 55.5 (och3) ; 61.7 (ch2) ; 108.7, 119.1, 122.0, 122.5, 124.7, 135.1 (ch) ; 76.8, 111.2, 111.8, 121.4, 141.1, 142.0, 145.1 146.0, 160.1 (c) ; 166.7 (c = o). c19h15in2o3 (446.24) ; calcd c 51.14, h 3.39, n 6.28, found c 51.21, h 3.41, n 6.01. ms (ei) : m / z (%) = 446 [m ] (22), 374 [m c3h4o2 ] (100). hplc (isocr) : 99.7% at 254 nm and 99.8% at 280 nm, tn = 7.6 min, tm = 1.0 min (acn / h2o 80:20). hplc (grad) : 99.7% at 254 nm, tn = 14.1 min, tm = 1.1 min (gradient, acn / h2o ; 0 min, 10/90 13 min, 90/10 (linear) ; 20 min, 90/10). crystals for x - ray structure analysis were prepared by crystallization from ethanol (96%). crystal data for 10oh2o at 100 k : orthorhombic, p212121, a = 6.36362(12), b = 15.0401(3), c = 18.3209(4), v = 1753.48, z = 4. a yellow irregular crystal 0.3 mm 0.2 mm 0.15 mm was used to record 76097 intensities, 5096 independent (rint = 0.036) on an oxford diffraction xcalibur e diffractometer using mo k radiation (= 0.710 73, 2max = 60). the structure was refined anisotropically on f using the program shelxl-97 to wr2 = 0.049, r1 = 0.020 for 249 parameters ; s = 1.12, max. crystallographic data have been deposited with the cambridge crystallographic data centre as supplementary publication no. buffer a consisted of 10 mm mgcl2, 1 mm egta, 1 mm dtt, 25 mm tris - hcl, ph 7.5, 50 g heparin / ml. buffer c consisted of 60 mm -glycerophosphate, 15 mm p - nitrophenylphosphate, 25 mm mops (ph 7.2), 15 mm egta, 15 mm mgcl2, 1 mm dtt, 1 mm sodium vanadate, 1 mm phenylphosphate. kinase activities were assayed in buffer a or c at 30 c at a final atp concentration of 15 mol / l. blank values were subtracted, and activities were expressed in percent of the maximal activity, i.e., in the absence of inhibitors. the gs-1, cks, cdk7/9 tide, and rs peptide substrates were obtained from proteogenix (oberhausbergen, france). cdk1/cyclin b (m phase starfish oocytes, native), cdk2/cyclin a, and cdk5/p25 (human, recombinant) were prepared as previously described. their kinase activity was assayed in buffer a, with 1 mg of histone h1/ml, in the presence of 15 mol / l [-33p ] atp (3000 ci / mmol ; 10 mci / ml) in a final volume of 30 l. after a 30 min incubation at 30 c, the reaction was stopped by harvesting onto p81 phosphocellulose supernatant (whatman) using a filtermate harvester (packard) and washing in 1% phosphoric acid. cdk9/cyclin t (human, recombinant, expressed in insect cells) was assayed as described for cdk1/cyclin b but using cdk7/9 tide (ysptspsysptspsysptspskkkk) (8.1 g / assay) as a substrate. gsk-3 (porcine brain, native, affinity purified on axin1-sepharose beads) was assayed, as described for cdk1, with 0.5 mg of bsa / ml + 1 mm dtt and using a gsk-3 specific substrate (gs-1, yrraavppspslsrhssphqspedeee) (ps stands for phosphorylated serine). ck1/ (porcine brain, native, affinity purified on axin2-sepharose beads) was assayed as described for cdk1 but in buffer c and using 25 m cks peptide (rrkhaaigpsaysita), a ck1-specific substrate. clk1, -2, -3, and -4 (mouse, recombinant, expressed in e. coli as gst fusion proteins) were assayed as described for cdk1/cyclin b with 0.5 mg bsa / ml + 1 mm dtt and rs peptide (grsrsrsrsrsr) (1 g / assay) as a substrate. dyrk1a, -1b, -2, -3 (human, recombinant, expressed in e. coli as gst fusion proteins) and clk1, -2, -3, and -4 (mouse, recombinant, expressed in e. coli as gst fusion proteins) were assayed in buffer a (supplemented extemporaneously with 0.15 mg of bsa / ml + 1 mm dtt) with 1 g of rs peptide (grsrsrsrsrsr) as a substrate. response curves were recorded in triplicate. typically, the standard deviation of single data points was below 10%. the assay for inhibition of sf3b1 phosphorylation was performed as described previously. for the assay of tau phosphorylation, we used a hek293 subclone with regulatable expression of gfp - dyrk1a and constitutive expression of gfp - tau (hek293-tau - dyrk1a) that was kindly provided by dr. matthias engel (department of pharmaceutical and medicinal chemistry, saarland university, saarbrcken, germany). cells were grown overnight in six - well plates before expression of gfp - dyrk1a was induced with 2 g / ml doxycyclin. the inhibitors were then added from stock solutions in dmso to the desired final concentration and cells were further incubated for 20 h. cells were lysed in sds lysis buffer (20 mm tris - hcl, ph 7.4, 1% sds). samples were sonicated and cleared by centrifugation before sds page and immunoblotting with a goat antibody for gfp (no. 600 - 101 - 215, rockland immunochemicals, gilbertsville, pa, usa) and a phosphorylation state specific antibody directed against pthr212 in the tau protein (no. immunoreactivities were detected by enhanced chemiluminescence using hrp - coupled secondary antibodies and quantified using the aida image analyzer 5.0 program (raytest, straubenhardt, germany). relative tau phosphorylation was calculated by normalization to total tau expression, as determined from gfp immunoreactivity. to calculate relative dyrk1a activity, the basal pt212 signal in control cells not treated with doxycyclin was subtracted from all values, and the phosphorylation in dyrk1a expressing cells not treated with inhibitors was set to 100%. curve fitting for ic50 determination was done with the help of the graphpad prism 5.0 program (graphpad software, la jolla, ca, usa). recombinant dyrk1a was purified as previously described and was treated with tev protease to remove the n - terminal his6 tag. the kinase at 1315 mg / ml in 50 mm hepes, ph 7.5, 500 mm nacl and 5 mm dtt was incubated with the inhibitors at 1 mm prior to crystallization. crystals were obtained using the sitting drop vapor diffusion method at 4 c using either 2 m ammonium sulfate, 0.2 m na / k tartrate, 0.1 m citrate, ph 5.6 (for 5s and 5 t), or 37% peg 400, 0.2 m lithium sulfate, 0.1 m tris, ph 8.8 (for 5j), as the reservoir solutions. diffraction data collected at diamond light source, beamline i04 - 1, were processed with xds or mosflm and subsequently scaled with scala from ccp4 suite. structures were determined by molecular replacement method using phaser and the coordinates of dyrk1a structure as a search model. iterative cycles of manual model building in coot alternated with refinement using refmac, and a tls model calculated from tlsmd server was performed. dyrk1a - ligand complexes (pdb i d) : dyrk1a-5j (4ylj) ; dyrk1a-5s (4ylk) ; dyrk1a-5 t (4yll). molecular docking was performed using gold, version 4.0, running under linux ubuntu dapper drake. the poses obtained with gold 4.0 were later reproduced using gold, version 5.1. the pdb file 2wo6 was downloaded from the protein data bank. only the b chain present in the structure hydrogen atoms were added, and the protonation / tautomerization status of amino acid side chains within the atp binding site was checked and adjusted manually, if necessary. a zone of 10 around the original ligand was defined as relevant for binding. the options flip side chains of asn / gln and alter tautomers of his were switched off. ligands were constructed with moe, energy minimized, and saved as mol2 files. the option redocking of the originally bound ligand djm2005 into chain b of the protein reproduced the original pose of the inhibitor, albeit not as highest ranked pose. | the protein kinase dyrk1a has been suggested to act as one of the intracellular regulators contributing to neurological alterations found in individuals with down syndrome. for an assessment of the role of dyrk1a, selective synthetic inhibitors are valuable pharmacological tools. however, the dyrk1a inhibitors described in the literature so far either are not sufficiently selective or have not been tested against closely related kinases from the dyrk and the clk protein kinase families. the aim of this study was the identification of dyrk1a inhibitors exhibiting selectivity versus the structurally and functionally closely related dyrk and clk isoforms. structure modification of the screening hit 11h - indolo[3,2-c]quinoline-6-carboxylic acid revealed structure activity relationships for kinase inhibition and enabled the design of 10-iodo - substituted derivatives as very potent dyrk1a inhibitors with considerable selectivity against clks. x - ray structure determination of three 11h - indolo[3,2-c]quinoline-6-carboxylic acids cocrystallized with dyrk1a confirmed the predicted binding mode within the atp binding site. |
world - wide, 13 million people develop sepsis each year, and as many as 4 million people have died. in 1996, there were 4.774 patients admitted to a teaching hospital in surabaya, indonesia, and 504 patients were diagnosed as having sepsis, with a mortality rate of 70.2%. in a study at a teaching hospital in yogyakarta, indonesia, there were 631 cases of sepsis in 2007, with a 48.96% mortality rate. a global effort is needed to improve the therapeutic management of sepsis because of its high prevalence and mortality rate. the therapeutic management of sepsis, including septic shock, requires a comprehensive and systematic approach that includes a diagnostic method, the initiation of empirical antibiotic use and administration of supportive therapy. empirical antibiotic therapy must also consider the site of infection, the common pathogen that caused sepsis and antibiotic sensitivity based on local patterns of antibiotic resistance. failed to define the source of infection will potentially lead to wrong pathogen identified, and will also lead to inappropriate antibiotic selection. the global escalation in both community- and hospital - acquired antimicrobial - resistant bacteria is increasingly compromising effective antimicrobial therapy, particularly when it comes to empiric antimicrobial selection. the appropriate use of an empirical antibiotic is critical to decrease the mortality rate of sepsis and should be started within 1 - 2 h after the diagnosis of severe sepsis. in this study, we analyzed the pattern of antibiotic use in septic patients and the pattern of microbial resistance based on the results of various cultures of microbial specimens from the sepsis patients. the information gained will be critical as a reference for pathogen identification, selection of empirical antibiotic therapy, and policies to control antibiotic resistance, especially in sepsis patients. a retrospective observational study was conducted in a hospital in bandung, indonesia during may to august 2012. adult patients aged 18 - 59 years, who were diagnosed with sepsis when admitted to the hospital from january 1 to december 31, 2011, met the inclusion criteria for the study. the data were collected from the medical records department of the hospital, including the patient identity, diagnosis, co - morbidities, source of infection, results of microbial culture, results of antimicrobial sensitivity testing, antibiotic use, length of stay and clinical outcome. the level of antibiotic resistance was obtained from the results of the microbial cultures and antibiotic sensitivity testing that were conducted at the time of hospitalization from the subject population. the data of antibiotic use were obtained from the medical records of the subject population. culture and sensitivity test procedures were based on the principles of test that published by world health organization. sterile specimen such as blood and pleura fluid, processed by using two medium enrichment (tryptic soy broth and brain heart infusion), then incubated with bact / alert instrument. the specimens which non - steril, such as sputum, pus, and swab were not processed with enrichment medium and incubatation process by bact / alert instrument, but directly to the next step incubation. the next step was incubation process with two different medium (macconkey agar and blood agar) in the temperature 35 - 37c for 18 - 24 h. colonies from the each medium isolated and processed with the vitek 2 compact automated instrument to identified microbe and susceptibility test to antibiotics. manual method was using to anticipate the error of automatic method with modified kirby bauer method. the determination of antibiotics types and sensitivity level of antibiotics in the susceptibility test were based on clsi standard. determination of contaminants or pathogens from the microbial results was based on the clinician 's decision by considering of infection source, clinical condition and microbial results that was not performed in this study. this study was approved by ethics committee of faculty of medicine universitas padjadjaran, and also ethics committee of hasan sadikin hospital, indonesia. a total of 192 patients, 103 males and 89 females, were diagnosed with sepsis during the study period, and 76 patients met the criteria for the study. the sepsis incidence rate was highest in the 55 - 59-year age range with 15 patients, followed by the 45 - 50-year age range with 14 patients. the incidence of sepsis was higher in females than males and the mortality rate from sepsis reached 53.95%. comparing the mortality rate in males and females in the > 50 years age group, the study showed a higher mortality rate in males (40%) than in females (38.46%). in contrast, in the 15 - 50-year age group, the mortality rate in females (65.38%) was higher than in males (51.8%). it 's showed 15 subjects population had discharged against medical advice due to cost reasoning and 1 subject population transferred to other hospital. lungs infection, renal failure, malignancy, diabetes mellitus and intraabdominal infection is the highest co - morbidities in the subject population. in the lung infection groups, the major problem are hospital acquired pneumonia, community acquired pneumonia (cap) and tuberculosis. the highest mortality showed in the subject population who got systemic lupus erythematous, hepatitis, meningitis, myocarditis, and human immunodeficiency virus infection. characteristics of the subject population (n=76) there were 6 sites of infection that developed into sepsis. lungs infection (49%) were the most common source of infection for sepsis in the subject population, followed intraabdominal (20%), skin and soft - tissue (11%), unknown resource (11%), urinary tract (8%), then central nervous system (1%). microbial cultures of blood, sputum, a wound swab, pus, abscess, feces, ascites fluid, and urine from each patient were performed. a total of 78 microbial cultures (n = 78) were conducted in the subject population, resulting in 47 (66.3%) positive and 31 (33.7%) negative cultures. the results of the microbial cultures suggest that a patient could be infected by more than one microbe. klebsiella pneumoniae, escherichia coli, staphylococcus hominis, candida albicans and candida non - albicans were the organisms most frequently detected by microbial culture. the other culture results were limited to show gram stain features and features of an acid fast stain. the results showed two organisms that were acid - fast bacilli, 11 organisms that were gram - negative cocci and 12 organisms that were gram - positive cocci. the pattern of the organisms isolated from the various specimens can be observed in table 2. organisms isolated from various specimens (n=78) we conducted 342 susceptibility tests (n = 342) of 25 antibiotics. a total of 14 antibiotics showed a resistance level 50% and 9 antibiotics showed a resistance level of 50%. the level of antibiotic resistance based on susceptibility testing of the subject population a total of 46 antibiotics were administered to the subject population with 255 episodes of use. the classes of antibiotics administered were penicillins, cephalosporins, carbapenems, quinolones, aminoglycosides, macrolides, glycopeptides, sulfonamides, polymyxins, antituberculosis agents, anthracyclines, antifungals, and others. the pattern of antibiotic use in the subject population can be observed in figure 1. pattern of antibiotic use at an indonesian hospital (n = 255), other antibiotics include amikacin, cotrimoxazole, fosfomycin, gentamycin, ketoconazole, pyrazinamide, rifampicin, teicoplanin, vancomycin at 0.78%, lamivudine, alostil, amoxicillin, amoxicillin / clavulanate, benzyl penicillin, bleomycin, cefazoline, cefpirome, clarithromycin, clindamycine, colistin, doripenem, doxorubicin, fluconazole, ganciclovir, imipenem - cilastatin, nystatin, streptomycin, sulbactam, and tenofovir at 0.39% a total of 192 patients, 103 males and 89 females, were diagnosed with sepsis during the study period, and 76 patients met the criteria for the study. the sepsis incidence rate was highest in the 55 - 59-year age range with 15 patients, followed by the 45 - 50-year age range with 14 patients. the incidence of sepsis was higher in females than males and the mortality rate from sepsis reached 53.95%. comparing the mortality rate in males and females in the > 50 years age group, the study showed a higher mortality rate in males (40%) than in females (38.46%). in contrast, in the 15 - 50-year age group, the mortality rate in females (65.38%) was higher than in males (51.8%). it 's showed 15 subjects population had discharged against medical advice due to cost reasoning and 1 subject population transferred to other hospital. lungs infection, renal failure, malignancy, diabetes mellitus and intraabdominal infection is the highest co - morbidities in the subject population. in the lung infection groups, the major problem are hospital acquired pneumonia, community acquired pneumonia (cap) and tuberculosis. the highest mortality showed in the subject population who got systemic lupus erythematous, hepatitis, meningitis, myocarditis, and human immunodeficiency virus infection. lungs infection (49%) were the most common source of infection for sepsis in the subject population, followed intraabdominal (20%), skin and soft - tissue (11%), unknown resource (11%), urinary tract (8%), then central nervous system (1%). microbial cultures of blood, sputum, a wound swab, pus, abscess, feces, ascites fluid, and urine from each patient were performed. a total of 78 microbial cultures (n = 78) were conducted in the subject population, resulting in 47 (66.3%) positive and 31 (33.7%) negative cultures. the results of the microbial cultures suggest that a patient could be infected by more than one microbe. klebsiella pneumoniae, escherichia coli, staphylococcus hominis, candida albicans and candida non - albicans were the organisms most frequently detected by microbial culture. the other culture results were limited to show gram stain features and features of an acid fast stain. the results showed two organisms that were acid - fast bacilli, 11 organisms that were gram - negative cocci and 12 organisms that were gram - positive cocci. the pattern of the organisms isolated from the various specimens can be observed in table 2. organisms isolated from various specimens (n=78) we conducted 342 susceptibility tests (n = 342) of 25 antibiotics. a total of 14 antibiotics showed a resistance level 50% and 9 antibiotics showed a resistance level of 50%. the antibiotic resistance pattern is shown in table 3. the level of antibiotic resistance based on susceptibility testing of the subject population a total of 46 antibiotics were administered to the subject population with 255 episodes of use. the classes of antibiotics administered were penicillins, cephalosporins, carbapenems, quinolones, aminoglycosides, macrolides, glycopeptides, sulfonamides, polymyxins, antituberculosis agents, anthracyclines, antifungals, and others. the pattern of antibiotic use in the subject population can be observed in figure 1. pattern of antibiotic use at an indonesian hospital (n = 255), other antibiotics include amikacin, cotrimoxazole, fosfomycin, gentamycin, ketoconazole, pyrazinamide, rifampicin, teicoplanin, vancomycin at 0.78%, lamivudine, alostil, amoxicillin, amoxicillin / clavulanate, benzyl penicillin, bleomycin, cefazoline, cefpirome, clarithromycin, clindamycine, colistin, doripenem, doxorubicin, fluconazole, ganciclovir, imipenem - cilastatin, nystatin, streptomycin, sulbactam, and tenofovir at 0.39% mortality rate in the sepsis patients affected by several factors, including early initiation and appropriateness of antimicrobial and non - antimicrobial therapy, severity, age, gender, and co - morbidities. in contras, the mortality rate of this study in the age group of 15 - 50 years were higher than the older (58.5% vs. 39.1%). controlling factors that may affect mortality is important to understand the relationship between age and mortality. in this study, it is difficult to know this relationship, because several factors that affected in the mortality rate are uncontrolled. furthermore, we also found 21.05% of the subject population had discharged against medical advise. although in this study it is difficult to know the relationship between age and mortality, carbajal - guerrero., have showed the co - morbidities in the elderly group (> 65 years) is higher than the younger groups and their co - morbidities associated to the mortality rate. other studies have evaluated the influence of gender on survival in patients with sepsis with conflicting results. various studies show that, in surgical units, survival was better in women, better in men or similar in men and women. concluded that in a group of severe sepsis patients of 50 years of age, women have a lower mortality risk than men. the difference between men and women in the risk of septicemia is due to differences in the immune response. women have more estrogen production than men, which influences greater activity of the immune system. increasing age and body mass index in women can affect the production of estrogen by increasing aromatase activity in adipose tissue, increasing estrogen, which provides better protection through the action of the immune system. other factors that influenced the immune system are non - hormonal factors such as the production of interleukin-6 and lipopolysaccharide - stimulated tumor necrosis, social factors, economic factors, levels of physical activity, the source of infection, and hormonal modification factors. one of the marker that can be used to detect sepsis is procalcitonin, as shown by azevedo. in adult subjects and also by nnanna. in infant populations. showed a higher level of procalcitonin in sepsis and severe sepsis in adult patients were related to increase risk mortality. in the neonatus population, the level of procalcitonin can be used as a marker for early detection of sepsis in the intensive care unit. as a sepsis marker in the neonatus, procalcitonin is better than c - reactive protein (crp) ; however, crp can be used as a marker for bacterial co - infection in the viral - induced bronchiolitis infant populations. the most commonly found a source of infection for sepsis in this study was the lungs. this finding concurs with previous studies that reported that lung infections were the highest source of infection for sepsis development. the common causes of lung infection that developed into sepsis are hospital - acquired pneumonia and cap. wang. suggested that as a source of infection, lung infections may contribute to 15.6 - 69% of the incidence of sepsis. a separate study showed that from 1963 to 1998, the predominant site of infection that develops to sepsis changed from intra - abdominal infections to lung infections. knowledge of the common pathogens that develop into sepsis based on the site of infection will help us determine a rational empirical antibiotic to use. the common pathogens that cause sepsis based on the site of infection are shown in table 4. common pathogens that can develop into sepsis based on the source of infection based on the results of the bacterial cultures, k. pneumoniae was the microbe most commonly detected in the specimens (sputum, blood, throat swab). k. pneumoniae is the common pathogen in lung infections and intra - abdominal infections that develop into sepsis. another microbe detected in cultures was e. coli, which can develop into sepsis from many initial sites of infection. in this study, e. coli pathogens were found in pus specimens from 4 septic patients who had diabetes mellitus. e. coli and s. aureus were the most common agents isolated from the diabetic patients. e. coli is also the causative pathogen in infections in immunosuppressed patients, patients with severe burns, cancer patients and patients using catheters, antibiotics or corticosteroids. cons are common organisms in nosocomial bacteremia due to the increases in medical device use including intravenous catheters, vascular grafts, prosthetic heart valves, and devices used in the treatment of joint disease. the presence of cons in blood cultures can not directly determine that the species is pathogenic, because in 85% isolate cons found as a contaminant. the most commonly used antibiotics varied among institutions, but were typically composed of drugs that have levels of high resistance from some bacteria, such as pseudomonas, e. coli, k. pneumoniae, acinetobacter sp. and s. aureus. making a microbiological diagnosis is mandatory. a multicenter randomized trial showed lower mortality using a microbiological - based approach (after adequate empirical treatment) compared to a clinical only approach (hazard ratio : 1.54, confidence interval : 1.1 - 2.16, p = 0.01). negative result and contaminant result from the microbial cultures requires an evaluation to increase quality of microbiology diagnosis. nwose have showed the difference results of culture and susceptibility test in some clinical laboratories ; therefore, the program of quality assurance and quality control should be made available through the availability of standard operational procedures and improving the competency and skills of personnel 's. the most frequently used (74.5%) were levofloxacin, ceftazidime, metronidazole, ciprofloxacin, cefotaxime, meropenem, ceftriaxone, erythromycin, and ampicillin / sulbactam. in our study, six of these nine antibiotics, levofloxacin, ceftazidime, ciprofloxacin, cefotaxime, ceftriaxone, and erythromycin, showed resistance rates above 50%. based on the results of the microbial cultures, antibiotic susceptibility tests and patterns of antibiotic use, 61.35% of the antibiotics used showed resistance rates of more than 50%. a total of 10 antibiotics with resistance rates below 50% were cefoxitin, cefoperazone / sulbactam, gentamicin, piperacillin / tazobactam, ampicillin / sulbactam, meropenem, amoxicillin / clavulanate, vancomycin, linezolide and amikacin. the high sensitivity of these antibiotics contributes to their use as an option in empirical antibiotic therapy, but the selection of which antibiotic to use should consider the location of the infection source and factors specific to the patient. the pattern of antibiotic use with high resistance rates can be observed in figure 2. pattern of antibiotic use with their resistance level at an indonesian hospital the high frequency of use of antibiotics with high levels of resistance required special attention. patients who received appropriate initial antimicrobial treatment have lower mortality than those of who did nt. the early administration of appropriate antibiotic therapy for serious infection is associated with lower mortality, shorter duration of hospitalization, and lower health care cost. in other hand, wrong or inappropriate use of antibiotic will contributed to the development of antibiotic resistance and multi drug resistance (mdr). the high incidence of mdr can reduce the opportunities of patients to get the appropriate antimicrobial that can affect to increase the risk of death. raymond in his study have suggested a high mortality cases founded in the patients with mdr and the study also showed that the patients get inappropriate empirical antibiotic and severity of co - morbid. the emergence of microbial resistances were not by the availability of novel antimicrobial agents, which is marked by only four new classes of antibacterials have been discovered in the last 11 years. the strategies for limiting or modifying antibiotic use are needed to control resistance growth and to improve the rational use of antibiotics. the seven strategies to prevent antibiotic resistance that were suggested by kollef in 2005 are as follows : (1) establishment of a formal protocol and guidelines, (2) hospital formulary restrictions, (3) use of narrow spectrum antibiotics when supported by clinical situation and culture data, (4) combination antibiotic therapy, (5) shorter courses of antibiotic treatment, (6) antibiotic heterogeneity, and (7) optimization of pharmacokinetic / pharmacodynamic principles. there are three option that can be used in antibiotic heterogeneity strategies, namely antibiotic cycling / rotation, scheduled antibiotic changes, and antibiotic mixing. antibiotic cycling / rotation can be used with a fixed temporal pattern for predominant use of antibiotic class or classes, followed by their repeated and reintroduction over time. in contrast with scheduled antibiotic changes, it has a predetermined and scheduled change in the predominant antimicrobial agent employed. the changes of antibiotic classes are often based on changing patterns of antimicrobial sensitivities and not simply time based. the others antibiotics heterogeneity strategy is antibiotic mixing, a strategy whereby all or most available antimicrobial classes are employed to minimize undue pressure for the emergence of resistance from having single or limited number of antibiotic classes available. broad spectrum antibiotics can be used in the critical ill patients to avoid inappropriateness of antibiotics which can be fatality. the modification broad spectrum for initial therapy is needed based on clinical condition of patient, microbial culture, and antibiotics susceptibility test. modification of the initial antibiotics regimen should include decreasing the number and or spectrum antibiotics. shortening the duration of therapy in patients with uncomplicated infections who are demonstrating signs of clinical improvement or discontinuing antibiotics altogether in patients who have a non - infectious etiology identified for the patient 's signs and symptoms. the long duration of broad spectrum antibiotic used will lead to the development of antibiotics resistance ; therefore, it is very important to know the local pattern of pathogen based on the infection site and microbial sensitivity to minimize use of broad spectrum antibiotic and inappropriateness of empirical antibiotic use. carbapenem is a broad spectrum antibiotic, which came in to use in 1985, since then, due to their good intrinsic bacterial activity and stability to most of the prevalent beta lactamase, they have been a drug of choice for extended spectrum beta lactamase - producing organism. restricted use of specific antibiotics has generally been applied to those drugs with a broad spectrum of action (e.g., carbapenems), rapid emergence of antibiotic resistance (e.g., cephalosporins), and drugs with readily identified toxicity (e.g., aminoglycosides). in the hospital setting, restrictions on the use of antibiotics are administered through the hospital formulary and treatment guidelines and policies. an evaluation of an antibiotic used and its susceptibility should be monitored periodically to control the alteration of susceptibility. the most successful strategies to combat antibiotic resistance will be multidisciplinary, involving cooperation from the pharmacy, infection control, nursing staff, treating physicians, microbiology laboratory personnel, and infectious disease consultants. such programs should also focus on promoting infection control practices and employing rational antibiotic utilization aimed at minimizing future emergence of resistance. mortality rate in the sepsis patients affected by several factors, including early initiation and appropriateness of antimicrobial and non - antimicrobial therapy, severity, age, gender, and co - morbidities. in contras, the mortality rate of this study in the age group of 15 - 50 years were higher than the older (58.5% vs. 39.1%). controlling factors that may affect mortality is important to understand the relationship between age and mortality. in this study, it is difficult to know this relationship, because several factors that affected in the mortality rate are uncontrolled. furthermore, we also found 21.05% of the subject population had discharged against medical advise. although in this study it is difficult to know the relationship between age and mortality, carbajal - guerrero., have showed the co - morbidities in the elderly group (> 65 years) is higher than the younger groups and their co - morbidities associated to the mortality rate. other studies have evaluated the influence of gender on survival in patients with sepsis with conflicting results. various studies show that, in surgical units, survival was better in women, better in men or similar in men and women. concluded that in a group of severe sepsis patients of 50 years of age, women have a lower mortality risk than men. the difference between men and women in the risk of septicemia is due to differences in the immune response. women have more estrogen production than men, which influences greater activity of the immune system. increasing age and body mass index in women can affect the production of estrogen by increasing aromatase activity in adipose tissue, increasing estrogen, which provides better protection through the action of the immune system. other factors that influenced the immune system are non - hormonal factors such as the production of interleukin-6 and lipopolysaccharide - stimulated tumor necrosis, social factors, economic factors, levels of physical activity, the source of infection, and hormonal modification factors. one of the marker that can be used to detect sepsis is procalcitonin, as shown by azevedo. in adult subjects and also by nnanna. in infant populations. showed a higher level of procalcitonin in sepsis and severe sepsis in adult patients were related to increase risk mortality. in the neonatus population, the level of procalcitonin can be used as a marker for early detection of sepsis in the intensive care unit. as a sepsis marker in the neonatus, procalcitonin is better than c - reactive protein (crp) ; however, crp can be used as a marker for bacterial co - infection in the viral - induced bronchiolitis infant populations. the most commonly found a source of infection for sepsis in this study was the lungs. this finding concurs with previous studies that reported that lung infections were the highest source of infection for sepsis development. the common causes of lung infection that developed into sepsis are hospital - acquired pneumonia and cap. wang. suggested that as a source of infection, lung infections may contribute to 15.6 - 69% of the incidence of sepsis. a separate study showed that from 1963 to 1998, the predominant site of infection that develops to sepsis changed from intra - abdominal infections to lung infections. knowledge of the common pathogens that develop into sepsis based on the site of infection will help us determine a rational empirical antibiotic to use. the common pathogens that cause sepsis based on the site of infection are shown in table 4. based on the results of the bacterial cultures, k. pneumoniae was the microbe most commonly detected in the specimens (sputum, blood, throat swab). k. pneumoniae is the common pathogen in lung infections and intra - abdominal infections that develop into sepsis. another microbe detected in cultures was e. coli, which can develop into sepsis from many initial sites of infection. in this study, e. coli pathogens were found in pus specimens from 4 septic patients who had diabetes mellitus. e. coli and s. aureus were the most common agents isolated from the diabetic patients. e. coli is also the causative pathogen in infections in immunosuppressed patients, patients with severe burns, cancer patients and patients using catheters, antibiotics or corticosteroids. cons are common organisms in nosocomial bacteremia due to the increases in medical device use including intravenous catheters, vascular grafts, prosthetic heart valves, and devices used in the treatment of joint disease. the presence of cons in blood cultures can not directly determine that the species is pathogenic, because in 85% isolate cons found as a contaminant. the most commonly used antibiotics varied among institutions, but were typically composed of drugs that have levels of high resistance from some bacteria, such as pseudomonas, e. coli, k. pneumoniae, acinetobacter sp. and s. aureus. making a microbiological diagnosis is mandatory. a multicenter randomized trial showed lower mortality using a microbiological - based approach (after adequate empirical treatment) compared to a clinical only approach (hazard ratio : 1.54, confidence interval : 1.1 - 2.16, p = 0.01). negative result and contaminant result from the microbial cultures requires an evaluation to increase quality of microbiology diagnosis. nwose have showed the difference results of culture and susceptibility test in some clinical laboratories ; therefore, the program of quality assurance and quality control should be made available through the availability of standard operational procedures and improving the competency and skills of personnel 's. the most frequently used (74.5%) were levofloxacin, ceftazidime, metronidazole, ciprofloxacin, cefotaxime, meropenem, ceftriaxone, erythromycin, and ampicillin / sulbactam. in our study, six of these nine antibiotics, levofloxacin, ceftazidime, ciprofloxacin, cefotaxime, ceftriaxone, and erythromycin, showed resistance rates above 50%. based on the results of the microbial cultures, antibiotic susceptibility tests and patterns of antibiotic use, 61.35% of the antibiotics used showed resistance rates of more than 50%. a total of 10 antibiotics with resistance rates below 50% were cefoxitin, cefoperazone / sulbactam, gentamicin, piperacillin / tazobactam, ampicillin / sulbactam, meropenem, amoxicillin / clavulanate, vancomycin, linezolide and amikacin. the high sensitivity of these antibiotics contributes to their use as an option in empirical antibiotic therapy, but the selection of which antibiotic to use should consider the location of the infection source and factors specific to the patient. the pattern of antibiotic use with high resistance rates can be observed in figure 2. pattern of antibiotic use with their resistance level at an indonesian hospital the high frequency of use of antibiotics with high levels of resistance required special attention. patients who received appropriate initial antimicrobial treatment have lower mortality than those of who did nt. the early administration of appropriate antibiotic therapy for serious infection is associated with lower mortality, shorter duration of hospitalization, and lower health care cost. in other hand, wrong or inappropriate use of antibiotic will contributed to the development of antibiotic resistance and multi drug resistance (mdr). the high incidence of mdr can reduce the opportunities of patients to get the appropriate antimicrobial that can affect to increase the risk of death. raymond in his study have suggested a high mortality cases founded in the patients with mdr and the study also showed that the patients get inappropriate empirical antibiotic and severity of co - morbid. the emergence of microbial resistances were not by the availability of novel antimicrobial agents, which is marked by only four new classes of antibacterials have been discovered in the last 11 years. the strategies for limiting or modifying antibiotic use are needed to control resistance growth and to improve the rational use of antibiotics. the seven strategies to prevent antibiotic resistance that were suggested by kollef in 2005 are as follows : (1) establishment of a formal protocol and guidelines, (2) hospital formulary restrictions, (3) use of narrow spectrum antibiotics when supported by clinical situation and culture data, (4) combination antibiotic therapy, (5) shorter courses of antibiotic treatment, (6) antibiotic heterogeneity, and (7) optimization of pharmacokinetic / pharmacodynamic principles. there are three option that can be used in antibiotic heterogeneity strategies, namely antibiotic cycling / rotation, scheduled antibiotic changes, and antibiotic mixing. antibiotic cycling / rotation can be used with a fixed temporal pattern for predominant use of antibiotic class or classes, followed by their repeated and reintroduction over time. in contrast with scheduled antibiotic changes, it has a predetermined and scheduled change in the predominant antimicrobial agent employed. the changes of antibiotic classes are often based on changing patterns of antimicrobial sensitivities and not simply time based. the others antibiotics heterogeneity strategy is antibiotic mixing, a strategy whereby all or most available antimicrobial classes are employed to minimize undue pressure for the emergence of resistance from having single or limited number of antibiotic classes available. broad spectrum antibiotics can be used in the critical ill patients to avoid inappropriateness of antibiotics which can be fatality. the modification broad spectrum for initial therapy is needed based on clinical condition of patient, microbial culture, and antibiotics susceptibility test. modification of the initial antibiotics regimen should include decreasing the number and or spectrum antibiotics. shortening the duration of therapy in patients with uncomplicated infections who are demonstrating signs of clinical improvement or discontinuing antibiotics altogether in patients who have a non - infectious etiology identified for the patient 's signs and symptoms. the long duration of broad spectrum antibiotic used will lead to the development of antibiotics resistance ; therefore, it is very important to know the local pattern of pathogen based on the infection site and microbial sensitivity to minimize use of broad spectrum antibiotic and inappropriateness of empirical antibiotic use. carbapenem is a broad spectrum antibiotic, which came in to use in 1985, since then, due to their good intrinsic bacterial activity and stability to most of the prevalent beta lactamase, they have been a drug of choice for extended spectrum beta lactamase - producing organism. restricted use of specific antibiotics has generally been applied to those drugs with a broad spectrum of action (e.g., carbapenems), rapid emergence of antibiotic resistance (e.g., cephalosporins), and drugs with readily identified toxicity (e.g., aminoglycosides). in the hospital setting, restrictions on the use of antibiotics are administered through the hospital formulary and treatment guidelines and policies. an evaluation of an antibiotic used and its susceptibility should be monitored periodically to control the alteration of susceptibility. the most successful strategies to combat antibiotic resistance will be multidisciplinary, involving cooperation from the pharmacy, infection control, nursing staff, treating physicians, microbiology laboratory personnel, and infectious disease consultants. such programs should also focus on promoting infection control practices and employing rational antibiotic utilization aimed at minimizing future emergence of resistance. k. pneumoniae, e. coli and s. hominis is the most widely isolated organisms that were detected in septic patients. the high use of antibiotics with high levels of resistance such as levofloxacin, ceftazidime, ciprofloxacin, cefotaxime, ceftriaxone, and erythromycin requires a policy to control the use of antibiotics. microbial culture and resistance pattern were obtained from the local sepsis patients can be used as data to choose appropriatness of empirical antibiotic therapy for reducing mortality and morbidity in the sepsis patients. | background : the appropriate selection of empirical antibiotics based on the pattern of local antibiotic resistance can reduce the mortality rate and increase the rational use of antibiotics.aims:we analyze the pattern of antibiotic use and the sensitivity patterns of antibiotics to support the rational use of antibiotics in patients with sepsis.materials and methods : a retrospective observational study was conducted in adult sepsis patient at one of indonesian hospital during january - december 2011. data were collected from the hospital medical record department. descriptive analysis was used in the processing and interpretation of data.results:a total of 76 patients were included as research subjects. lung infection was the highest source of infection. in the 66.3% of clinical specimens that were culture positive for microbes, klebsiella pneumoniae, escherichia coli, staphylococcus hominis were detected with the highest frequency. the six most frequently used antibiotics, levofloxacin, ceftazidime, ciprofloxacin, cefotaxime, ceftriaxone, and erythromycin, showed an average resistance above 50%.conclusions : the high use of antibiotic with a high level resistance requires a policy to support its rational use. local microbial pattern based on site infection and pattern of antibiotics sensitivity test can be used as supporting data to optimize appropriateness of empirical antibiotics therapy in sepsis patients. |
daytime running lights (drls) are a safety feature intended to reduce crashes by increasing the contrast between vehicles and the background. currently, finland, sweden, norway, canada, denmark, hungary, and iceland all require vehicle lights during daytime hours. finland was the first to institute drl legislation in rural areas, and literature reports a 27% crash rate reduction. in 1977, sweden started requiring the use of daytime vehicle lights on all roads, and reduction of crash rates from 9 to 21% were reported by andersson and nilsson. norway began to require installation of drls in all new cars beginning in 1985 and use of daytime lights on all vehicles by 1988. a 15% crash rate reduction for crashes involving more than one vehicle was later reported by elvik. lastly, denmark has required use of drls on all roads since 1990, with a statistically significant 37% rate reduction for crashes involving a left turn in a study by hansen. a 1995 paper by theeuwes and riemersma criticized the odds ratio methodology of all these early studies. in response, a meta - analysis of 17 studies by elvik estimated a decrease in crash rate of 1015% for multi - vehicle crashes and total crash reduction of 312%. the first studies of drls in north america were done on fleet vehicles. in a study by stein, corporate fleet vehicles in the usa equipped with drls had 7% fewer relevant crashes compared to the group of fleet vehicles without drls during 19831984. sparks. reported 15% crash reduction in government fleet vehicles in canada equipped with drls. by december 1989 all newly manufactured vehicles in canada were required to be equipped with drls, and within 4 years, arora. reported a statistically significant 8% reduction in relevant collisions. drls in non - fleet passenger vehicles have been introduced more recently in the usa. in 1995, volvo and saab were first to install drls on all their new cars sold in the usa. by 1997, all new suzuki, volkswagen, and general motors models included drls. yet a decade later, only a few studies and reports have been published regarding the use of daytime headlights in the usa. farmer and williams used a case - control method to analyze multiple vehicle daytime crashes in nine states for a group of vehicles equipped with drls. the national highway traffic safety administration (nhtsa) reported a preliminary assessment in june 2000. using the fatality analysis reporting system (fars), they analyzed fatal crashes in four states from 1995 to 1997. they found no significant difference in risk of two vehicle opposite - direction crashes comparing vehicles with drls to vehicles without drls. however, using the state data system (sds) from florida, maryland, missouri, and pennsylvania, a statistically significant 7% reduction in risk for relevant (including crash subtypes presumably affected by drls, such as opposite - direction) nonfatal crashes was identified, and drl - equipped vehicles were associated with 28% fewer pedestrian fatalities. in this study, we tested the hypothesis that passenger vehicles in the usa equipped with drls are associated with decreased crash rates compared to those without drls under high test weather (daylight and optimal visibility) and road (dry) conditions. this was a retrospective study using the minnesota department of transportation (mndot) crash database from 1995 to 2002. vehicle crashes, for which police reports were filed, were cross - verified and matched against the nhtsa archival registry maintained for research purposes. definitions of crash and fatality were based on the terminology referenced by mndot traffic accident report (form version : ps-32003 - 10) as documented by police authorities at the time of the actual accident. specifically, fatalities recorded were for any scene deaths immediately related to the motor vehicle collision. crash reports included in the analyses were limited to crashes involving automobiles, pickups, and vans and crashes that occurred under high test weather and road conditions all defined a priori. the high test conditions included : (1) temporal limitations to daylight, defined as dawn to dusk, (2) optimal visibility, defined as clear or cloudy, and (3) road surface identified as dry. studied vehicles were also limited to models 1995 and newer, since prior models did not have drls. the vehicle identification number (vin) of vehicles involved in crashes was used to determine the specific make, model, and year. this information was cross - referenced with a nhtsa table of manufacturer listed drl conditions to determine each vehicle drl status. crash rates for vehicles with standard drl and without drl feature were calculated as relative to the number of all registered vehicles in minnesota with or without the drl feature, respectively. the number of registered vehicles in minnesota was determined from the mndot vehicle registration file obtained in 2004 for models 19952002. in 2004, the number of these vehicles, with and without standard drls, was 788,840 and 1,763,134, respectively. therefore, the only total number of vehicles which can be obtained is a number in real time. use of this single - year denominator assumes that the proportion of vehicles with and without the standard drl feature was constant over the years of this study. although the rates will be overestimated since the denominators represent a single year, the rate ratios will be appropriate if the previous assumption holds. ninety - five percent confidence intervals (ci) for the rates were constructed using a poisson error distribution. the two rates were compared using a two - sided f test for the ratio of two poisson random variants. during the 7-year study period, 184,637 vehicles (1995 or newer) had identifiable vins and were involved in accidents that occurred under the specified test conditions. of these vehicles, 37,909 were determined to have standard drls and 146,728 were determined to be models without drls (fig. 1). the standard drl group had a higher percentage of automobiles vs pickups and vans (78.5%) than the group without standard drls (66.3%). other accident characteristics were similar between the standard vs nonstandard drl groups (table 1). drl, year 1995 +, n = 37,909 (%) no std. drl, year 1995 +, n = 146,728 (%) vehicle typeautomobile29,750 (78.5)97,317 (66.3)pickup5,600 (14.8)30,959 (21.1)van2,559 (6.8)18,452 (12.6)type of accidentcollision with vehicle34,475 (90.9)133,892 (91.3)collision with train15 (< 0.1)30 (< 0.1)collision with bike358 (0.9)1,379 (0.9)collision with pedestrian230 (0.6)911 (0.6)diagramrear end13,721 (36.2)52,700 (35.9)sideswipe passing2,396 (6.3)9,379 (6.4)left turn into oncoming2,412 (6.4)9,723 (6.6)ran off road, left side539 (1.4)2,145 (1.5)right angle7,979 (21)30,347 (20.7)right turn into cross traffic218 (0.6)814 (0.6)ran off road, right side728 (1.9)3,019 (2.1)head on499 (1.3)2,147 (1.5)sideswipe opposing406 (1.1)1,612 (1.1)road descriptionfreeway (including ramps)5,701 (15)21,698 (14.8)other divided highway6,054 (16)22,640 (15.4)one - way street793 (2.1)3,409 (2.3)46 lane undivided, 23 each7,063 (18.6)27,380 (18.7)3 lanes undivided482 (1.3)1,735 (1.2)2 lanes, 1 each way11,894 (31.4)45,577 (31.1)alley, driveway149 (0.4)603 (0.4)private property153 (0.4)486 (0.3)functional classrural6,717 (17.7)23,685 (16.1)urban30,152 (79.5)118,558 (80.8)where percentages do not equal 100, categories labeled not applicable and other were incomplete and therefore not included in the data set summary of vehicles identified for analysis vehicle characteristics by drl status where percentages do not equal 100, categories labeled not applicable and other were incomplete and therefore not included in the data set the crash rate per 10,000 vehicles among vehicles with standard drls was 481 (37,909/788,840 ; 95% ci : 476485). for vehicles without standard drls the crash rate per 10,000 was 832 (146,728/1,763,134 ; 95% ci : 828836). 0.001) (table 2). table 2crash rate ratios : vehicles with drls versus vehicles without drlsall vehicles1.74 vehicles involved in fatal crashes1.48 vehicles involved : in collisions with other vehicles1.74 in collisions with pedestrians1.77 in collisions with bicycles1.72 crash rate ratios : vehicles with drls versus vehicles without drls crashes were also analyzed based on whether a fatality was reported. the rate of fatal vehicle crashes for vehicles with standard drls in minnesota between 1995 and 2002 was 2.0 per 10,000 (158/788,840 ; 95% ci : 1.72.3). the rate of fatal vehicle crashes for vehicles without standard drls was 3.0 per 10,000 (521/1,763,134 ; 95% ci : 2.73.2). vehicle crashes were divided by the type of collision, including collisions with other vehicles, pedestrians, and bicycles. (table 1). of the 37,909 vehicles with standard drls involved in accidents, this is a crash rate of 437 per 10,000 vehicles (95% ci : 432442). of the 146,728 vehicles without standard drls involved in accidents, this is a crash rate of 759 per 10,000 vehicles (95% ci : 755764). the rate ratio for vehicles involved in collisions with other vehicles was 1.74 (95% ci : 1.721.76 ; p < 0.001) (table 2). a total of 230 vehicles with standard drls were involved in collisions with pedestrians, which is a crash rate of 2.9 per 10,000 vehicles (95% ci : 2.53.3). in comparison, a total of 911 vehicles without standard drl were involved in collisions with pedestrians, which is a crash rate of 5.2 per 10,000 vehicles (95% ci : 4.85.5) (table 2). the rate ratio for vehicles involved in collisions with pedestrians was 1.77 (95% ci : 1.532.05 ; p < 0.001). finally, for collisions with a bicycle, there were 358 vehicles with standard drls involved in such collisions for a crash rate of 4.5 per 10,000 vehicles. without standard drls, 1,379 vehicles were involved in collisions with bicycles for a crash rate of 7.8 per 10,000 vehicles. the rate ratio for vehicles involved in collisions with bicycles is 1.72 (95% ci : 1.541.94 ; p < 0.001. based on our study results, drls had an association with vehicle crash reduction in motor vehicle collisions, consistent with two previous studies. farmer and williams showed that vehicles equipped with drls were involved in 3.2% fewer crashes. our crude crash rate reduction as reflected by the rate ratio was notably higher than in both of these previous studies. this may be due to the fact that our study was a retrospective study of all vehicle crashes in minnesota during the time period, whereas the preceding studies cited employed a case - control methodology to compare specific subsets of vehicles with and without drls. our study shows a statistically significant reduction in fatal crashes for vehicles with drls versus those without drls. this latter finding may be attributable to the relatively low numbers of vehicles involved in fatal crashes compared to all crashes reflected in the nhtsa study denominator. larger studies with greater numbers of fatal crashes would be helpful to further delineate the impact of drls in fatal crashes where causation is likely multifactorial. vehicles that collided with other vehicles showed lower crash rate in vehicles with standard drls compared to those without drls. this is a subtype of crashes that would expectedly be impacted by the drl feature, as increased visibility of other vehicles would likely decrease collisions. in addition, the rate of vehicles colliding with pedestrians may also be predictably lowered by the use of drls because these vehicles may be increasingly visible to pedestrians. our study does demonstrate a reduction in vehicle - pedestrian crashes not inconsistent with the 28% reduction rate reported by the nhtsa. to our knowledge, no traffic law revisions, such as lower speed limitations, or newer primary seat belt stop legislations, affected our crash rates. specifically, there were no traffic law changes in minnesota identified during the study period. first, unknown drl status excluded vehicles from analysis, and incremental value of layered standards or options in crash prevention is not quantified. second, snapshots of data streams may fail to demonstrate the whole picture in complex large volume relationships over time. we used a denominator from vehicles registered in 2004 and assumed a similar proportion of vehicles with drl standard to those without drl standard for all of the study years. we believe the proportion of vehicles remained reasonably constant over the study period, but there is no retrospective database to confirm this. third, use of best - case scenario assumptions to disprove the null hypothesis may limit capture of other significant differences between groups. lastly, confounders related to the driver or vehicle parameters such as age, experience, or safety record may significantly affect associations. driver and vehicle files containing private or privileged information (insurance status, license qualifications, organ donor information, health outcomes, etc.) first, unknown drl status excluded vehicles from analysis, and incremental value of layered standards or options in crash prevention is not quantified. second, snapshots of data streams may fail to demonstrate the whole picture in complex large volume relationships over time. we used a denominator from vehicles registered in 2004 and assumed a similar proportion of vehicles with drl standard to those without drl standard for all of the study years. we believe the proportion of vehicles remained reasonably constant over the study period, but there is no retrospective database to confirm this. third, use of best - case scenario assumptions to disprove the null hypothesis may limit capture of other significant differences between groups. lastly, confounders related to the driver or vehicle parameters such as age, experience, or safety record may significantly affect associations. driver and vehicle files containing private or privileged information (insurance status, license qualifications, organ donor information, health outcomes, etc.) minnesota vehicles equipped with drls were associated with a statistically significant decrease in crash rates compared to vehicles without drls, model year 1995 or newer, from 1995 to 2002. | backgrounddaytime running lights (drls) are a safety feature intended to reduce crashes by increasing the contrast between vehicles and the background.aimsthe purpose of this study was to determine whether there is an association between vehicles in the usa being equipped with drls and crash rates.methodsthis was a retrospective study using the minnesota department of transportation (mndot) crash database from 1995 to 2002. crash reports included in the analyses were limited to accidents involving vehicles 1995 or newer (drls not available on prior models) and limited to ideal conditions : (1) daylight, (2) optimal visibility, and (3) dry road surface. the vehicle identification number (vin) was used to determine the make, model, and year. this information was cross - referenced with a national highway traffic safety administration table of manufacturer listed drl conditions to determine vehicle drl status. crude crash rates for vehicles were calculated relative to the number of all registered vehicles in minnesota in 2004, for models 19952002. ninety - five percent confidence intervals (ci) for the rates were constructed assuming a poisson error distribution.resultsduring 19952002, there were 184,637 vehicles (1995 or newer) with identifiable vins involved in accidents which occurred under the specified test conditions. of these vehicles, 37,909 were determined to have standard drls and 146,728 were determined to be models without drls (including those listed as drl optional). the crash rate among vehicles without standard drls was 1.73 (95% ci : 1.711.75) times higher than the rate for vehicles with standard drls. the rate ratio was also significant for fatal vehicle crash rates 1.48 (95% ci : 1.231.76).conclusionminnesota vehicles equipped with drls were associated with a statistically significant lower crash rate compared to vehicles without drls from 1995 to 2002. |
although prostate cancer is the second leading cause of cancer - related deaths in men in the us, early detection with serum prostate - specific antigen (psa) has led to the over - detection and over - treatment of indolent prostate cancer(13). recently, active surveillance, where men newly diagnosed with prostate cancer undergo serial biopsy, psa and/or imaging to delay intervention until prompted by clinicopathological evidence of disease progression (or patient decision), has emerged as management strategy for low risk prostate cancer that does not significantly decrease prostate cancer specific mortality compared to immediate treatment(46). despite advances in imaging and prognostic expression / protein assays, serum psa and clinicopathological parameters are the only factors routinely used to assess prognosis at diagnosis (4, 611). although pathological inclusion criteria for active surveillance protocols vary by institution or group, almost all include gleason score, number (or percentage) of positive cores and the tumor measurement / length or maximum percentage of tumor involvement in any core (4, 5, 1115). in particular, more than 50% of tumor involvement in any given core is commonly considered as an exclusion criterion for most active surveillance protocols (4, 5, 1115). however, when two or more foci of prostate cancer separated by intervening benign tissue are present in a single core biopsy, there is currently no consensus dictating the optimal method to report such a tumor involvement percentage. the pathologist can either 1) measure discontinuous foci as if they were one continuous tumor by including the benign intervening tissue, assuming they represent two sections of a unique tumor, or 2) only measure the areas actually involved by a tumor focus. the first option, which would report a higher percentage of tumor involvement of a core, has been proposed to be the optimal method by suggesting it is more representative of tumor volume at prostatectomy (16). prostate cancer is known to be a multifocal disease (17), with most radical prostatectomy specimens actually harboring clonally distinct tumor foci, as supported by single marker molecular subtyping and next generation sequencing (1824). of note, approximately 50% of psa - screened prostate cancer foci in predominantly caucasian populations harbor chromosomal rearrangements that result in the fusion of the 5 untranslated region of tmprss2 to erg, an ets transcription factor, which can be detected by fluorescence in situ hybridization (fish) or immunohistochemistry (2527). additionally, about 10% of prostate cancers show marked over - expression of spink1, which can be evaluated concurrently with erg by dual immunohistochemistry (28, 29). importantly, in these studies, erg fusion status has been shown to be clonal in a given tumor focus, and spink1 and erg show essentially mutually exclusive expression. hence, dual erg / spink1 immunohistochemistry represents a simple, rapid and inexpensive method to assess tumor clonality in routine specimens. in this study, we hypothesized that spatially distinct tumor foci in a given biopsy core may arise from separate clones, and sought to determine the frequency of such multiclonality. thus, we used erg / spink1 dual immunohistochemistry to determine whether discontinuous cancer foci truly represent the same tumor clone (uniform erg / spink1 status in separate foci), or multiclonal disease (discordant erg / spink1 status between foci). results of this pilot study demonstrate multiclonality in 25% of discontinuously involved cores, supporting additional studies on whether clonality impacts the prognostic ability of tumor volume at biopsy and prostatectomy. surgical pathology databases were searched for prostatic needle core biopsies containing discontinuous prostatic adenocarcinoma diagnosed between 2010 and 2013 at two institutions, weill medical college of cornell university and university of michigan. two genitourinary pathologists reviewed the h&e - stained slides for each case ; biopsies with confirmed discontinuous foci of prostatic adenocarcinoma were selected. only biopsies with intact cores (measuring at least 1 cm), in this study we considered biopsies with tumor foci separated by at least 2.5 mm or 25% of total core volume as discontinuously involved (figure 1a c). for each core, the gleason score was verified, and we determined the maximum percentage of tumor involvement by 1) routine histomorphology (inclusive of discontinuous foci), as well as 2) tumor involvement measured after evaluating erg / spink1 status (if discontinuously staining foci were present, we added the percentage of both foci [figure 1a c ]. dual erg / spink1 immunohistochemistry staining (28) was performed on 5m - thick unstained slides. these corresponded to intervening unstained levels generated as part of the standard diagnostic workup, or de - stained h&e slides when unstained slides were not available or tissue was exhausted from the block. immunohistochemistry was performed either on the benchmark ultra or the discovery xt automated staining systems (ventana medical system, inc., tucson, az). a monoclonal rabbit anti - erg primary antibody from ventana medical systems (epr3864) and a mouse primary anti - spink1 4d4 antibody (abnova, taiwan) were used, as previously described (26, 2830). tumor foci with intense (2 + or 3 +) and diffuse (at least 90% of cancer cells) nuclear erg staining were considered as erg positive (erg). tumor foci with intense (2 + or 3 +) cytoplasmic spink1 staining in more than 10% of cancer cells were considered spink1 positive (spink1). biopsies were considered multiclonal if they harbored spatially distinct foci with discrepant erg and/or spink1 status (i.e. one erg / spink1 focus and one erg / spink1 focus ; one erg / spink1 focus and one erg / spink1 focus ; or one erg / spink1 focus and one erg / spink1 focus). biopsies with uniform erg or spink1 staining of tumor foci were considered clonal (figure 1a d). surgical pathology databases were searched for prostatic needle core biopsies containing discontinuous prostatic adenocarcinoma diagnosed between 2010 and 2013 at two institutions, weill medical college of cornell university and university of michigan. two genitourinary pathologists reviewed the h&e - stained slides for each case ; biopsies with confirmed discontinuous foci of prostatic adenocarcinoma were selected. only biopsies with intact cores (measuring at least 1 cm), in this study we considered biopsies with tumor foci separated by at least 2.5 mm or 25% of total core volume as discontinuously involved (figure 1a c). for each core, the gleason score was verified, and we determined the maximum percentage of tumor involvement by 1) routine histomorphology (inclusive of discontinuous foci), as well as 2) tumor involvement measured after evaluating erg / spink1 status (if discontinuously staining foci were present, we added the percentage of both foci [figure 1a c ]. dual erg / spink1 immunohistochemistry staining (28) was performed on 5m - thick unstained slides. these corresponded to intervening unstained levels generated as part of the standard diagnostic workup, or de - stained h&e slides when unstained slides were not available or tissue was exhausted from the block. immunohistochemistry was performed either on the benchmark ultra or the discovery xt automated staining systems (ventana medical system, inc., tucson, az). a monoclonal rabbit anti - erg primary antibody from ventana medical systems (epr3864) and a mouse primary anti - spink1 4d4 antibody (abnova, taiwan) were used, as previously described (26, 2830). tumor foci with intense (2 + or 3 +) and diffuse (at least 90% of cancer cells) nuclear erg staining were considered as erg positive (erg). tumor foci with intense (2 + or 3 +) cytoplasmic spink1 staining in more than 10% of cancer cells were considered spink1 positive (spink1). biopsies were considered multiclonal if they harbored spatially distinct foci with discrepant erg and/or spink1 status (i.e. one erg / spink1 focus and one erg / spink1 focus ; one erg / spink1 focus and one erg / spink1 focus ; or one erg / spink1 focus and one erg / spink1 focus). biopsies with uniform erg or spink1 staining of tumor foci were considered clonal (figure 1a d). ninety - seven prostate needle biopsies from 80 patients fulfilled our criteria for discontinuous involvement by prostatic adenocarcinoma (table 1).. maximum tumor involvement, including benign intervening tissue, ranged from 20 to 100% of core length. overall, erg and spink1 frequency (assessed per core) was 38% (37/97) and 12% (12/97), respectively. erg / spink1 immunohistochemistry status, gleason score, and maximum tumor involvement for all evaluated cores are shown in a heatmap in figure 1e. benign prostate tissue was negative for erg or spink1 expression, consistent previous reports of exceptionally rare staining for either antigen in benign prostate tissue(23, 24, 26, 28, 30). of the 97 biopsies with discontinuous tumor, 24 (25%) showed discrepant erg / spink1 status between spatially distinct foci (table 2). within these 24 multiclonal biopsies, 14 (58%) harbored one erg / spink focus and one erg / spink1 focus ; 7 (29%) harbored one erg / spink1 focus and one erg / spink1 focus ; and 3 (13%) showed one erg / spink1 focus and one erg / spink1 focus (examples of discordant foci are shown in figure 2). when determining clonality by only considering erg status, 17 (17.5%) had discordant erg status between foci, consistent with multiclonal disease. in the 24 multiclonal biopsies, gleason scores were 3 + 3=6 in 54% (13/24), 3 + 4=7 in 21% (5/24), 4 + 3=7 in 17% (4/24) and 4 + 4=8 in 12% (2/24) of cores. maximum tumor involvement percentage in multiclonal biopsies, including intervening benign tissue, ranged from 20 to 100%. after incorporating multiclonal assessment (collapsing involvement to the summed tumor lengths of foci with discordant erg / spink1 status), maximum tumor involvement percentage fell under under 50% in 22/24 biopsies (92%). recent consensus statements recommend reporting the amount of cancer in prostate biopsies by estimating the percentage of tumor involvement in the core with the greatest tumor extent (11, 31). importantly, this pathology parameter has been shown to be correlated with tumor volume at prostatectomy (32). although measuring the percentage of involvement in a core may seem straightforward, it can be challenging for the pathologist when assessing biopsy cores with discontinuous tumor separated by benign intervening tissue, which is a well - recognized finding on prostate biopsies. there is currently no consensus on the best method including or excluding intervening benign tissue when reporting tumor percent involvement in a discontinuously involved core. the choice of method remains controversial between pathologists, and both techniques are being utilized clinically (3335). brimo. reported no significant difference using either tumor content estimation technique on predicting pathologic outcome at prostatectomy, although they included cases in which the amount of intervening benign tissue was less than 5 mm (34). however, recent studies propose reporting the maximum tumor involvement, including the benign intervening tissue, assuming that the separate foci represent the same tumor coming in and out of the core section. showed that this measurement method better correlated with stage and margin positivity in cases with gleason 6 on biopsy with no upgrade at radical prostatectomy (16). shultz. further supported this finding in a private practice setting (35). multiclonality / multifocality is well recognized at prostatectomy, as spatially distinct tumor foci are routinely appreciated. importantly, molecular evidence supporting true multiclonality of distinct tumor foci has been demonstrated by our groups and others using erg status (by fish or immunohistochemistry as a surrogate) to identify distinct foci (1820, 23, 24). a recent study assessed clonality between biopsies in patients with multiple positive biopsy cores, using erg immunohistochemistry as a clonal marker. in that study, mertz. showed that approximately 12% of prostate needle biopsy sets in their cohort of patients with indolent prostate cancer showed heterogeneous erg staining (36). in this study, we show that 25% of discontinuously involved prostate biopsies harbor clonally distinct tumors, revealed by discrepant erg and/or spink1 immunohistochemistry status between foci. to our knowledge, this represents the first study to interrogate prostate cancer clonality in core needle biopsies with discontinuous tumor involvement. although associations with disease burden at prostatectomy must be established in cases where the discontinuously involved core drives management, we hypothesize that reporting tumor volume by excluding intervening benign tissue (instead of including intervening benign tissue) will be more appropriate in cores with multiclonal discontinuous involvement. hence, erg / spink1 evaluation in such cases would be expected to increase the number of men eligible for active surveillance by identifying those with multiclonal involvement. in our study, the frequency of erg and spink1 overexpression was comparable to previous studies at 38% (37/97) and 12% (12/97), respectively. as expected, erg status was therefore the major driver in determining clonality, since 17/24 (71%) of multiclonal cases showed at least one erg focus. however, incorporating spink1 in a dual staining with erg adds further data to support distinct clonal origin, since erg and spink1 represent distinct molecular prostate cancer subclasses and their expression is essentially mutually exclusive (23, 29, 3739). although other molecular subtypes have been identified (e.g. spop mutated), routine assays to identify them in situ (or surrogate tissue - based markers) are not yet available (40, 41). although heterogeneous spink1 status has been observed within the same tumor focus (42), in our experience with prostatectomy specimens this usually represents discrepant staining between the leading edge and center of a given focus (23, 28). thus, although we interpret discordant spink1 staining between two discontinuous foci on biopsy as evidence of multiclonality (as a leading edge of both can be appreciated), this will require further validation using alternative techniques. given that this study represents, to our knowledge, the first assessment of multiclonality in discontinuously involved cores, we included cores from cases where tumor content of the discontinuous core would not have impacted potential active surveillance inclusion (e.g. the discontinuously involved core or other core had high grade / volume disease or numerous other cores were positive). however, our cohort did include cases where evaluation of the discontinuously involved core may have played a key role in clinical management. for example, one core was from a patient diagnosed with gleason 3 + 3=6 prostate cancer in 1 of 12 cores, with cancer discontinuously involved 80% of the core (figure 3). this case was reviewed by an expert genitourinary pathologist at an outside institution, who agreed with high volume discontinuous involvement, and the patient underwent surgery. at radical prostatectomy, only a single, small (0.7 cm in greatest dimension) focus of gleason score 6, organ confined prostate cancer was present, consistent with clinically insignificant disease by standard epstein criteria (43). erg / spink1 dual immunohistochemistry on the involved biopsy core demonstrated discordant erg / spink1 status between the foci (figure 3), and hence clonality assessment would support this patient as having two small foci (occupying a total of ~20% [~15% and 6, there were multiple other positive cores or other high volume involved cores). our study is a proof of principle study assessing the frequency of multiclonality across prostate biopsies with discontinuous tumor foci. therefore, further validation on larger study sets including prospectively collected cohorts (e.g. active surveillance) is essential. confirmation of our hypothesis would provide compelling evidence that multifocal disease is common in biopsies with discontinuous tumor, supporting the opportunity to refine key histopathological parameters through incorporating multiclonality assessment to provide more accurate prognostic information. likewise, assessment of the prognostic impact on assigned gleason scores in discontinuously involved cores may also be of interest (e.g. a discontinuous gleason score 3 + 4=7 foci may actually represent separate gleason score 4 + 4=8 and 3 + 3=6 foci). we expect that assessment of erg / spink1 for clonality may also have utility in selected scenarios at prostatectomy. for example, we are assessing whether incorporation of erg / spink1 in gleason score 7 tumors may improve the prognostic ability of routine pathologic parameters (e.g. gleason score and tumor size) through identifying tumors that are collisions of foci with distinct erg / spink1 status. examples of prostatectomy cases where a single tumor focus by morphology are composed of foci with discordant erg / spink1 status are shown in figure s1. in summary, in our study, nearly 25% of prostate core biopsies with discontinuous tumor involvement harbored spatially molecularly distinct cancer foci, as indicated by discrepant erg and/or spink1 status. here we demonstrate that assessment of tumor clonality by erg / spink1 dual immunohistochemistry in biopsies with discontinuous involvement of prostate cancer is an effective tool to refine histopathological parameters, specifically estimation of tumor involvement when discontinuous foci are present in prostate biopsies. incorporation of this inexpensive ancillary test may have significant impact on routine practice as it could potentially increase the number of eligible patients for active surveillance. clinical application of erg / spink1 dual immunohistochemistry is an important area for continued study. | the presence of two or more prostate cancer foci separated by intervening benign tissue in a single core is a well - recognized finding on prostate biopsy. cancer involvement can be measured by including intervening benign tissue or only including the actual cancer involved area. importantly, this parameter is a common enrollment criterion for active surveillance protocols. we hypothesized that spatially distinct prostate cancer foci in biopsies may arise from separate clones, impacting cancer involvement assessment. hence, we used dual erg / spink1 immunohistochemistry to determine the frequency of separate clones when separate tumor foci showed discordant erg and/or spink1 status in discontinuously involved prostate biopsy cores from two academic institutions. in our cohort of 97 prostate biopsy cores with spatially discrete tumor foci (from 80 patients), discontinuous cancer involvement including intervening tissue ranged from 20 to 100% and gleason scores ranged from 6 to 9. twenty four (25%) of 97 discontinuously involved cores harbored clonally distinct cancer foci by discordant erg and/or spink1 expression status : 58% (14/24) had one erg+ focus, and one erg/spink1 focus ; 29% (7/24) had one spink1 + focus and one erg/spink1 focus ; and 13% (3/24) had one erg+ focus and one spink1 + focus. erg and spink1 overexpression were mutually exclusive in all tumor foci. in summary, our results demonstrate that ~25% of discontinuously involved prostate biopsy cores showed tumor foci with discordant erg / spink1 status, consistent with multiclonal disease. the relatively frequent presence of multiclonality in discontinuously involved prostate biopsy cores warrants studies on the potential clinical impact of clonality assessment, particularly in cases where tumor volume in a discontinuous core may impact active surveillance eligibility. |
the leptomeninges are composed of the pia and arachnoid mater connected by strands termed arachnoid trabeculae. in the young, the leptomeninges and dura mater have been traditionally thought to contain few, if any, inflammatory cells, and any increase in cellularity is potentially equated to a pathologic condition, including inflicted trauma [1, 2 ]. at present, we do not know what constitutes normal cellularity of infant leptomeninges and if inflammatory or iron containing cells should be present at all in non - forensic settings. however, when indeed present under suspicious circumstances, they are often linked to inflicted trauma, such as in cases of the shaken baby syndrome. in order to recognise and characterise the pathologic findings in infant brains, it is important to have an understanding of the normal constituents of the various intracranial compartments. while some studies in the past, largely in rodent pups, have sought to evaluate and characterise the leptomeningeal cellular constituents, up until now, a rigorous analysis of the inflammatory cellular constituents of the leptomeninges has not been performed in human late - foetal and infant brains. this characterisation will serve as a baseline for comparison with brains of similarly aged children in forensic settings. therefore, in addition to determining the inflammatory cellular composition and presence of iron in foetal and infant leptomeninges associated with natural disease processes and in the absence of physical trauma beyond that accompanying vaginal birth, this study aims to formulate a basis of comparison of leptomeningeal cellular constituents in forensic settings, based on rigorous histological analyses of hospital - derived autopsies. a total of 33 foetal and infant autopsies in which neuropathologic examinations had been performed at stanford university medical center / lucille packard children s hospital were identified utilising the department of pathology database program. the cases for study were chosen in concert with the attending neuropathologist responsible for rendering the original diagnoses. the criteria for study inclusion were cases between 2005 and 2008, the age bracket of late third trimester and 1 year of post - natal life, and availability of formalin - fixed paraffin - embedded tissue samples harvested from at least two different brain sites including the cerebral cortex, cerebellum and brainstem. there were three cases within these 33 total cases which did not conform to these criteria but were included. each sample slide was screened on microscopy by the attending neuropathologist, and only sections of brains reflecting a wide (5.0 mm) sampling of the leptomeninges were chosen. slides of routinely processed formalin - fixed, paraffin - embedded sections in each case were prepared and stained with antibodies to cd45 (dilution 1:100), cd68 (dilution 1:100) and cd163 (dilution 1:200). cd45, also known as leucocyte common antigen, is uniquely expressed on the surface of all leucocytes and their progenitor cells these include neutrophils, eosinophils, basophils, lymphocytes and monocytes. cd163 was used as an additional stain for cells of monocyte / macrophage lineage and microglia (although the density of microglia was not specifically evaluated in this study). immunoperoxidase staining was performed, following microwave antigen retrieval in citrate buffer at ph6, on an automatic stainer (dako autostainer, universal staining system). iron was detected in sections utilising the standard perl s staining method. in order to reduce inter - observer variation, the number of variously immunoreactive or iron containing cells was quantified by a single observer and representative slides reviewed for accuracy by a second observer. at a microscopic magnification of 20x, immunoreactive cells within leptomeninges were counted and recorded. as the length of leptomeninges evaluated varied between slides, the length of leptomeninges scored was measured in millimetres and results recorded as immunoreactive cells / millimetre. only leptomeninges on gyral surfaces were scored as it was impossible to accurately measure the length of the leptomeninges in the sulci. the samples were divided into infants who died beyond 33 post - natal days up to 1 year (group 1) and newborns who survived up to 33 days (group 2)these represent pre- and post - natal leptomeninges. the mean number of cd45, cd68 and cd163 positive immunoreactive cells / millimetre, occurring in the cerebellum, cortex and brain stem, was calculated for groups 1 and 2, respectively. iron was recorded as being either present or absent. to test for significant differences between the numbers of immunoreactive cells between groups 1 and 2, an unpaired a total of 33 foetal and infant autopsies in which neuropathologic examinations had been performed at stanford university medical center / lucille packard children s hospital were identified utilising the department of pathology database program. the cases for study were chosen in concert with the attending neuropathologist responsible for rendering the original diagnoses. the criteria for study inclusion were cases between 2005 and 2008, the age bracket of late third trimester and 1 year of post - natal life, and availability of formalin - fixed paraffin - embedded tissue samples harvested from at least two different brain sites including the cerebral cortex, cerebellum and brainstem. there were three cases within these 33 total cases which did not conform to these criteria but were included. each sample slide was screened on microscopy by the attending neuropathologist, and only sections of brains reflecting a wide (5.0 mm) sampling of the leptomeninges were chosen. slides of routinely processed formalin - fixed, paraffin - embedded sections in each case were prepared and stained with antibodies to cd45 (dilution 1:100), cd68 (dilution 1:100) and cd163 (dilution 1:200). cd45, also known as leucocyte common antigen, is uniquely expressed on the surface of all leucocytes and their progenitor cells these include neutrophils, eosinophils, basophils, lymphocytes and monocytes. cd163 was used as an additional stain for cells of monocyte / macrophage lineage and microglia (although the density of microglia was not specifically evaluated in this study). immunoperoxidase staining was performed, following microwave antigen retrieval in citrate buffer at ph6, on an automatic stainer (dako autostainer, universal staining system). in order to reduce inter - observer variation, the number of variously immunoreactive or iron containing cells was quantified by a single observer and representative slides reviewed for accuracy by a second observer. at a microscopic magnification of 20x, immunoreactive cells within leptomeninges were counted and recorded. as the length of leptomeninges evaluated varied between slides, the length of leptomeninges scored was measured in millimetres and results recorded as immunoreactive cells / millimetre. only leptomeninges on gyral surfaces were scored as it was impossible to accurately measure the length of the leptomeninges in the sulci. the samples were divided into infants who died beyond 33 post - natal days up to 1 year (group 1) and newborns who survived up to 33 days (group 2)these represent pre- and post - natal leptomeninges. the mean number of cd45, cd68 and cd163 positive immunoreactive cells / millimetre, occurring in the cerebellum, cortex and brain stem, was calculated for groups 1 and 2, respectively. iron was recorded as being either present or absent. to test for significant differences between the numbers of immunoreactive cells between groups 1 and 2, an unpaired, sixteen cases involved infants who died beyond the post - natal age of 33 days (group 1), and 17 cases represented either foetuses or newborns who survived up to 33 post - natal days (group 2). one child (number 7) who survived to 16 months of age was included in group 1. there were two cases (numbers 25 and 30) involving foetuses in the 26th and 28th weeks of gestation which were included in group 2. the general autopsy and neuropathology findings of both groups overlapped, and these included congestive heart failure, noonan s syndrome, micrencephaly and pontosubicular neuronal necrosis (tables 1 and 2).table 1general autopsy and neuropathologic findings for infants with a post - natal age greater than 33 days (group 1)case nogeneral autopsy findingsneuropathology findings1chronic aspiration / pneumonitis with mosfoedema with herniation ; hieseizure disorder4noonan s syndromemicrencephaly ; pontosubicular neuronal necrosis5trisomy 21 with chdhaemorrhagic infarct (left periventricular)7chdmicrencephaly8interstitial pneumonitis (cultures negative)pvl9heterotaxy asplenia syndromemicrencephaly ; multiple cortical and wm infarcts11congenital pulmonary malformationmultifocal acute hi changes12pulmonary hypoplasiaremote and focal acute hi changes14noneagenesis of cc(brain only)17chd with infarctionnone22cardiomyopathymultiple cerebral infarctionssepsis with mosf23foetal hydropspvl ; right occipital infarction26noonan s syndromemicrencephaly ; hypomyelination ; organising sah, sdh27liver failuremetabolic astrogliosis ; neuronal pyknosis ; bilateral subdural membranes28rsv bronchiolitisremote occipital infarct ; subacute edhpna33chdcystic infarct (frontal) cc corpus callosum, chd congenital heart disease, edh epidural haemorrhage, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, mosf multiple organ system failure, pna pneumonia, pvl periventricular leukomalacia, rsv respiratory syncytial virus, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white mattertable 2general autopsy and neuropathologic findings for foetuses and infants with a post - gestational age up to 33 days (group 2)case no.general autopsy findingsneuropathologic findings2chdnone3chdmicrencephaly;arhinencephaly;neuronal necrosis, subiculum6harlequin ichthyosisnone10diaphragmatic herniaomphalocoelepsnn13pna (strep)metabolic encephalopathy;hydrocephalus;partial agenesis of cc15chdthoracic meningomyelocoele;hydrocephalus ; possible partial agenesis of cc16chdorganising sdh18cardiomyopathyacute hi change (focal);digeorge syndromepsnn;remote dural haemorrhage19pulmonary htnpsnn20foetal hydropsgm haemorrhage extending to sa;diffuse hie21tetralogy of fallotpsnn ; bg infarct24chdfocal sah;small wm haemorrhages;psnn25chdpsnn;focal hi, cerebellum;pvl;parietal infarct;organising sdh, ivh, sah29none (brain only)diffuse hie30nec (mosf)meningoencephalitis;psnn31multiple congenital abnormalities including chdgm haemorrhage with ivh and sah ; psnn;arhinencephaly32pulmonary hypoplasia / htngm haemorrhage with iv and sah ; psnn bg basal ganglia, cc corpus callosum, chd congenital heart disease, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, htn hypertension, iv intraventricular, mosf multiple organ system failure, nec necrotizing enterocolitis, pna pneumonia, pvl periventricular leukomalacia, sa subarachnoid, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white matter, psnn pontosubicular neuronal necrosis general autopsy and neuropathologic findings for infants with a post - natal age greater than 33 days (group 1) cc corpus callosum, chd congenital heart disease, edh epidural haemorrhage, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, mosf multiple organ system failure, pna pneumonia, pvl periventricular leukomalacia, rsv respiratory syncytial virus, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white matter general autopsy and neuropathologic findings for foetuses and infants with a post - gestational age up to 33 days (group 2) bg basal ganglia, cc corpus callosum, chd congenital heart disease, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, htn hypertension, iv intraventricular, mosf multiple organ system failure, nec necrotizing enterocolitis, pna pneumonia, pvl periventricular leukomalacia, sa subarachnoid, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white matter, psnn pontosubicular neuronal necrosis table 3 demonstrates the number of slides from each brain region and the range of length, mean and standard deviation of associated leptomeninges scored per slide. overall, 39 sites were sampled from cerebral cortices, 37 from brainstems and 30 from cerebella. overall, no statistically significant differences between the mean number of immunoreactive cells in the cerebellum, brain stem and cortex were observed between groups 1 and 2 (table 4).table 3site and range, mean and standard deviations of lengths of leptomeninges in all casessitesno. of sites sampledleptomeningeal length (mm)mean (mm) + / sdcerebral cortex3954118.8 + / 11.1table 4test for statistically significant differences between the mean number of immunoreactive cells in the cerebellum, brain stem and cortex between groups 1 and 2comparison (group 1 vs. group 2) p - valuecerebellumcd450.623cd680.235cd1630.457brain stemcd450.689cd680.337cd1630.172cerebral cortexcd450.247cd680.149cd1630.204 site and range, mean and standard deviations of lengths of leptomeninges in all cases test for statistically significant differences between the mean number of immunoreactive cells in the cerebellum, brain stem and cortex between groups 1 and 2 looking specifically at the cases in group 1 (table 1 and fig. 1), four had congenital heart disease (chd) and two were diagnosed with noonan s syndrome. the mean number of cd45, cd68 and cd163 immunoreactive cells per mm of leptomeninges of the former group was 14.4, 17.5 and 17.9 cells / mm, respectively, and of the latter group 22.3, 18.6 and 21 cells / mm, respectively. two cases in which sepsis or significant infection was documented (cases 22 and 8) had mean cd45, cd68 and cd163 counts of 19.3, 27.4 and 34.1 cells / mm, respectively. in group 1, 11 of 17 cases had some form of hypoxic / ischaemic event (pontosubicular neuronal necrosis, hypoxic - ischaemic encephalopathy, infarction, periventricular leukomalacia and hypoxic ischaemic changes). the mean cd45, cd68 and cd163 positive cells / mm for this subgroup was 11.4, 15.5 and 19.0 cells / mm, respectively. cases in the hypoxic / ischaemic subgroup incorporated cases in each of the other subgroups. two cases had evidence of organising haemorrhages (epidural, subdural or subarachnoid ; cases 26 and 28). the mean cd45, cd68 and cd163 cell counts / mm in this subgroup were 33, 35.8 and 40.7, respectively. these three cases had the highest mean cd45, cd68 and cd163 counts within group 1. these three cases also contained cells in the leptomeninges with stainable iron in at least one section. an additional 12 cases in group 1 demonstrated some degree of iron staining in at least one brain section.fig. 1mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 1) mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 1) overall within group 1, the mean number of cd45, cd68 and cd163 cells in the cerebellum and brain stem was similar, with slightly higher numbers being observed in the cortex (fig. 1) looking specifically at the cases in group 2 (table 2 and fig.. the mean density of cd45, cd68 and cd163 immunoreactive cells / mm was 9.4, 16.6 and 23.2, respectively. two cases in which sepsis or significant infection was documented had mean cd45, cd68 and cd163 counts of 31.8, 28 and 33 cells / mm, respectively. in group 2, 10 of 16 cases had some form of hypoxic / ischaemic event as noted above with cd45, cd68 and cd163 positive cells of 18, 20.5 and 24.6 cells / mm, respectively. seven cases had some form of haemorrhage involving the dural surface or extending into the subarachnoid space. the number of cd45, cd68 and cd163 immunoreactive cells / mm in this subgroup was 13.5, 25.5 and 32.4 cells / mm, respectively. five cases of seven in this subset with associated haemorrhage also had evidence of iron staining. two cases with no reported neuropathologic findings had cd45, cd68 and cd163 counts of 5.9, 6.2 and 11.5 cells / mm, respectively. an additional eight cases in group 2 demonstrated some degree of iron staining in at least one brain section.fig. 2mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 2) mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 2) overall within group 2, the mean number of cd45, cd68 and cd163 was similar across the locations sampled (fig. 2). in three cases within both groups without neuropathologic abnormalities, all classes of inflammatory cells were found in the leptomeninges (case 17 of group 1 ; cases 2 and 6 of group 2). of the 19 cases in which caesarean sections were performed, 16 had positive iron findings, whereas 8 cases of the 13 vaginal births were positive for iron. fifteen cases of caesarean section births were associated with some form of hypoxic / ischaemic event in contrast to seven cases involving vaginal births. sixteen cases involved infants who died beyond the post - natal age of 33 days (group 1), and 17 cases represented either foetuses or newborns who survived up to 33 post - natal days (group 2). one child (number 7) who survived to 16 months of age was included in group 1. there were two cases (numbers 25 and 30) involving foetuses in the 26th and 28th weeks of gestation which were included in group 2. the general autopsy and neuropathology findings of both groups overlapped, and these included congestive heart failure, noonan s syndrome, micrencephaly and pontosubicular neuronal necrosis (tables 1 and 2).table 1general autopsy and neuropathologic findings for infants with a post - natal age greater than 33 days (group 1)case nogeneral autopsy findingsneuropathology findings1chronic aspiration / pneumonitis with mosfoedema with herniation ; hieseizure disorder4noonan s syndromemicrencephaly ; pontosubicular neuronal necrosis5trisomy 21 with chdhaemorrhagic infarct (left periventricular)7chdmicrencephaly8interstitial pneumonitis (cultures negative)pvl9heterotaxy asplenia syndromemicrencephaly ; multiple cortical and wm infarcts11congenital pulmonary malformationmultifocal acute hi changes12pulmonary hypoplasiaremote and focal acute hi changes14noneagenesis of cc(brain only)17chd with infarctionnone22cardiomyopathymultiple cerebral infarctionssepsis with mosf23foetal hydropspvl ; right occipital infarction26noonan s syndromemicrencephaly ; hypomyelination ; organising sah, sdh27liver failuremetabolic astrogliosis ; neuronal pyknosis ; bilateral subdural membranes28rsv bronchiolitisremote occipital infarct ; subacute edhpna33chdcystic infarct (frontal) cc corpus callosum, chd congenital heart disease, edh epidural haemorrhage, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, mosf multiple organ system failure, pna pneumonia, pvl periventricular leukomalacia, rsv respiratory syncytial virus, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white mattertable 2general autopsy and neuropathologic findings for foetuses and infants with a post - gestational age up to 33 days (group 2)case no.general autopsy findingsneuropathologic findings2chdnone3chdmicrencephaly;arhinencephaly;neuronal necrosis, subiculum6harlequin ichthyosisnone10diaphragmatic herniaomphalocoelepsnn13pna (strep)metabolic encephalopathy;hydrocephalus;partial agenesis of cc15chdthoracic meningomyelocoele;hydrocephalus ; possible partial agenesis of cc16chdorganising sdh18cardiomyopathyacute hi change (focal);digeorge syndromepsnn;remote dural haemorrhage19pulmonary htnpsnn20foetal hydropsgm haemorrhage extending to sa;diffuse hie21tetralogy of fallotpsnn ; bg infarct24chdfocal sah;small wm haemorrhages;psnn25chdpsnn;focal hi, cerebellum;pvl;parietal infarct;organising sdh, ivh, sah29none (brain only)diffuse hie30nec (mosf)meningoencephalitis;psnn31multiple congenital abnormalities including chdgm haemorrhage with ivh and sah ; psnn;arhinencephaly32pulmonary hypoplasia / htngm haemorrhage with iv and sah ; psnn bg basal ganglia, cc corpus callosum, chd congenital heart disease, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, htn hypertension, iv intraventricular, mosf multiple organ system failure, nec necrotizing enterocolitis, pna pneumonia, pvl periventricular leukomalacia, sa subarachnoid, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white matter, psnn pontosubicular neuronal necrosis general autopsy and neuropathologic findings for infants with a post - natal age greater than 33 days (group 1) cc corpus callosum, chd congenital heart disease, edh epidural haemorrhage, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, mosf multiple organ system failure, pna pneumonia, pvl periventricular leukomalacia, rsv respiratory syncytial virus, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white matter general autopsy and neuropathologic findings for foetuses and infants with a post - gestational age up to 33 days (group 2) bg basal ganglia, cc corpus callosum, chd congenital heart disease, gm germinal matrix, hi hypoxic / ischaemic, hie hypoxic / ischaemic encephalopathy, htn hypertension, iv intraventricular, mosf multiple organ system failure, nec necrotizing enterocolitis, pna pneumonia, pvl periventricular leukomalacia, sa subarachnoid, sah subarachnoid haemorrhage, sdh subdural haemorrhage, wm white matter, psnn pontosubicular neuronal necrosis table 3 demonstrates the number of slides from each brain region and the range of length, mean and standard deviation of associated leptomeninges scored per slide. overall, 39 sites were sampled from cerebral cortices, 37 from brainstems and 30 from cerebella. overall, no statistically significant differences between the mean number of immunoreactive cells in the cerebellum, brain stem and cortex were observed between groups 1 and 2 (table 4).table 3site and range, mean and standard deviations of lengths of leptomeninges in all casessitesno. of sites sampledleptomeningeal length (mm)mean (mm) + / sdcerebral cortex3954118.8 + / 11.1table 4test for statistically significant differences between the mean number of immunoreactive cells in the cerebellum, brain stem and cortex between groups 1 and 2comparison (group 1 vs. group 2) p - valuecerebellumcd450.623cd680.235cd1630.457brain stemcd450.689cd680.337cd1630.172cerebral cortexcd450.247cd680.149cd1630.204 site and range, mean and standard deviations of lengths of leptomeninges in all cases test for statistically significant differences between the mean number of immunoreactive cells in the cerebellum, brain stem and cortex between groups 1 and 2 looking specifically at the cases in group 1 (table 1 and fig. 1), four had congenital heart disease (chd) and two were diagnosed with noonan s syndrome. the mean number of cd45, cd68 and cd163 immunoreactive cells per mm of leptomeninges of the former group was 14.4, 17.5 and 17.9 cells / mm, respectively, and of the latter group 22.3, 18.6 and 21 cells / mm, respectively. two cases in which sepsis or significant infection was documented (cases 22 and 8) had mean cd45, cd68 and cd163 counts of 19.3, 27.4 and 34.1 cells / mm, respectively. in group 1, 11 of 17 cases had some form of hypoxic / ischaemic event (pontosubicular neuronal necrosis, hypoxic - ischaemic encephalopathy, infarction, periventricular leukomalacia and hypoxic ischaemic changes). the mean cd45, cd68 and cd163 positive cells / mm for this subgroup was 11.4, 15.5 and 19.0 cells / mm, respectively. cases in the hypoxic / ischaemic subgroup incorporated cases in each of the other subgroups. two cases had evidence of organising haemorrhages (epidural, subdural or subarachnoid ; cases 26 and 28). the mean cd45, cd68 and cd163 cell counts / mm in this subgroup were 33, 35.8 and 40.7, respectively. these three cases had the highest mean cd45, cd68 and cd163 counts within group 1. these three cases also contained cells in the leptomeninges with stainable iron in at least one section. an additional 12 cases in group 1 demonstrated some degree of iron staining in at least one brain section.fig. 1mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 1) mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 1) overall within group 1, the mean number of cd45, cd68 and cd163 cells in the cerebellum and brain stem was similar, with slightly higher numbers being observed in the cortex (fig. looking specifically at the cases in group 2 (table 2 and fig. the mean density of cd45, cd68 and cd163 immunoreactive cells / mm was 9.4, 16.6 and 23.2, respectively. two cases in which sepsis or significant infection was documented had mean cd45, cd68 and cd163 counts of 31.8, 28 and 33 cells / mm, respectively. in group 2, 10 of 16 cases had some form of hypoxic / ischaemic event as noted above with cd45, cd68 and cd163 positive cells of 18, 20.5 and 24.6 cells / mm, respectively. seven cases had some form of haemorrhage involving the dural surface or extending into the subarachnoid space. the number of cd45, cd68 and cd163 immunoreactive cells / mm in this subgroup was 13.5, 25.5 and 32.4 cells / mm, respectively. five cases of seven in this subset with associated haemorrhage also had evidence of iron staining. two cases with no reported neuropathologic findings had cd45, cd68 and cd163 counts of 5.9, 6.2 and 11.5 cells / mm, respectively. an additional eight cases in group 2 demonstrated some degree of iron staining in at least one brain section.fig. 2mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 2) mean immunoreactive cells / mm in cerebral cortex, brainstem and cerebellum (group 2) overall within group 2, the mean number of cd45, cd68 and cd163 was similar across the locations sampled (fig. 2). in three cases within both groups without neuropathologic abnormalities, all classes of inflammatory cells were found in the leptomeninges (case 17 of group 1 ; cases 2 and 6 of group 2). of the 19 cases in which caesarean sections were performed, 16 had positive iron findings, whereas 8 cases of the 13 vaginal births were positive for iron. fifteen cases of caesarean section births were associated with some form of hypoxic / ischaemic event in contrast to seven cases involving vaginal births. in the current study, we found the presence of inflammatory cells in the leptomeninges, both overall and when segregated into two groups by age (foetal and early post - natal vs. infants beyond 33 days post - natal life) and by anatomic and neuropathologic conditions (fig. a notable finding is that even in foetuses and infants with no neuropathologic abnormalities, inflammatory cells and occasionally iron was identified in the leptomeninges. this is in contrast to the widely held belief that the leptomeninges should be largely devoid of inflammatory cells and iron in children with no reported neuropathology. in addition, there was a positive correlation between the mean number of cd163 positive immunoreactive cells in cases with edh, sdh or sah pooled from both groups 1 and 2, when compared to those without these hemorrhages (mean immunoreactive cd163 cells / mm 30.34 vs. 15.12 ; p = 0.022). interestingly, this correlation was not detected for the number of cd45 or cd68 positive cells and probably reflects the higher sensitivity of cd163 as a marker for monocytic cells (fig. cells in the leptomeninges demonstrating appropriate positive staining are indicated with red arrows, and the cells and other constituents demonstrating non - specific staining are indicated with blunt black arrows. a vascular lumen containing some cells with appropriate staining is indicated by a yellow star. 4cd68 (panel a) and cd163 (panel b) immunostained sections (600) from same case depicted in fig. 3. in each panel, cerebral cortex is on the right side. in panel a, portions of two vessels (yellow stars) containing cd68 immunoreactive cells are also seen representative case with cd45 immunostain (600). cells in the leptomeninges demonstrating appropriate positive staining are indicated with red arrows, and the cells and other constituents demonstrating non - specific staining are indicated with blunt black arrows. a vascular lumen containing some cells with appropriate staining is indicated by a yellow star. the brown blush elsewhere within the vessel represents additional non - specific staining cd68 (panel a) and cd163 (panel b) immunostained sections (600) from same case depicted in fig. a, portions of two vessels (yellow stars) containing cd68 immunoreactive cells are also seen caesarean section deliveries and vaginal births were associated with a variety of anatomic and neuropathologic diagnoses, with hypoxic / ischaemic events commonly found in both modes of delivery. both modes of delivery were also associated with iron deposition in the leptomeninges and haemorrhage - related neuropathology (fig. 5example of iron staining in the leptomeninges (600) in a different case than shown in figs. 3 and 4. the leptomeninges are diagonally oriented downward from left to right and contain a large number of cells with an appropriate granular intracellular blue reaction product. in the upper right corner are some cells with non - specific staining example of iron staining in the leptomeninges (600) in a different case than shown in figs. 3 and 4. the leptomeninges are diagonally oriented downward from left to right and contain a large number of cells with an appropriate granular intracellular blue reaction product. in the upper right corner are some cells with non - specific staining accordingly, it appears that the presence of iron in the leptomeninges does not necessarily equate to traumatic haemorrhage but may be found in completely naturally occurring processes and occurs irrespective of the mode of delivery. there also seemed to be no recurring pattern allowing us to associate the presence of iron to a single anatomic or neuropathologic diagnosis. there are multiple mechanisms which allow the brain to sense inflammatory signals from systemic circulation, including interactions with circulating molecules in areas in the brain devoid of the brain early migration of microglia from the ventricular and meningeal surfaces has been observed as early as 4.5 weeks of gestation in humans. animal studies have demonstrated that inflammation, either introduced systemically or within the brain, causes microglial activation along with cytokine release. accordingly, there is evidence to suggest that infection distant from the brain may damage developing foetal brain. the activation of neuroinflammatory responses may also sensitise the brain to the damaging effects of other insults, such as hypoxia / ischaemia, and amplify the effects of the latter. thus, there are multiple possible reasons to account for the presence of inflammatory cells within the leptomeninges early in gestation in humans outside of inflicted trauma. we conclude that all patients with various natural disease processes in this hospital - based population had measureable numbers of cd45, cd68 and cd163 immunoreactive cells (and a subset had iron containing cells) in the leptomeninges overall and when segregated by age and anatomic and neuropathologic diagnoses. although we studied a larger number of cases than the dura study by croft., our numbers of total cases are relatively small, particularly in consideration of the varied anatomic and neuropathologic diagnoses. our cases were derived only from hospital autopsies, and these observations require comparison to actual forensic cases involving both traumatic injuries and natural disease processes. clearly, the presence of inflammatory cells and iron in the leptomeninges can occur commonly, and in significant numbers, in non - traumatic neuropathologic conditions. as this study evaluated only non - forensic cases, we did not define a threshold level of inflammatory or iron containing cells above which a non - natural disease process (including inflicted injury) would be implicated. thus, these findings support the recommendation of cautious interpretation of the findings of leptomeningeal inflammation and iron in forensic cases. | objectivesnotwithstanding the lack of definitive evidence from studies conducted to date, inflammatory infiltrates and iron deposition in the leptomeninges are routinely used as forensic markers of traumatic brain injury. we investigated the presence of these forensic markers of trauma in neonates and infants, with the objective of determining their suitability for use in forensic cases.methodsleptomeninges derived from non - traumatic deaths were studied. thirty - three cases were divided into groups 1 and 2, according to set age groups. inflammatory cells and iron in these groups were quantified.resultscd45, cd68 and cd163 positive inflammatory cells were identified in the leptomeninges of sections of the cerebellum, brain stem and cortex of all 33 cases of non - traumatic infant deaths surveyed in this study. there were no significant differences between the two groups. iron was found in the leptomeninges in several cases, even those without recent haemorrhage. overall within the two subgroups, the numbers of inflammatory cells and iron containing cells were not significantly different.conclusionthese findings demonstrate that inflammatory cells and iron in the leptomeninges can be found in natural and non - traumatic conditions. further, two cases with no reported neuropathology demonstrated the presence of inflammatory cells and iron. thus, cautious interpretation of the presence of inflammatory cells and iron containing cells in forensic paediatric cases is recommended. |
their occurrence was first described in 1670 by thilesus. however, at that time fistulas were a common complication of chronic and untreated cholecystitis. according to a 2005 study, 226 cases have been reported in total, with fewer than 25 in the last 50 years. the reduced incidence in current times can be attributed to more rapid diagnosis and treatment with antibiotics or surgery. although occurring in acalculous cholecystitis and carcinoma of the gallbladder, fistulas are still most commonly associated with gallstones [3, 4 ]. obstruction of the cystic duct leads to an increase in gallbladder pressure and reduced perfusion with necrosis, which consequently causes gallbladder perforation. the contents of the gallbladder may then empty into the peritoneal cavity and an abscess may form or a fistula may develop through adherence to the duodenum, colon or abdominal wall, often via the fundus of the gallbladder. the right upper quadrant is the most common location for the exit tract of the fistula, but locations such as the gluteal region, umbilicus and right groin have also been documented. cholecystocutaneous fistulas are most often seen in elderly women over the age of 60, likely due to coexistent disease and non - specific symptoms interfering with diagnosis. a white 85-year - old female with hypertension and a previous history of breast biopsy underwent endoscopic retrograde cholangiopancreatography with sphincterotomy after initially presenting on may 3, 2011 with common duct stones. the patient was initially seen in the emergency department complaining of a 3-day history of sharp intermittent epigastric and right upper quadrant pain radiating to the central back. mild scleral icterus was noted, but there were no signs of jaundice or lymphadenopathy. her abdomen was soft, non - distended and mildly tender to palpation with a positive murphy 's sign. routine blood work demonstrated an elevated white blood cell count of 16.1, no abnormalities on sma7, elevated lipase > 3,000, and elevated liver function testing including an alkaline phosphatase of 215, a bilirubin of 41, an ast of 100, a ggt of 305 and an alt of 194. clinical evidence of mild jaundice accompanied by blood work abnormalities and positive radiological signs led to the diagnosis of acute calculous cholecystitis, common bile duct stones up to 7 mm in size and biliary gallstone pancreatitis. she was treated conservatively with intravenous antibiotics and underwent endoscopic retrograde cholangiopancreatography with sphincterotomy for removal of several stones of various sizes. percutaneous cholecystostomy was then carried out for drainage of the gallbladder after development and medical control of atrial fibrillation. on june 1, 2011 she was re - admitted to the hospital with a left lower lobe pulmonary embolism. on june 27, 2011 the percutaneous drain was removed at her request. in early august 2011, she re - developed right upper quadrant discomfort ; furthermore, she noted some purulent drainage from the percutaneous drain site and extrusion of approximately 30 gallstones. she had several follow - up ultrasounds which identified a fistulous tract measuring 0.78 cm in diameter communicating with the external opening in the right upper quadrant (fig. an irregular hypoechoic area just inside the subcutaneous tissue measuring 4.1 2.7 cm was presumed to represent a contracted gallbladder. plans were made for laparoscopic cholecystectomy and management of her cholecystocutaneous fistula on february 22, 2012, once she finished her coumadin regiment. in the morning of the operation, on february 22, 2012, the patient 's inr was still elevated at 1.8 and the surgery was re - scheduled for a month later. on april 18, 2012 the patient underwent laparoscopic cholecystectomy and fistula division (fig. 2). three additional gallstones were found in the gallbladder at the time of the operation. we present the case of an 85-year - old white female who was diagnosed with a cholecystocutaneous fistula that developed as a complication following removal of a percutaneous drain that was used to treat her acute cholecystitis. re - occurrence of her cholecystitis after drain removal and the presence of gallstones promoted the production of a fistula along the pre - existing tract of the drain. her concurrent treatment with anticoagulants for a pulmonary embolism delayed the definitive management of her cholecystitis and fistula. fortunately, the patient remained in reasonably good health throughout the waiting period from time of fistula diagnosis to surgery. more conservative approaches such as percutaneous cholecystotomy have been used in high - risk patients, leading to spontaneous closure of the fistula. however, in this case the fistula developed through the old drain tract, so surgical intervention was employed. as with uncomplicated cholecystitis, laparoscopic techniques are favorable compared to open surgery and thus a laparoscopic cholecystectomy was undertaken in this case. the gallstones removed during cholecystectomy were of orange - brown color consistent with cholesterol stones. although fistula formation is now a rare complication of cholecystitis, it remains a possibility and should be considered in the differential diagnosis of any fistulous tract in the right abdominal wall. we have demonstrated that previous percutaneous drainage of an acute gallbladder infection can promote the formation of such a fistula if the infection is not properly dealt with or re - occurs. physicians should be prepared to recognize this complication in patients after drain removal and prior to definitive surgery. | cases of cholecystocutaneous fistulas are now a rare occurrence as a result of rapid diagnosis and treatment. we present a case of cholecystocutaneous fistula developing after the removal of a percutaneous drain for the treatment of acute cholecystitis. re - occurring infection and presence of gallstones led to fistulization of the gallbladder fundus and the development of a tract along the path created by the drain. the patient presented with re - occurring right upper quadrant abdominal pain, purulent discharge from the fistulous opening and expulsion of multiple gallstones. she underwent laparoscopic cholecystectomy and fistula excision. |
tropical fevers are defined as infections that are prevalent in or are unique to tropical and subtropical regions. they occur in different parts of the indian subcontinent throughout the year, especially in rainy and postrainy season. these fevers include leptospirosis, enteric fever, malaria, dengue, scrub typhus (can be remembered as mnemonic lemds) and some other fevers. they have overlapping clinical presentation, multi - system involvement, require intensive care unit (icu) care, and are associated with high morbidity and mortality. clinical picture of these diseases is usually so overlapping that it is not always possible to achieve differential diagnosis in emergency and icu settings. a syndromic approach is usually followed for diagnosis and treatment of these infections. the major five clinical syndromes are undifferentiated fever, fever with rash or thrombocytopenia, fever with acute respiratory distress syndrome, fever with encephalopathy, and fever with multi - organ dysfunction syndrome (mods). here, we report a case presented in rainy season with fever, rash, and mods, which was initially investigated and managed as tropical fever, but later, a diagnosis of drug rash with eosinophilia and systemic symptoms (dress) was established. a 32-year - old woman presented with fever, skin rash, dyspnea, jaundice, and decreased urine output in the emergency department on july 5, 2015. the patient had been well until 10 days before admission when low - grade fever developed. two days after the onset of fever, she went to a local practitioner and was prescribed oral ciprofloxacin. next day, she became afebrile but she developed maculopapular skin rash with itching, which started on the bilateral upper limbs and progressed to involve whole body in the next 2448 h [figure 1 ]. the same day, she developed shortness of breath which progressed to dyspnea at rest within 2 days with dry cough and orthopnea. three days before admission, the patient developed jaundice with high - colored urine without cholestatic symptoms, and her urine output reduced to 500 ml / day. her menstrual cycles were normal, and she was married with two children without abortions. on examination, she was conscious and oriented with blood pressure of 110/56 mm of hg, pulse rate of 110/min, respiratory rate of 26/min, temperature of 39c, and oxygen saturation 96% on oxygen supplementation with fio2 0.5 and flow 12 l / min. she had icterus, facial and bilateral hand swelling, as well as bilateral symmetrical pitting pedal edema. there were nontender discrete lymph nodes in the bilateral cervical regions ; the largest was on the right side and measured 2 cm in diameter. skin rash was diffuse maculopapular involving whole body (> 90% body surface area), nonblanching with occasional pustules. a presumptive diagnosis of tropical fever with rash and multi - organ involvement was made, and the patient was empirically started of ceftriaxone and doxycycline. laboratory investigation showed hemoglobin of 9.1 g / dl ; total leukocyte count of 21.2 10/l with 48% neutrophils, 15% lymphocytes, 1% monocytes, and 36% eosinophils (absolute eosinophil count 7.6 10/l) ; platelet counts of 281 10/l ; creatinine level of 2.4 mg / dl ; total bilirubin of 7.3 mg / dl with conjugated fraction of 6.7 mg / dl ; aspartate aminotransferase (ast), alanine aminotransferase (alt), and alkaline phosphatase (alp) of 1048 iu / l, 965 iu / l, and 332 iu / l, respectively ; international normalized ratio of 1.8 ; and serum albumin of 2.0 g / dl. serum lipase and amylase levels were 381 u / l and 375 u / l, respectively. chest x - ray showed bilateral mild pleural effusion and ultrasonography abdomen revealed hepatosplenomegaly with moderate ascites. rapid kit test and peripheral smear for malaria, serological tests for dengue (ns1 ag, igm and igg antibody), leptospirosis (igm antibody), scrub typhus (igm antibody), widal, viral hepatitis (hepatitis a virus igm, hepatitis e virus igm, hepatitis b surface antigen, anti - hepatitis c virus antibody), hiv elisa, and antinuclear antibody gave negative results. a skin biopsy was done which was suggestive of drug rash with thinned out epidermis, flattening of rete pegs, spongiosis and vacuolization of dermoepidermal junction, presence of apoptotic keratinocytes, and dermis showing perivascular lymphocytic inflammatory infiltrates. a diagnosis of dress secondary to ciprofloxacin with hepatic, hematological, renal, and pulmonary involvement was made. antibiotics were stopped and she was started on prednisolone 1 mg / kg / day, following which she had a rapid improvement in systemic symptoms and eosinophilia. at discharge on july 17, absolute eosinophil count was 0.35 10/l, serum creatinine 0.43 mg / dl, total bilirubin 2.0 mg / dl, and ast, alt, and alp 166, 335, and 200, iu / l respectively. on outpatient department follow - up, the patient had flare of skin rash on steroid withdrawal. common culprit drugs are aromatic anticonvulsants, antidepressants, allopurinol, antimicrobials, sulfonamides and sulfones, and nonsteroidal anti - inflammatory drugs. antimicrobial commonly involved are abacavir, nevirapine, minocycline, linezolid, doxycycline, nitrofurantoin, metronidazole, piperacillin - tazobactam, and ceftriaxone. rash is symmetric, confluent, pruritic, maculopapular and affects face, upper trunk, upper extremities first, followed by lower extremities. hepatic involvement occurs in 50%70% cases, which presents as tender hepatomegaly, transaminitis, granulomatous hepatitis, and even fulminant hepatic failure. renal involvement is seen in 10%30% patients and manifests as hematuria, mild proteinuria, interstitial nephritis, or acute renal failure. hematological abnormalities occur as eosinophilia, atypical lymphocytosis, and lymphadenopathy. lung (interstitial pneumonitis, pleural effusion), heart (eosinophilic myocarditis, pericarditis), pancreas (pancreatitis), muscle (myositis), and other organs may be involved. the regiscar scoring system is most commonly used criteria to diagnose dress [table 1 ]. our case was diagnosed as a definite case of dress with score 8 (fever > 38c, lymphadenopathy, eosinophilia 36%, typical rash, multi - organ involvement with excluding other potential causes and resolution in > 15 days). regiscar diagnosis score for drug rash with eosinophilia and systemic symptoms prompt diagnosis and withdrawal of the offending drug are important. high - dose corticosteroids (0.51 mg / kg / day) are the cornerstone of therapy. a dramatic improvement is seen soon after corticosteroids ; however, it should be slowly tapered while the function of affected organs is closely monitored to minimize recurrences. | we report a case of ciprofloxacin - related drug rash with eosinophilia and systemic symptoms (dress) which was initially diagnosed and managed on the line of tropical fever. later, a diagnosis of definite case of dress was made according to the regiscar scoring system and the patient was managed with the removal of ciprofloxacin along with steroids. dress is a great masquerader. the diagnosis should be highly suspected in the presence of fever, skin rash, liver involvement, and hypereosinophilia. |
angiogenesis, the formation of new vessels from existing vessels, plays an important role in pathological conditions in various organs. pathological angiogenesis in the eye is the most common cause of blindness in all age groups. retinopathy of prematurity (rop) occurs in children, diabetic retinopathy (dr) in young adults, and age - related macular degeneration (amd) in the elderly. it is important to understand the mechanism of underlying pathological neovascularization to identify new targets to treat these diseases. vascular endothelial growth factor (vegf) is thought to be the major angiogenesis factor in rop, dr, and amd. recently, some evidence indicates that chronic inflammation is also implicated in the pathogenesis of retinal neovascularization [4, 5 ]. the high - mobility group box-1 (hmgb1) protein was initially discovered as a nuclear chromatin - binding protein that stabilizes nucleosome formation and facilitates transcription. hmgb1 can be actively secreted by various cell types, including activated monocytes and macrophages, and endothelial cells, after inflammatory stimuli [7, 8 ]. extracellular hmgb1 functions as a proinflammatory cytokine [6, 9 ] and exhibits angiogenic effects [10, 11 ]. hmgb1 signals through the receptor for advanced glycation end products (rage) leading to the activation of the transcription factor nuclear factor kappa b (nf-b) and induces the expression of various leukocyte adhesion molecules proinflammatory cytokines, chemokines, and angiogenic factors [6, 9 ]. in a previous report, increased levels of hmgb1 were found in vitreous samples from patients with proliferative dr, and hmgb1 expression was upregulated in the retinas of diabetic mice. ethyl pyruvate (ep) is derived from pyruvate by the addition of an aliphatic ester group and is more stable and safer than pyruvate in inhibiting the generation of reactive oxygen species (ros) and inflammation. moreover, ep inhibits tumor angiogenesis and intracerebral hemorrhage - induced angiogenesis [16, 17 ]. however, the pathogenic role of hmgb1 and the effect of its inhibitor, ep, in pathological retinal neovascularization have remained uncertain. in the present study, we examined whether ep has a preventive effect against pathological retinal neovascularization and inhibits hmgb1 expression in a mouse model of oxygen - induced retinopathy (oir). oir was induced in mouse (c57bl/6) pups according to smith. with some modifications. briefly, postnatal day 7 (p7) mice, with their nursing mothers, were exposed to hyperoxia (75 2% oxygen) for five days (p7p11) to produce retinal vasoobliteration and then returned to normoxia (room air) for five days (p12p16) to induce ischemic retinal neovascularization. at p12, after the pups were exposed to 75% oxygen, they were randomly assigned to three groups : ep-50 (50 mg / kg / day), ep-100 (100 mg / kg / day), and the oir. the normal group was maintained in room air from birth until postnatal day 17 (p17). louis, mo, usa) was diluted with hartmann 's solution (6.0 g nacl, 0.3 g kcl, 0.2 g cacl22h2o, and 3.1 g c3h5nao3/l, choongwae pharma, republic of korea). the pups were injected in 100 l intraperitoneally once a day for five days. in the normal group, hartmann 's solution was injected. at p17, after five days of intraperitoneal injection, the mice were anesthetized and sacrificed. all of the experiments were approved by the korean institute of oriental medicine institutional animal care and use committee. at p17, the mice were deeply anesthetized and then 0.1 ml of phosphate - buffered saline (pbs) containing 5 mg of fluorescein - dextran (fd40s, sigma, st. the retinas were dissected, flat mounted onto glass slides, and viewed by fluorescence microscopy (bx51, olympus, tokyo, japan). quantification of the central nonperfused area of the retina was performed using the imagej software (nih, md, usa). flat - mounted retinas were fixed in 4% paraformaldehyde for 3 h at room temperature. the retinas were washed with pbs and then incubated for 3 h on an orbital shaker at room temperature with 5% triton x-100 and 1% bsa. the retinas were washed with pbs and incubated overnight at 4c with bandeiraea simplicifolia isolectin b4 (1 : 50, sigma - aldrich, st. louis, mo) diluted in pbs. the retinas were washed with 0.05% tween 20 in pbs followed by incubation with streptavidin tritc (1 : 500, serotec, oxford, uk) for 4 h at 37c. the samples were added in extraction buffer, 20 mm tris hcl buffers (ph 4) with 2% sds, incubated on ice for 5 min, and mixed by vortexing then boiled at 100c for 20 min followed by an optional incubation at 80c for 2 hours. after protein extraction, any remaining unsolubilized material was pelleted at 14000 g for 20 min, and protein concentration of total protein extracted was determined by the bca protein assay (pierce chemicals co., rockford, il, usa). the protein was separated by sds - polyacrylamide gel electrophoresis and transferred to pvdf membrane (biorad, ca, usa). membrane was probed with anti - hmgb1 antibody (abcam, ma, usa) and anti -actin antibody (sigma, mo, usa), and then the immune complexes were visualized with an enhanced chemiluminescence detection system (amersham bioscience, nj, usa). the eyes were removed and fixed with davidson 's solution at room temperature for 24 h and embedded in paraffin. the paraffin sections were deparaffinized, hydrated with water, and stained with hematoxylin and eosin. for immunohistochemical hmgb1 staining, paraffin sections were deparaffinized, rinsed with 3% hydrogen peroxide, and boiled in citrate buffer (ph 6.0) for 15 min. these sections were washed with pbs, blocked with cas - block (invitrogen, ca, usa) for 30 min, and subsequently incubated overnight at 4c with a rabbit anti - hmgb1 antibody (1 : 1000, epitomics, ca, usa). after washing with pbs, the sections were incubated with polymer peroxidase - conjugated rabbit anti - igg for 30 min at room temperature and were visualized using a 3,3-diaminobenzidine tetrahydrochloride (immpact dab, vector, ca, usa) solution and hematoxylin (vector, ca, usa). the data were expressed as the mean se and analyzed by one - way analysis of variance (anova) followed by tukey 's multiple comparison test or by an unpaired student 's t - test using graphpad prism 6.0 software (graphpad, ca, usa). hmgb1 has been recognized as a proinflammatory cytokine and more recently as a proangiogenic factor [6, 9 ]. we therefore determined the expression levels of hmgb1 and its distribution in the retina of oir mice by immunohistochemistry. first, retinal neovascularization was qualitatively analyzed using fluorescein angiography and quantitatively analyzed by counting neovascular tufts using isolectin b4 and h&e staining. the oir mice showed a characteristic loss of central retinal vessels by p12, followed by hypoxia - induced regeneration of the central vascular plexus and the development of preretinal neovascularization. in the oir group, the neovascular response occurred predominantly at the junction between the nonperfused retina and perfused retina. the retinas of oir mice had an area of multiple neovascular tufts (figures 1 and 2). in western blot analysis and immunohistochemical staining for hmgb1, we found that hmgb1 was increased in the retinas of oir mice compared with the control group (figures 3 and 4). hmgb1 is highly expressed in ganglion cells, inner nuclear layers, outer nuclear layer, and retinal vasculatures, and hmgb1 levels were higher in the inner nuclear layer than the outer nuclear layer in oir mice (figure 4). elevated expression of hmgb1 was detected in the nuclei as well as in the cytoplasm in oir mice. these results suggest that hmgb1 is highly produced and translocates into the cytoplasm in retina of oir mice. it was previously reported that a dramatic alteration of retinal gene expression occurred in oir mice, and a high level of retinal hmgb1 expression might be related to active gene transcription. thus, an increase in hmgb1 might be related to active angiogenic - related gene transcription., we examined whether ep has a preventive effect against pathological retinal neovascularization and inhibits hmgb1 expression. to test whether pharmacological inhibition of hmgb1 decreases pathological retinal neovascularization, the oir mice induced the formation of neovascular tufts that penetrate the internal limiting membrane of the retina (figure 2). five days after the injection (at p17), there were dose - dependently fewer neovascular tufts in the ep - injected eyes compared to the oir group (figure 4, p < 0.05). moreover, the treatment with ep significantly reduced retinal hmgb1 expression in oir mice (figures 3 and 4). it thus seemed that hmgb1 in the retina of oir mice has a proangiogenic role and ep has an antiangiogenic effect via inhibition of the production and release of hmgb1. in addition, the retinas of oir mice had an area of nonperfusion in the center, tortuous vessels (figure 1). however, the ep (100 mg / kg)-treated mice had a significantly smaller nonperfused area compared to controls (figure 1, p < 0.01). retinal neovascularization (rnv) is a major cause of blindness associated with ischemic retinopathy [2, 23 ]. oir is a well - established animal model of proliferative ischemic retinopathy. in the oir model, revascularization of the vasoobliteration (vo) area has progressed farther and pathologic neovascularization formation is at its maximum at p17. the vo area is then fully revascularized in all of the layers, and neovascularization is completely resolved at p25. in this study, to verify the pathogenic role of hmgb1 in retinal neovascularization, we determined the expression levels of hmgb1 and its distribution in the retina of oir mice and examined whether ep, a well - known hmgb1 inhibitor, could prevent this retinal neovascularization in oir mice. we showed that nuclear and cytoplasmic overexpression of hmgb1 and subsequent angiogenesis occurred in oir mice. however, the treatment with ep dramatically promoted stable vascular growth and blocked retinal pathologic neovascularization. based on these results, it is likely that the retinal pathogenic angiogenesis that occurs in ischemic retinopathy could also be linked to increases in hmgb1 expression. several angiogenic growth factors and cytokines are involved in the pathogenesis of retinal neovascularization [2527 ]. a critical role for hmgb1 in angiogenesis has been recently suggested by wake. who reported that histidine - rich glycoproteins block hmgb1-heparin complex - induced vessel sprouting in matrigel plugs. hmgb1 signals through the rage leading to activation of the transcription fact or nuclear factor kappa b (nf-b) and induces the expression of various angiogenic factors [9, 29 ]. administration significantly increases the levels of growth factors, including vegf, released by cultured human cardiac fibroblasts. although concrete evidence of hmgb1-induced retinal angiogenesis is lacking, our findings suggest that hmgb1 might provide the mechanistic link between ischemic retinopathy and pathogenic angiogenesis. in oir, ros are increased in the retina and nicotinamide adenine dinucleotide phosphate (nadph) oxidase is activated, causing apoptosis in endothelial cells, which contributes to avascular retinas. nadph oxidase activation is exacerbated, contributing to angiogenic blood vessel growth into the vitreous. ros are increased in the retina, which directly correlates with vegf expression and angiogenesis. in addition, oxidative stress in various cells is induced in ischemic injury, and then hmgb1 is actively secreted into the extracellular environment under these conditions. hydrogen peroxide (h2o2) and the loss of superoxide dismutase 1 (sod1) mediated oxidative stress promote cytosolic hmgb1 expression and extracellular release. although we did not test the retinal oxidative status in our oir mice, these findings suggest that increased retinal hmgb1 expression might be mediated through retinal ros generation. in this study, we showed that ep treatment prevents the overexpression of nuclear and cytoplasmic hmgb1 and retinal pathogenic angiogenesis in oir mice. ep has salutary effects in lethal sepsis and systemic inflammation, hemorrhagic shock, and stroke and pressure - induced retinal damage and ameliorates murine colitis and renal ischemia and reperfusion injury. the pharmacological effects of ep include amelioration of redox - mediated damage to cells and tissues and the scavenging of ros [16, 42 ]. ep regulates the activation of the expression of inflammatory proteins such as il-6 and tnf- and the inflammatory transcription factor nf-b [43, 44 ]. this pharmacological agent has been shown to interfere specifically with hmgb1 released from the nucleus into the extracellular space. in a previous study, ep prevented the increase of hmgb1 mrna expression after raw264.7 cells were stimulated with lps. although ep is expected to exert an antioxidant effect, ep could be a potent hmgb1 inhibitor. this observation suggests that the antiangiogenic effect of ep is attributable, at least in part, to not only its antioxidative but also hmgb1-inhibitory properties. therefore, to confirm a proangiogenic role of hmgb1, we also performed a similar animal study using a selective inhibitor of hmgb1, glycyrrhizin (gl). gl is a triterpenoid saponin glycoside of glycyrrhizic acid and its mechanism of action is different from ep. ep inhibits the expression and cytoplasmic release of hmgb1 but do not bind directly to hmgb1 and thus can not block its extracellular functions. in contrast, gl does not interfere with hmgb1 release, but directly inhibit its extracellular cytokine activities. gl treatment also significantly reduced the area of neovascularization in retinas compared with oir (see supplementary data in supplementary material available online at http://dx.doi.org/10.1155/2013/245271). this observation also strongly supports the proangiogenic role of hmgb1 in oir mice. in conclusion, our study shows for the first time that ep inhibits the retinal pathogenic angiogenesis induced by ischemic retinopathy in oir mice. in addition, hmgb1 overexpression induced by ischemic retinopathy was significantly inhibited by treatment with ep. these observations suggest that ep acts through an anti - hmgb1 mechanism to protect against retinal pathogenic angiogenesis. taken together, these results indicate that treatment with ep could be a valuable therapeutic approach in the treatment or prevention of ischemic retinopathy. | retinal pathogenic angiogenesis in the eyes is a causative factor in retinopathy of prematurity, diabetic retinopathy, and age - related macular degeneration. this study was designed to examine the pathogenic role of the high - mobility group box-1 (hmgb1) protein and the inhibitory effect of ethyl pyruvate (ep), a well - known antioxidant substance, in retinal pathogenic angiogenesis in mice with oxygen - induced retinopathy (oir), one of the animal models of proliferative ischemic retinopathy. the oir mouse model was used for our in vivo studies. the mice were exposed to 75% oxygen from postnatal day 7 (p7) to p11, after which the mice were brought to room air and intraperitoneally injected with ep (50 mg / kg, or 100 mg / kg) for five days. at p17, the mice were perfused with fluorescein isothiocyanate - dextran, and flat - mounted retinas were used to measure nonperfused and neovascular tufts. in oir mice, an intraperitoneal injection of ep reduced the nonperfused retinal area in the treatment group and significantly reduced the retinal neovascular tufts. in addition, ep inhibited the overexpression of hmgb1 in the retinas of oir mice. these data suggest that ep could serve as an innovative pharmaceutical agent to prevent retinal neovascularization through inhibiting hmgb1 expression. |
smallholder agriculture in semiarid areas is hampered by accelerated soil erosion, induced soil moisture deficits, soil fertility depletion, and soil crusting and compaction. the soil crusting and compaction problem is attributed to inherent soil properties and poor tillage practices. the semiarid areas of kenya are characterized by temporal and spatial variability of rainfall which is not suitable for sustainable rainfed agriculture [1, 3 ]. thus, resource - poor farmers in these areas who entirely rely on rainfed agriculture are subject to various hydrological constraints. the rainfall occurrence is bimodal with two distinct rainy seasons : the short and long rains. in kenya 's semiarid areas, the short rains (october to december) are more reliable and are evenly distributed and more often support plant growth. however, very high soil moisture deficits experienced in these areas usually result in significant decreases in crop yields under rainfed conditions. deficit of soil moisture in these areas is attributed to low infiltration rates caused by surface sealing, crusting, low organic matter content, and subsequent high runoff rates [1, 5, 6 ]. soil moisture conservation under such conditions requires appropriate tillage practices that not only improve rainwater infiltration but also conserve adequate soil moisture for plant growth. soil tillage, as a necessary practice in crop production, can affect the soil physical properties that are important for plant growth [8, 9 ]. improvements of root penetration, water infiltration and soil moisture storage, weed control, and supply of nutrients from rapid decomposition of organic matter are considered the most beneficial contributions of tillage to crop production [1012 ]. conventional tillage involves the mechanical soil manipulation of an entire field, by ploughing (inverting the soil) followed by one or more harrowings. the degree of soil disturbance depends on the type of implement used, the number of passes, type of soil, and intended crop type. the most common conventional tillage practiced in kenya involves the use of hand hoes, ox - drawn mould board ploughs, tractor - drawn disc ploughs, and harrows combined with straw collection and burning during land preparation [2, 14 ]. during the operation, the soils are cut, inverted, and pulverized burying most of the residues underneath. consequently, in many areas, especially semiarid areas, conventional agriculture has led to a decline in crop yields and profitability when compared to those realized from areas with higher rainfall. other sustainable techniques advocated in the semiarid areas for soil and water conservation have included tied ridging, cover cropping, mulching, subsoiling and ripping [16, 17 ]. these techniques are referred to as conservation tillage and have the potential of soil moisture retention and mitigation of intraseasonal dry spells that often result in low productivity and crop failure, reduce soil evaporation, and enable organic matter build - up which enhances water holding capacity of the soil [1820 ]. conservation tillage practices are particularly important for increasing the productivity of water through reducing runoff and evaporation and improving soil water storage. in kenya, promotion of animal - drawn conservation tillage tools such as rippers and subsoilers among smallholder farmers has resulted in improved water productivity and crop yields [21, 22 ]. although conservation tillage is highly advocated, there is strong evidence that this kind of tillage may not be good with soils prone to surface crusting and sealing, a characteristic of most of the soils in the semiarid areas of kenya [2326 ]. therefore, the local biophysical conditions in the smallholder farming systems in these semiarid areas need to be considered and deliberate adaptation efforts made. the success of any tillage practices is directly related to the improvement of the soil physical properties which in turn may affect the growth and yield of crops due to the different soil conditions created. in a review on the effect of soil physical properties on soil moisture, strudley. showed that bulk density, porosity, soil surface sealing and crusting, surface roughness, hydraulic conductivity, and infiltration rates were highly influenced by different tillage. therefore, the choice of any tillage system is too critical for maintenance of the soil physical properties necessary for crop growth. however, the resulting effect of tillage on selected physical properties depends on the site - specific biophysical environment such as seasonal variability in rainfall, inherent soil fertility status, or the prevailing climatic conditions. few studies have measured these soil physical properties, especially in semiarid eastern kenya, hence the essence for the study reported herein. recent efforts have focused on minimum tillage practices as a soil and water conservation measure [30, 31 ]. nonetheless, maize - legume cropping systems are popular in improving land use efficiency and economic returns. in order to exploit these practices, there is a need to understand the influence of tillage and cropping systems on selected soil physical properties in dryland cropping systems for sustained productivity. the objective of this study was to investigate the effect of selected tillage practices and cropping systems on selected soil physical properties in semiarid mwala district, eastern kenya. specifically, their effects on soil moisture, soil surface roughness, crust strength, saturated hydraulic conductivity, bulk density and porosity. the study was conducted in mbiuni location, mwala district, machakos county, eastern kenya (115s, 3725e). the area is characterized by low, erratic, and poorly distributed bimodal rainfall that makes crop production difficult under rainfed conditions. the long rains commence in mid - march and end in may while short rains start in mid - october and end in late november. pulses are grown in the district and the predominant ones are beans, pigeon peas, cowpeas, green grams, and chickpeas. soil chemical and physical properties at the site are given in table 1. the trials were established in 2012 and ran for four seasons during the long (lr) (march may) and short rains (sr) (october december) (i.e., lr 2012, sr 2012, lr 2013, and sr 2013). the treatments consisted of six tillage practices : disc ploughing (dp), disc ploughing and harrowing (dph), ox - ploughing (ox), hand hoeing with tied ridges (htr), hand hoeing only (h) and subsoiling - ripping (ssr). the cropping systems treatments were sole maize, sole bean, and maize - bean intercrop. the treatment factors were arranged in a split - plot design with tillage practices as the main plots and the cropping system as the subplots and replicated four times in plot sizes of 25 m. time in weeks after planting (wap) and season were considered as experimental factors to test the changes within a growing season and across the different cropping seasons. soil moisture was monitored from crop emergence to harvesting at depths of 020 cm and 2040 cm using the gravimetric method. monitoring of soil moisture was done at the 040 cm depth due to the concentration of active roots at this level and less below this depth range. soil surface roughness was measured immediately after the tillage operations and before weeding was done. readings were taken from three randomly selected positions in each tillage plot. a microrelief meter similar to that described by kuipers crust strength (penetration resistance) was measured at the soil surface (010 cm depth) using a hand - held penetrometer (eijkelkamp equipment type 1b) in three seasons (sr 2012, lr 2013, and sr 2013). ten soil crust strength measurements were taken at randomly selected positions in each plot. the penetrometer springs and cone sizes crust strength was calculated as (1)cr = i csac, where cr is the cone resistance (n cm), i is the impression on the scale (cm), cs is the spring constant (n cm), and ac is the area of the cone (cm). saturated hydraulic conductivity (ksat) determinations were done in the laboratory using the constant head method as described by klute and dirksen. undisturbed soil core rings (5 cm depth, volume 98.13 cm) collected from the field were carefully trimmed to the size of the core ring. a piece of muslin cloth held with a rubber band was used on one side of the core ring to protect the soil from spilling but to allow water to pass through. the volume of water that passed through the soil sample was measured and recorded until a constant average was achieved. the ksat was then calculated as (2)ksat=qlath, where q is the discharge or percolate through the soil (cm or ml), l is the length of the soil core (cm), a is the cross - sectional area of the soil core (cm), t is the time taken (hours), and h is the hydraulic head difference (cm). the bulk density was determined using undisturbed core samples from each plot. soil core samples were carefully trimmed at both sides to the size of the core ring. the soil core was then oven - dried at 105c to a constant mass and then weighed. bulk density was then calculated as the mass of the dry soil divided by the core ring volume. total porosity was then calculated from the bulk density as (3)total porosity=1bs, where b is the bulk density and s is the average particle density (2.65 mg / m). the ksat, bulk density, and total porosity were determined at one or two intervals in a growing season (the beginning of the season (3 wap) and at harvest). a dryland maize variety (dh 02) and beans (rose coco : glp 2) were used as the test crops. the maize was planted at a spacing of 90 30 cm in pure stands while the bean plants were planted at a spacing of 45 15 cm. in the tied ridging plots, maize and beans thinning was done to a single plant per hill for maize and two plants for the legume, four weeks after germination. in each cropping system, the nitrogen was applied at 60 kg n ha (dap 18 : 46 : 0) at planting to the maize and additional 60 kg n ha (can 26 : 0 : 0) was top - dressed when maize was knee - high (4 wap). all plots were hand - weeded using a hand hoe as practiced by the farmers during the cropping periods. no crop rotation was done during the study. in analysis of the soil properties, analysis of variance (anova) with repeated measures mean separation was done using lsd at 5% level of probability where the anova f - values were significant. tillage practices resulted in significant differences in moisture content in lr 2012 (p = 0.019), lr 2013 (p = 0.003), and sr 2013 (p = 0.001) but not in sr 2012 (p = 0.158) (table 2). however, in sr 2012, higher moisture levels were observed in ox (15.3%) and ssr (14.4%). the ox and ssr plots maintained high moisture levels in the sr 2012 and lr 2013. subsoiling - ripping (ssr) and ox - ploughing (ox) enhance water penetration into deeper soil layers and allow deeper rooting of crops that benefit from stored water and nutrients as they exploit larger soil volumes. steiner and rockstrom made similar observations. in lr 2012, the htr plots had high moisture levels (15.6%) due to the microbasin formation that traps rainwater and allowed more time for infiltration and storage. in contrast to lr 2012, htr had the lowest moisture (8.42%) in the lr 2013 season. similar findings are supported by guzha, who found that the higher moisture was stored in ridges and this was associated with higher roughness resulting from ridge configuration. tied ridges create rectangular basins between ridges, which increase surface retention capacity and decrease runoff, thus improving soil moisture content and ultimately crop growth and yields. found that tied ridges retained significantly higher moisture than conventional tillage (ploughing) especially during the dry months. while gicheru., found that tied ridging conserved the lowest amount of moisture and attributed this to no runoff to impound and high evaporation losses due to increased soil surface area exposure in the semiarid areas of laikipia district, kenya.. in semiarid turkey found that ridge tillage accelerated drying of the seed zone ; thus, low moisture contents were observed. some water logging was observed in the lr 2012 season only and could explain the high moisture by htr (15.61%) noted and hence the lowest moisture contents in the succeeding seasons (table 2). therefore, the effectiveness of tied ridges depends on the rainfall received and climatic conditions within a season. the soil moisture decreased over time (wap) during the four growing seasons (p ssr > dph > h > htr > dp (p dph > h. soil surface roughness soon after tillage was significantly higher among the tillage practices (p ox > dph > h > dp > htr with values ranging from 1.03 to 1.18 mpa (table 4). the crust formed on the ridges was weaker than other tillage practices as the season progressed. a probable cause of this was the inversion and mixing of top soil when constructing the tied ridges and this could have affected the structure of the top soil. however, at the base of the ridge basins, the crust strength was higher in all the seasons because of the excessive drying of the soil in the basin owing to direct insolation. the crust strength trend observed during the lr 2013 season was in the decreasing order of ssr > ox > dp > htr > h > dph with average values ranging from 1.42 to 1.50 mpa (table 4). lower crust strength in h and dph could be due to the loosening effect of the tillage used. during the sr 2013 season, a trend of ssr > ox > h > dp > dph > htr was observed with values ranging from 1.14 to 1.22 mpa. a three - season average trend shows a decreasing trend of ssr > ox > h > dp > dph > htr with values ranging from 1.21 to 1.30 mpa. these results are in line with those reported by khurshid. where soil under conventional tillage (use of rigger in ridge tillage and use of a cultivator in deep tillage plots) had lower penetration resistance than minimum tillage (dibbling) treatments in the semiarid faisalabad, pakistan. therefore, intensive tillage as observed in dp, dph, and htr causes a breakdown of soil surface sealing resulting in the weak crust strength observed (table 4). the high crust strength observed in the ssr plots could be attributed to nonploughing of the soils between the planting rows. this premise is supported by miriti. who observed higher crust in subsoiling - ripping (0.47 mpa) followed by tied ridging (0.40 mpa) and ox - ploughing (0.15 mpa), respectively, working in makueni district, eastern kenya. the cropping systems did not significantly affect (p = 0.379) the crust strength but an overall average trend of intercrop (1.263 mpa) > sole maize (1.258 mpa) > sole bean (1.251 mpa) was noted. penetrometer readings greater than 2 mpa are generally reported to produce a significant root growth reduction which was not observed in this study. according to lampurlans and cantero - martnez, penetration resistance measured with a penetrometer is usually two to eight times greater than that actually undergone by the root tip, owing to the different way in which roots and probes penetrate the soil. however, roots can grow at a speed greater than the penetrometer reading because they can elongate through the biopores and interaggregate spaces. thus, the crust strength obtained in this study in all treatments and seasons is not expected to limit root penetration and plant growth. the bulk densities of the soils ranged from 1.20 mg m to 1.42 mg m across the seasons which are within the acceptable range for a clay loam (table 1). the current study shows variation across the seasons (p ssr > h > ox > htr > dp. the cropping systems did not influence the bulk densities in the four seasons (p = 0.502). in the sr 2012 season, the bulk density increased (1.34 mg m) at the end of the season from 1.27 mg m at the beginning of that season, while, in the sr 2013, higher bulk density (1.55 mg m) was noted at the beginning of sr 2013 and was lower at the end of that season (1.35 mg m). according to husnjak., tillage at the beginning of the growing season temporarily decreases soil bulk density but subsequent trips in the field for agronomic practices, rainfall events, and other disturbances activities can recompact the soil. lower bulk density at the end of the growing season could be attributed to the short term loosening effect of the tillage method used. the high densities in dph and ssr could be attributed to the second passes of soil manipulation (harrowing and ripping) to the initial tillage method of disc ploughing and subsoiling in the respective plots. agbede found high bulk densities in the ploughing plus harrowing plots and attributed that to tractor wheel traffic and implement passes and lower macroporosity. the plough layer gets compacted as the tillage implement keeps passing the same depth season after season, thus increasing the bulk density. eventually, such structural degradation can lead to a formation of a surface seal that further reduces infiltration and hinders seed germination. the increase in bulk density in the ssr plots especially at the beginning of a season could also be attributed to the compacted unploughed sections during the land preparation. nonsignificance of tillage effect on bulk density over time has also been observed in other studies by lampurlans and cantero - martnez, anken. [62, 63 ] reported higher bulk density with minimum tillage and lower bulk density with deep tillage. mcvay. reported that bulk density was greater in no - till than conventional tillage in a long term study at manhattan and ashland bottoms sites in kansas, usa. the bulk density is dependent on soil texture and the densities of soil minerals as well as their packing arrangement. gomez. realized that it took five years before changes in some of the soil physical properties (structure and aggregate stability, which are indicators of bulk density) could be detected as a result of the soil management practices. full effect of tillage is observed after four to five years and this could not be obtained in our short term study. total porosity values of the soils varied across the seasons (p ox > dph > dp > h > ssr with values ranging from 0.66 cm / h to 0.98 cm / h. there were no significant interactions observed among the cropping systems (p = 0.744). the low ksat values in ssr plots have been reported elsewhere by miriti. in makueni district, kenya, who attributed it to the restricted lateral movement of water due to the low porosity in the unploughed sections. non - significant results on saturated hydraulic conductivity (ksat) over time have also been observed by chang and lindwall after 20 years of tillage in a clay loam soil in alberta. furthermore, inversion tillage (ploughing) makes the aggregates unstable during wetting which could cause lower ksat. pikul jr and aase demonstrated that continuous tillage for 11 years in a dryland site in eastern montana, usa, resulted in low ksat due to a compacted 1015 cm layer that impeded water movement. a soil profile description done at this study site showed a high bulk density of 1.54 mg / m in the ap horizon (010 cm depth) that impeded water infiltration as indicated by the low ksat value of 0.37 cm / h obtained. green. also reported that the effects of tillage on the soil hydraulic properties under different tillage treatments are not always consistent across locations, soils and experiment designs. the overall low ksat values observed imply low infiltration rates and thus low rainwater intake by the soil. the low ksat values could also be influenced by the soil particle size which is reflected in the texture of the soil. this soil has a clay loam texture (39% clay), which has smaller grains and thus lower hydraulic conductivity. these low values observed in this study also indicate the presence of a hardpan and thus deep subsoiling is required to improve the soil water uptake [55, 71 ]. soil moisture conservation is important during land preparation and crop growth due to climate change, which affects the amount of rainfall and the rainfall seasons. the soil moisture trends by tillage and cropping systems were inconsistent in each cropping season. however, on average, the ox, ssr, and the dph conserved the highest moisture. nonsignificant results by tillage and cropping systems were also observed on bulk density, porosity and ksat values. the dp, htr, and ox plots had numerically greater porosity (> 45%) due to the lower bulk density achieved by the tillage method used. based on the soil physical properties measured in this study, there was no significant advantage of a tillage practice over the other as the soil physical properties developed slowly after initiation of tillage practices. this suggests that long term tillage experiments (> 4 seasons) under similar environmental and soil conditions are required to detect changes in soil physical properties as a result of the soil management practices. the long term studies will thus provide site - specific recommendations of the appropriate tillage practices for adoption in these semiarid areas. | a field study was carried out to evaluate the effects of tillage practices on soil physical properties in mwala district, eastern kenya, during the long rains (lr) and short rains (sr) of 2012/13. the treatments were disc ploughing (dp), disc ploughing and harrowing (dph), ox - ploughing (ox), subsoiling - ripping (ssr), hand hoeing with tied ridges (htr), hand hoeing only (h). these were investigated under three cropping systems of sole maize, sole bean, and maize - bean intercrop in a split - plot design with four replications. soil physical properties were monitored at different weeks after planting (wap) throughout the growing seasons. a four - season average shows that soil moisture content was significantly (p ssr > dph > h > htr > dp with values ranging from 13.1 to 14.1%. soil surface roughness and crust strength varied significantly (p 4 seasons) would be required to detect changes in soil physical properties as a result of the soil management practices. |
pregabalin (pg) chemically is 3-amino methyl hexanoic acid, with the chemical formula c8h17no2. its structural and pharmacological features correspond to the mammalian neurotransmitter gamma - aminobutyric acid (gaba), and it is primarily used as anticonvulsant drug. however, its effects are much broader being analgesic, antiepileptic, antidiabetic, and anti - inflammatory drug. further more, it has been recommended for gastrointestinal damage, alcoholism, and insomnia. exact mechanism is not known ; however, it has been an established fact that it binds to calcium channel in the central nervous system which decreases calcium influx at nerve endings and therefore reduces the liberation of several associated neurotransmitters which subsequently results in the above - mentioned activities. tranexamic acid (txa) its significant use is to treat ovarian tumors and it is recommended to manage pregnancy and reduce blood lose in surgery. it is considered as the best substitute to surgery in cases of menorrhagia [3, 4 ]. however, analytical method reported for pg includes spectrophotometry [59 ], spectrofluorimetry, and high pressure liquid chromatography with uv detection, with mass detection, and with fluorescence detection. txa quantification has been reported through colorimetric spectrophotometry [1418 ], hplc, and spectrofluorometry. although chromatographic method offers high level of selectivity and specificity, the associated expenses are alarming and can not be shouldered by every pharmaceutical industry. moreover, the molecules of txa and pg lack uv absorbing chromospheres, so in case of hplc high load on column will deteriorate and reduce its life which turned out to be huge burden on low and middle economy - based industries. amino acids like txa and pg are highly polar and can not be easily volatilized, so gas chromatographic method would not be easy or straightforward. all these suggest that the proposed analytical methods are highly important from quality control point of view. the colorimetric reagents used in this study are trinitrophenol (tnp) and dinitrophenol (dnp). these reagents have wide range utility as quality colorimetric analyzing agents in pharmaceutical industry [2127 ]. a glance over the literature revealed few reports regarding charge transfer complexes for pg and txa. a structural analogous of pg and txa gabapentin has been determined by colorimetry - visible spectrophotometry by reacting with iodine, chloranil, chloranilic acid, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, tetracyanoethylene, methyl orange, hydroxy benzaldehyde, picric acid and with ninhydrin [2830 ]. colorimetric spectrophotometric methods for pg include reaction with 1,2-naphthoquinone-4-sulphonate sodium and 2,4-dinitrofluorobenzene, ascorbic acid and salicylaldehyde, 7,7,8,8-tetracyanoquinodimethane, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone, tetracyanoethylene and 2,3,5,6-tetrachloro-1,4-benzoquinone, quinalizarin and alizarin, and vanillin, acetyl acetone, and formaldehyde. colorimetric quantification reagent for tx includes ninhydrine [14, 15 ], ferric chloride, 1,2-naphthoquinone-4-sulphonate sodium, ascorbic acid, and azo dye. there is no report on txa and pg determination which utilizes tnp or dnp reagents. most of the reported spectrometric methods are insensitive or need complicated extractions and heating / cooling procedures or use reagents which do not produce linear response. moreover, most of the methods are based on the absorbance in near uv region and thus specificity is questioned. the method under proposal is highly selective and extremely simple, so it can be usefully adopted for routine analysis in quality control laboratories. the proposed methods are accompanied by the reaction of amine group of amino acid with hydroxyl group, activated by neighboring nitro groups of both tnp and dnp. as a result, yellow color complexes are formed on simple addition of the two reagents and no extra process is required. thermodynamics features in respect of association constant and standard free energy changes have been evaluated for both reactions. both of methods have been proved to be very useful from routine quality control prospective and can be considered as superior to most of the published methods with respect to pace, ease, low cost, and sensitivity. a uv - visible shimadzu spectrophotometer 1601 with 1 cm path length quartz cells controlled by shimadzu uv probe 3.9 version software was used. ms excel sheet was used to evaluate f, t tests, and other statistical parameters. pregabalin and tranexamic acid pure drugs were a kind gift from a local pharmaceutical agency having 99% plus purity. tranex capsules 250 and 500 mg and syngab capsules 200 mg, 50 mg, and 100 mg (atco laboratories ltd., karachi, pakistan) were procured from the market. microcrystalline cellulose (avicel ph 101, maize starch, magnesium stearate, pvp, aerosil r-200, crospovidone - x, hpmc (606), and peg (6000)), titanium dioxide, and isopropyl alcohol were a gift from a local pharmaceutical agency. the entire chemicals were used according to their safety precautionary measures. reagents and standard stock solutions preparation. a 2.27 gl tnp and 1.84 gl 2,4-dnp solutions were prepared individually in dichloromethane corresponding to 10 mmol of the concerned reagent. 160 gml of pg and 155 gml of txa were prepared individually by taking 16 and 15.5 mg weights, respectively, in 100 ml calibrated volumetric flasks, dissolved in 10 ml of water, and diluted to the mark level with the same solvent. stoichiometric study. to study stoichiometry of the reaction, equimolar solution (1 mmol) of each of the coloring reagents and drugs were prepared. in a series of 10 ml calibrated amber volumetric flasks, solutions of pg and txa were prepared individually, comprising different complementary proportions (0 : 10, 1 : 9, 2 : 8, 3 : 7, 4 : 6, 5 : 5,, 9 : 1) each with both of the reagents (tnp and dnp) separately. after 10 minutes of reaction the absorbance of the complex was measured at the wavelength of maximum absorption against the reagent blanks treated similarly. dnp method. 3 ml of drug pg or txa stock solution was taken individually in 10 ml amber volumetric flasks to which 4 ml of the coloring reagent dnp was added. absorbance was measured after 10 minutes of reaction at 418 nm using appropriate blank treated similarly. tnp method. 3 ml of drug pg or txa stock solution was taken individually in 10 ml amber volumetric flasks to which 4 ml of the coloring reagent tnp was added. absorbance was measured after 10 minutes of reaction at 425 nm using appropriate blank treated similarly. bulk homogenous powder equivalent to 10 mg of pregabalin or tranexamic acid was dissolved in methanol by sonication for 15 min and shaking thoroughly for about 3040 min. the samples were cooled down and diluted up to the mark level with methanol, mixed well, and filtered using a whatman number 42 filter paper to give 100 gml of pg and txa each, individually. 10 mg of each pg and 10 mg of txa were spiked individually with common excipients like magnesium stearate, hpmc (hydroxypropyl methylcellulose), glucose, pyrrolidone, lactose, talc powder, and starch. this reaction is based on the proton transfer reaction from lewis acid such as tnp and dnp to lewis base such as txa and pg, which produces intensely yellow color ion - pair complex as depicted by saito and matsunaga. standard free energy changes (g) and association constant (k c) were determined for both of the methods to evaluate thermodynamic aspects. the reaction of tnp and dnp each with pg and txa was investigated for the association constants by the application of benesi - hildebrand equation : (1)caa=1+1kc1cb, where c a and c b are the concentrations of the drug (pg or txa) and coloring reagent (dnp or tnp) respectively, a is the absorbance of the complex, is the molar absorptivity of the complex, and k c is the association constant of the complex. utilizing the above equation, a was plotted against c a in both cases and straight lines were obtained. standard free energy changes were calculated by the following equation:(2)g=2.356rtlogkc, where g is free energy change associated with complex (kj mol), r is the thermodynamic gas constant (1.987 cal mol deg), t is the kelvin temperature (273+c), and k c is the equilibrium constant. least square procedure was adopted to develop the regression equations which showed linear relation of the concentration of complex with absorbance, complying lambert beer 's law. under these experimental set - ups, absorbance at the given wavelength was found to vary directly with the concentrations of both the donner and the acceptor molecules. accuracy of the analytical method is that the parameter confirms that the test results obtained with the method are close to the true or accepted values, while precision is the reproducibility of test result when the same homogenous sample is subjected to multiple testing. solutions containing three different concentrations of pregabalin and tranexamic acid were arranged and investigated in triplicate for accuracy and precision. specificity is the quantification of the analytes in presence of component mixtures, excipients, and additives. the empirical formulas 3/s and 10/s were used to establish lod and loq for the method, where s is the slope and is the standard deviation of the response statistically inferred from calibration curve. a signal to noise ratio of 3 : 1 was defined for lod and of 10 : 1 was defined for loq, respectively. the reaction of coloring reagent tnp or dnp as lewis acids with amino acid (pg and txa) as lewis base afforded intense yellow charge transfer complex. the intensity in color is attributed to the formation of phenolate ion as shown in reaction scheme in figure 2. in order to achieve optimum experimental condition, various factors were evaluated including time, temperature, solvent of choice, and concentration of drugs and reagent. those experimental factors which were affecting the absorbing abilities of the resulting complex were optimized to enhance selectivity and sensitivity. the reaction and stability of the complex were evaluated with respect to time in intervals at room temperature. maximum absorbance was obtained in 5- to 10-minute time period and prolonged period of time did not affect the absorbance of the complex. however, a negative impact was noted when reaction mixture was heated especially beyond 60c. similarly, effect of coloring reagent was evaluated by adding it to the fixed concentration of drug substance. to 100 gml of drug substance, reagents tnp and dnp were added in the range from 1 to 9 ml. the amount of coloring reagent linearly increased the absorbance up to equimolar stage beyond which the absorbance remains constant at maximum absorbance, which corresponds to 4.0 ml of solution of each dnp and tnp. different solvents systems including chloroform, acetone, 2-propanol, acetonitrile, and dichloromethane were evaluated for optimum results. water was used to prepare stock solution of pg and txa because of the free solubility of the drugs and working range solutions were prepared in acetonitrile. it has been known that methanol interferes in charge transfer complex formation so acetonitrile was used in working range solutions in subsequent analysis. the solutions were scanned in visible range (800 to 350 nm) by spectrophotometer using the corresponding blank. it was found that, for tnp complex, max was 425 nm, while for dnp it was 418 nm which justifies intensity in color of complex derived from tnp as compared to that of dnp as in figure 3. in view of these results, all working solutions to reaction were prepared in acetonitrile in sequence of drug - reagent solvent, maintained at 25 2c. absorbance is measured at 418 and 425 nm for dnp and tnp complexes, respectively, after 10 minutes of mixing. job 's continuous variations method suggested a 1 : 1 molar ratio for both reagents with both drugs. however, experimental design considered the drug molecules to be limiting reactants, so the chromogenic reagent was taken in slight excess in subsequent analysis in order to make the reaction drug concentration dependent and to counter any possible interference. (k c) was in the range of 1.511.62 10 for dnp, while it was in the range of 2.742.86 10 for tnp and standard free energy changes were 2.336 to 2.358 for complex derived from dnp and 2.498 to 2.508 for tnp - based complex. high wavelength, high association constant, and low standard free energy changes with complex involving tnp as compared to dnp justify the resonance phenomenon in the phenolate ion derived from tnp compared to dnp. after development method validation studies were conducted in line with the above - mentioned protocol. using linear regression equation, methods showed linear response in concentration range from 0.02 to 200 g ml with correlation coefficient of more than 0.9990 in both cases. in all the cases studied, slopes were found to be in the range of 0.01018 to 0.0146. small intercept and insignificant variation of slope are attributes of the excellent response of the methods. low loq values of (0.0950.109) g ml explained sensitivity of the method as in table 2. obtained results in tables 3 and 4 are very close to 100% label claim, indicating excellent accuracy, while very good precision is obvious from the % relative standard deviation which is less than 2. during spiking experiments no interference was found which indicated that none of those additives possess enough basicity to cause interference in analysis of txa and pg. in addition, good % recoveries in common excipients spiked samples that ranged from 98.3 to 101.4% further confirmed the specificity of the projected methods. general procedures described above were followed to determine content of capsule dosage pharmulations for both of the drugs. the projected colorimetric methods based on charge - transfer complexes were applied to determine pg and txa, along with the reference methods 9 and 15 for pg and txa, respectively, to evaluate the test results. t - test and f test were carried out with ms excel, and the proposed methods were found to be comparable to the referenced methods and no considerable variation was found between them which indicated similar precision and accuracy. to estimate the quantity of tranexamic acid and pregabalin in commercial products, two simple and sensitive colorimetric methods were developed and validated. picric acid and 2,4-dinitrophenol, the two coloring reagents, have been utilized to develop two simple, much more common but sensitive and selective visible range spectrophotometric methods for routine analysis of tranexamic and pregabalin in bulk raw material and finished or semifinished dosage form. the suggested methods are superior to the already established spectrophotometric methods in terms of simplicity. hence, the proposed methods can be eagerly adopted by pharmaceutical quality control laboratories for routine quantitative analysis. | this paper demonstrates colorimetric visible spectrophotometric quantification methods for amino acid, namely, tranexamic acid and pregabalin. both drugs contain the amino group, and when they are reacted with 2,4-dinitrophenol and 2,4,6-trinitrophenol, they give rise to yellow colored complexes showing absorption maximum at 418 nm and 425 nm, respectively, based on the lewis acid base reaction. detailed optimization process and stoichiometric studies were conducted along with investigation of thermodynamic features, that is, association constant and standard free energy changes. the method was linear over the concentration range of 0.02200 gml1 with correlation coefficient of more than 0.9990 in all of the cases. limit of detection was in range from 0.0041 to 0.0094 gml1 and limit of quantification was in the range from 0.0137 to 0.0302 gml1. excellent recovery in placebo spiked samples indicated that there is no interference from common excipients. the analytical methods under proposal were successfully applied to determine tranexamic acid and pregabalin in commercial products. t - test and f ratio were evaluated without noticeable difference between the proposed and reference methods. |
world health organization and unaids report about 39.5 million hiv - infected people worldwide and about 2 million people in latin america. in 2009, around 70,000 people were estimated to be infected in peru. the advent of haart has altered the course of hiv infection, transforming it from a fatal illness to a chronic condition and thereby reducing morbidity and mortality. however, antiretroviral therapy has also led to an increased incidence of metabolic problems such as ir, dyslipidemia, lipodystrophy and impaired glucose metabolism. haart comprises a combination of at least three active antiretroviral drugs against the virus which belong to different classes of drugs with different sites of action. the preferred initial regimen is the combination of two nucleoside reverse transcriptase inhibitors with a non - nucleoside reverse transcriptase inhibitor or with a pi. the antiretroviral drugs most frequently associated with the development of lipodystrophy and ir are the pis such as lopinavir, ritonavir and nelfinavir. ir, impaired glucose tolerance (igt) and type 2 diabetes mellitus (t2 dm) are part of a plurimetabolic syndrome associated with haart, thus, the detection of this syndrome should be systematic. guidelines recommend testing fpg at the time of hiv diagnosis, before initiating haart, and every 612 months during treatment. also, performing an oral glucose tolerance test when a patient presents with abnormal fasting glucose this strategy allows the detection of most cases of t2 dm ; however, it misses some cases of ir. an early diagnosis of ir could allow for lifestyle modification in order to prevent progression to t2 dm. the homa mathematical model is a clinical and epidemiological tool used to estimate ir based on plasma levels of fasting glucose and insulin. in order to investigate the prevalence of ir and the potentially implicated factors, we conducted a cross - sectional study in a cohort of peruvian hiv - patients on haart using the homa - ir. moreover, we described the prevalence of ms according to the criteria proposed by the national cholesterol education program s adult treatment panel iii report. the study was approved by the institutional review board of the universidad peruana cayetano heredia and of the hospital nacional cayetano heredia. authors take complete responsibility for the integrity of the data and the accuracy of the data analysis. a cross - sectional study of adult patients with hiv infection on haart was carried out between june and october 2012 at the institute of tropical medicine alexander patients recruited were adults over 18 years of age diagnosed with hiv infection on haart. we excluded patients with a known history of carbohydrate metabolism disorders (igt, t2 dm), diseases that alter insulin sensitivity (cushing syndrome, acromegaly, polycystic ovary syndrome), use of corticosteroids, and pregnancy. we obtained clinical records that included the following variables : age, gender, family history of diabetes in a first degree relative, smoking, blood pressure, weight, height, bmi, waist circumference, regimen and duration of haart. we obtained a fasting serum sample to measure levels of glucose, insulin, and lipids. insulin level determination was performed using the immunoradiometric assay based on the separation of antibody - coated tube (ins - irma diasource - belgium). this method has a detection limit, defined as the concentration resulting in two standard deviations above the average link of the zero calibrator, 1 uiu / ml. the coefficient of variation intra - assay and inter - assay were 1.5 % and 6.5 % respectively. hyperglycemia was considered as a fasting glucose value 100 mg / dl. total cholesterol, high - density lipoprotein cholesterol (hdl - c), and triglycerides were determined by enzymatic method. low - density lipoprotein cholesterol (ldl - c) was calculated according to the friedewald formula. the presence of ir was determined by the homa mathematical model using the following formula : [insulin (u / ml glucose (mmol / l)/22.5 ]. this cutoff point was determined in a previous study conducted at the hospital nacional cayetano heredia in subjects with normal glucose tolerance tests. continuous variables were summarized using means and standard deviations, and comparisons between groups with and without ir were made using the student s t - test. categorical variables were summarized by frequencies and percentages, and their association with the outcome variable (ir) was determined using fisher s exact test. generalized linear models with the backward stepwise method, removing variable with p values over 0.05. prevalence ratios, with their corresponding confidence intervals, were calculated as measures of excess risk. the analysis was performed with the statistical program stata se version 11 (college station, tx). the study was approved by the institutional review board of the universidad peruana cayetano heredia and of the hospital nacional cayetano heredia. authors take complete responsibility for the integrity of the data and the accuracy of the data analysis. a cross - sectional study of adult patients with hiv infection on haart was carried out between june and october 2012 at the institute of tropical medicine alexander patients recruited were adults over 18 years of age diagnosed with hiv infection on haart. we excluded patients with a known history of carbohydrate metabolism disorders (igt, t2 dm), diseases that alter insulin sensitivity (cushing syndrome, acromegaly, polycystic ovary syndrome), use of corticosteroids, and pregnancy. we obtained clinical records that included the following variables : age, gender, family history of diabetes in a first degree relative, smoking, blood pressure, weight, height, bmi, waist circumference, regimen and duration of haart. we obtained a fasting serum sample to measure levels of glucose, insulin, and lipids. insulin level determination was performed using the immunoradiometric assay based on the separation of antibody - coated tube (ins - irma diasource - belgium). this method has a detection limit, defined as the concentration resulting in two standard deviations above the average link of the zero calibrator, 1 uiu / ml. the coefficient of variation intra - assay and inter - assay were 1.5 % and 6.5 % respectively. hyperglycemia was considered as a fasting glucose value 100 mg / dl. total cholesterol, high - density lipoprotein cholesterol (hdl - c), and triglycerides were determined by enzymatic method. low - density lipoprotein cholesterol (ldl - c) was calculated according to the friedewald formula. the presence of ir was determined by the homa mathematical model using the following formula : [insulin (u / ml glucose (mmol / l)/22.5 ]. this cutoff point was determined in a previous study conducted at the hospital nacional cayetano heredia in subjects with normal glucose tolerance tests. continuous variables were summarized using means and standard deviations, and comparisons between groups with and without ir were made using the student s t - test. categorical variables were summarized by frequencies and percentages, and their association with the outcome variable (ir) was determined using fisher s exact test. generalized linear models with the backward stepwise method, removing variable with p values over 0.05. prevalence ratios, with their corresponding confidence intervals, were calculated as measures of excess risk. the analysis was performed with the statistical program stata se version 11 (college station, tx). the demographic information, family and social history of 219 subjects are presented in table 1. the mean age of the study population was 38.4 10.1 years. most of the participants were male (67.1 %), 23.3 % had a first - degree family history of t2 dm and 24.7 % patients were smokers. regarding the treatment, (76.3 %) subjects were on haart over a year and the most commonly used regimen did not include a pi (90.9 %).table 1comparison of general characteristics of hiv - infected patients on haart with and without insulin resistance (ir)general characteristics (n = 219)total (n = 219)no ir (n = 144)ir (n = 75)pr p male, n (%) 147 (67.1)96 (66.7)51 (68.0)1.0410.844age0.007 35 years, n (%) 95 (43.4)70 (48.6)25 (33.3)1.000reference 36 - 45 years, n (%) 74 (33.8)50 (34.7)24 (32.0)1.2320.398 46 years, n (%) 50 (22.8)24 (16.7)26 (34.7)1.9760.002hispanics, n (%) 190 (86.7)124 (86.1)66 (88.0)1.1190.709family history of t2 dm, n (%) 51 (23.3)34 (23.6)17 (22.7)0.9650.877cigarrette smoker, n (%) 54 (24.7)40 (27.8)14 (18.7)0.7010.186>1 year on haart, n (%) 167 (76.3)106 (73.6)61 (81.3)1.3570.249haart regimen with pi, n (%) 20 (9.1)11 (7.6)9 (12.0)1.3570.235abbreviations : haart highly active antiretroviral therapy ; ir insulin resistance ; pr prevalence ratio ; pi protease inhibitor ; t2 dm type 2 diabetes mellitus comparison of general characteristics of hiv - infected patients on haart with and without insulin resistance (ir) abbreviations : haart highly active antiretroviral therapy ; ir insulin resistance ; pr prevalence ratio ; pi protease inhibitor ; t2 dm type 2 diabetes mellitus the mean bmi was 24.5 3.6. most patients had a normal weight (55.3 %), 32.0 % overweight, 8.2 % obese and 4.6 % underweight. the mean abdominal circumference in men and women was 88.8 9.0 cm and 87.1 10.3 cm, respectively (table 2). laboratory results are shown in table 3.table 2clinical characteristics of hiv - infected patients on haart with and without insulin resistance (ir)clinical characteristicstotal (n = 219)no ir (n = 144)ir (n = 75) p sbp, mmhg104.7 11.9103.6 11.4106.8 12.60.060dbp, mmhg68.5 8.267.5 7.870.4 8.60.013weight, kg64.7 11.563.0 11.667.9 10.70.003bmi, kg / m 24.5 3.623.8 3.625.6 3.50.001bmi categories0.001 underweight, n (%) 10 (4.6)9 (6.3)1 (1.3)0.535 normal weight, n (%) 121 (55.3)91 (63.2)30 (40.0)reference overweight, n (%) 70 (32.0)35 (24.3)35 (46.7)0.011 obesity, n (%) 18 (8.2)9 (6.3)9 (12.0) 125 g / dl9 (4.1)1 (0.7)8 (10.7)2.750.004high blood pressure, n (%) 8 (3.7)4 (2.8)4 (5.3)1.4860.258abbreviations : hdl - c high - density lipoprotein cholesterol, ir insulin resistance comparison of metabolic syndrome and its components in hiv - infected patients on haart with and without ir abbreviations : hdl - c high - density lipoprotein cholesterol, ir insulin resistance hyperglycemia was found in 103 (47 %) subjects, 9 (4.1 %) of them had serum glucose > 125 mg / dl. the prevalence of hypertriglyceridemia and low hdl - c was 48.4 % and 57.5 % respectively (table 4). subjects with ir were older, had higher bmi, and were more likely to have hyperglycemia, central obesity and diastolic hypertension. with regard to the lipid profile, although the patients with ir had higher triglycerides levels, no significant differences were found when the variable was categorized as hypertriglyceridemia. there were no differences in gender, family history of t2 dm, cigarette smoking, duration of haart, using pi and systolic blood pressure (sbp). in multivariate analysis, the factors associated with ir were age 46 years (pr = 2.767, 95 % ci 1.325 to 5.780) and greater bmi (pr = 1.148, 95 % ci 1.054 to 1.250) (table 5).table 5multivariate logistic regression analysis for ir in hiv - infected patients on haartfactorpr p 95 % ciage (years)0.014 351.000reference 36 - 451.0700.8480.5332.150 462.7670.0071.3255.780bmi1.1480.0021.0541.250abbreviations : bmi body mass index, ci confidence interval, pr prevalence ratio multivariate logistic regression analysis for ir in hiv - infected patients on haart abbreviations : bmi body mass index, ci confidence interval, pr prevalence ratio the demographic information, family and social history of 219 subjects are presented in table 1. the mean age of the study population was 38.4 10.1 years. most of the participants were male (67.1 %), 23.3 % had a first - degree family history of t2 dm and 24.7 % patients were smokers. regarding the treatment, (76.3 %) subjects were on haart over a year and the most commonly used regimen did not include a pi (90.9 %).table 1comparison of general characteristics of hiv - infected patients on haart with and without insulin resistance (ir)general characteristics (n = 219)total (n = 219)no ir (n = 144)ir (n = 75)pr p male, n (%) 147 (67.1)96 (66.7)51 (68.0)1.0410.844age0.007 35 years, n (%) 95 (43.4)70 (48.6)25 (33.3)1.000reference 36 - 45 years, n (%) 74 (33.8)50 (34.7)24 (32.0)1.2320.398 46 years, n (%) 50 (22.8)24 (16.7)26 (34.7)1.9760.002hispanics, n (%) 190 (86.7)124 (86.1)66 (88.0)1.1190.709family history of t2 dm, n (%) 51 (23.3)34 (23.6)17 (22.7)0.9650.877cigarrette smoker, n (%) 54 (24.7)40 (27.8)14 (18.7)0.7010.186>1 year on haart, n (%) 167 (76.3)106 (73.6)61 (81.3)1.3570.249haart regimen with pi, n (%) 20 (9.1)11 (7.6)9 (12.0)1.3570.235abbreviations : haart highly active antiretroviral therapy ; ir insulin resistance ; pr prevalence ratio ; pi protease inhibitor ; t2 dm type 2 diabetes mellitus comparison of general characteristics of hiv - infected patients on haart with and without insulin resistance (ir) abbreviations : haart highly active antiretroviral therapy ; ir insulin resistance ; pr prevalence ratio ; pi protease inhibitor ; t2 dm type 2 diabetes mellitus most patients had a normal weight (55.3 %), 32.0 % overweight, 8.2 % obese and 4.6 % underweight. the mean abdominal circumference in men and women was 88.8 9.0 cm and 87.1 10.3 cm, respectively (table 2). laboratory results are shown in table 3.table 2clinical characteristics of hiv - infected patients on haart with and without insulin resistance (ir)clinical characteristicstotal (n = 219)no ir (n = 144)ir (n = 75) p sbp, mmhg104.7 11.9103.6 11.4106.8 12.60.060dbp, mmhg68.5 8.267.5 7.870.4 8.60.013weight, kg64.7 11.563.0 11.667.9 10.70.003bmi, kg / m 24.5 3.623.8 3.625.6 3.50.001bmi categories0.001 underweight, n (%) 10 (4.6)9 (6.3)1 (1.3)0.535 normal weight, n (%) 121 (55.3)91 (63.2)30 (40.0)reference overweight, n (%) 70 (32.0)35 (24.3)35 (46.7)0.011 obesity, n (%) 18 (8.2)9 (6.3)9 (12.0) 125 g / dl9 (4.1)1 (0.7)8 (10.7)2.750.004high blood pressure, n (%) 8 (3.7)4 (2.8)4 (5.3)1.4860.258abbreviations : hdl - c high - density lipoprotein cholesterol, ir insulin resistance comparison of metabolic syndrome and its components in hiv - infected patients on haart with and without ir abbreviations : hdl - c high - density lipoprotein cholesterol, ir insulin resistance hyperglycemia was found in 103 (47 %) subjects, 9 (4.1 %) of them had serum glucose > 125 mg / dl. 8 (3.7 %) patients had hypertension. the prevalence of hypertriglyceridemia and low hdl - c was 48.4 % and 57.5 % respectively (table 4). subjects with ir were older, had higher bmi, and were more likely to have hyperglycemia, central obesity and diastolic hypertension. with regard to the lipid profile, although the patients with ir had higher triglycerides levels, no significant differences were found when the variable was categorized as hypertriglyceridemia. there were no differences in gender, family history of t2 dm, cigarette smoking, duration of haart, using pi and systolic blood pressure (sbp). in multivariate analysis, the factors associated with ir were age 46 years (pr = 2.767, 95 % ci 1.325 to 5.780) and greater bmi (pr = 1.148, 95 % ci 1.054 to 1.250) (table 5).table 5multivariate logistic regression analysis for ir in hiv - infected patients on haartfactorpr p 95 % ciage (years)0.014 351.000reference 36 - 451.0700.8480.5332.150 462.7670.0071.3255.780bmi1.1480.0021.0541.250abbreviations : bmi body mass index, ci confidence interval, pr prevalence ratio multivariate logistic regression analysis for ir in hiv - infected patients on haart abbreviations : bmi body mass index, ci confidence interval, pr prevalence ratio in our study, the prevalence of ir was 34 %, two times higher than that of the non - hiv - infected population. the reported prevalence rates of ir among hiv - patients on haart are highly variable, ranging from 13 % to 45.7 %. because our study did not have a control group of patients without hiv infection, the homa - ir s cutoff was 2.1, which was based on a previous study conducted at the hospital nacional cayetano heredia in subjects without hiv infection and with similar geographical and ethnic characteristics. this value corresponds to the 75 percentile value determined by that study for subjects without hiv infection, normal ogtt and normal weight. it is higher than the cutoff point of a study made with peruvian andean adults living at 4100 m above sea level (homa = 1.4) and lower than the value found for peruvian metropolitan population living at sea level (homa = 3.56). through bivariate analysis, we found the following variables to be associated with ir : age, diastolic blood pressure, weight, bmi, abdominal circumference and fgp. all of these variables are components traditionally associated with the development of ir in the general population. our study did not find a significant association between ir and the use of pis, in contrast to other reports which showed a clear association [12, 13 ], this may be explained by the small number of patients [20 (9 %) ] using pi as part of their haart regimen included in our study. in addition, we found that 9 of the 20 patients on pi used atazanavir, which has not been associated with ir in published studies. on the other hand, the prevalence of ir was not significantly higher among patients who were more than one year on haart compared to those who were less time on treatment, suggesting that ir occurs early. however, our study does not allow us to assess if the ir worsens with the course of the hiv infection. jemsek jg. found that the alteration of insulin sensitivity in the early stages of antiretroviral therapy tends to normalize over time. in the multivariate analysis, through a logistic regression model using the variables that were significant in the bivariate analysis, we found that patients older than 46 years have almost three times more risk of ir compared with younger subjects. similarly, we found an increased risk of ir with higher bmi ; for each unit increase in bmi, the risk of ir increased by 15 %. the prevalence of ms in our series was 27 %, similar to the prevalence reported in american and latin american series in patients with hiv infection [16, 17 ], and higher than the prevalence found in the peruvian adult population without hiv infection (22 %). with regard to the components of ms, in our study, similar to other studies of ms in hiv patients, dyslipidemia and hyperglycemia predominant followed by obesity and hypertension. also, a high percentage of patients with ms had normal weight, which differs from previous reports about ms in the general population, where abdominal obesity and hypertension are the principal components. we found an alarmingly high prevalence of hyperglycemia (47 %), higher than that reported in other studies of hiv patients on haart (38 %), these results suggest that hiv treatment is an important factor in the development of ir and other metabolic disorders such as igt and t2 dm. unfortunately, our study did not perform oral glucose tolerance test so that there may be cases of t2 dm undiagnosed. the prevalence of hypertension was 3.7 %, lower than that found in a previous study (16 %). the study s limitations were the lack of a control group of hiv - uninfected subjects to define a more precise homa - ir s cutoff point and the clinical evaluation of lipodystrophy. however, el - sadr wm. has shown an inverse relationship between cd4 count and ir. these inconsistent associations, on regard the cd4 count, might reflect underlying immunological mechanisms that could affect the insulin sensitivity. therefore, those two variables must be included in future studies as possible underlying factors. in conclusion, this study shows a significant percentage of hiv - infected patients on haart present ir. since these patients have a high frequency of dyslipidemia and tobacco consumption, the detection of ir and its risk factors will allow for the assessment of each patient s metabolic risk and will also promote addressing modifiable risk factors to prevent the development of t2 dm and cardiovascular disease. we did not find any significant association between ir and protease inhibitors, which may be explained by the small number of patients using pi as part of their haart regimen included in our study. based on our results, we suggest following current guidelines for the use of antiretroviral therapy in hiv patients, which recommend performing laboratory tests, such as fasting glucose or ogtt, during follow - up. the use of homa - ir in these patients will require further longitudinal studies in order to demonstrate its usefulness in early predicting diabetes or igt in this population. | backgroundthe highly active antiretroviral therapy (haart) has altered the course of hiv infection, transforming it from a fatal illness to a chronic condition, reducing morbidity and mortality. however, this therapy has led to an increased incidence of metabolic problems such as insulin resistance, dyslipidemia, lipodystrophy and impaired glucose metabolism.the objectives of this study are to determine the prevalence of insulin resistance (ir) in a cohort of human immunodeficiency virus (hiv)-infected patients on highly active antiretroviral therapy (haart) and to investigate the potentially associated factors.methodswe conducted a cross - sectional study including 219 adult patients with hiv on haart. ir was determined through the homeostasis model assessment (homa - ir) mathematical model, using fasting plasma glucose (fpg) and insulin. bivariate and multivariate analyses were performed to assess the association between demographic information, clinical characteristics and laboratory results, and ir.results75 (34.2 %) [95 % confidence interval (ci) 28.940.9 ] hiv - patients on haart showed ir. 61 (81 %) of these patients were on haart for more than one year, which was mainly composed by non - protease inhibitors drugs (88 %). metabolic syndrome (ms) was found in 59 (26.9 %) subjects. in the multivariate analysis, the factors associated with ir were age 46 years (prevalence ratio = 2.767, 95 % ci 1.325 to 5.780) and greater body mass index (bmi) (prevalence ratio = 1.148, 95 % ci 1.054 to 1.250).conclusionsthe prevalence of ir was 34.2 %. factors associated with ir were age and bmi. we did not find any significant association between ir and protease inhibitors (pi), which may be explained by the small number of patients using pi as part of their haart regimen included in our study. |
the jichi medical school (jms) cohort study is a prospective, population - based study aimed at exploring the risk factors for cvd in 12 communities in japan. details regarding the jms cohort study design and additional descriptive data are available in the supplementary data or in our previous reports (1315). enrollment into the jms cohort study and baseline data collection were performed between april 1992 and november 1993. a total of 12,490 subjects (39.3% male [n = 4,911 ]), who were a mean sd age of 55.3 11.6 years, participated in the current study. of these, 12,393 (99.2%) gave us written informed consent to be prospectively followed up for study purposes and complete follow - up was achieved for 12,388 (99.9%). glucose parameters (i.e., plasma glucose levels and hemoglobin alc [hba1c ]) and responses to a self - administered questionnaire documenting their medical history of diabetes were available for 3,727 subjects (33.2% male [n = 1,240 ]), who were a mean age of 53.8 12.0 years. we excluded 86 participants who had insufficient data for at least one clinical parameter of age, sex, bmi, systolic or diastolic blood pressure (bp), habitual smoking, information on medical history of hypertension, myocardial infarction, stroke, and cancer, or data of circulating lipid parameters. ultimately, data from 3,641 subjects were analyzed in the current study (supplementary fig. the 3,641 subjects who were included in the present analysis were showed a younger age (53.7 12.1 vs. 55.9 11.4 years ; p < 0.001) and a lower prevalence of men (33.5% [n = 1,220 ] vs. 41.7% [n = 3,649 ] ; p < 0.001) compared with the 8,747 who were excluded from the analysis. to synchronize the methods of data collection, we established a central committee composed of the chief medical officers from the participating districts. information about lifestyle and medical history was gathered by means of a written questionnaire. in some subjects, information about physical activity (n = 3,615 [99% ]), educational level (n = 3,601 [99% ]), and marital status (n = 3,616 [99% ]) were also obtained (supplementary data). systolic and diastolic bp were measured with a fully automated sphygmomanometer, bp203rv - ii (nippon colin, komaki, japan), which was placed on the right arm of a subject who had rested in a sitting position for 5 min before measurement. hypertension was defined as systolic bp / diastolic bp 140/90 mmhg or self - reported usage of antihypertensive medication. blood samples were drawn from the antecubital vein of seated subjects, with minimal tourniquet use (details are described in the supplementary data). total cholesterol and triglycerides were measured using an enzymatic method (wako ; interassay coefficient of variation [cv ], 1.5% for total cholesterol and 1.7% for triglyceride). hdl cholesterol was measured using the phosphotungstate precipitation method (wako ; interassay cv, 1.9%). blood glucose was measured via an enzymatic method (kanto chemistry ; interassay cv, 1.9%), and the value for hba1c was estimated as a national glycohemoglobin standardization program equivalent value calculated with the following formula (16) : hbalc (%) = hbalc (japan diabetes society) (%) + 0.4%. the japan society for the study of obesity defines obesity as a bmi 25 kg / m and leanness as a bmi < 18.5 kg / m (2,17) therefore, in the current study, we defined obesity as bmi 25 kg / m (mean 27.0 2.4. however, if we had defined leanness as bmi < 18.5 kg / m, only 160 subjects (4% of total patients) would qualify. thus, in the current study, we defined leanness as the lowest quartile of bmi (range 14.221.1 ; mean 19.5 1.2 kg / m ; n = 910). as a consequence, we defined normal bmi as bmi ranging from the second quartile of bmi to 25 kg / m (mean 22.7 1.0 kg / m ; n = 1,535). diabetes was defined in accordance with the american diabetes association guidelines (18) as a fasting glucose concentration of 126 mg / dl or higher, casual blood glucose concentration of 200 mg / dl or higher, hba1c of 6.5% or higher, or self - reported use of antihyperglycemic drugs. as described in previous reports (1315), mortality data from the date of entry to 31 december 2002 were collected from the cause - of - death register at public health centers in each community with the permission of the agency of general affairs and the ministry of health, labor, and welfare. the follow - up period was 10.2 2.1 years (37,278 person - years). information on the cause of death was coded for participants who died using icd-10 codes. causes of death were classified as follows : 1) cvd death : heart disease including sudden death (i21i23, i46, i48i50, q20q28), cvd (i60, i61, i63, i69), and other cvd (i71) ; 2) cancer death (c02, c10, c14c20, c22c26, c30, c34, c41, c50, c53, c54, c61, c64, c65, c71, c74, c76, c81c85, c90c93) ; and 3) other causes, such as infection and suicide (a41, b1519, d65, g12, g21, g93, j10j18, j43, j84, j96, k72, m62, n00n08, r57, r54, r64, s06, t58, x60x84, y85y87, w75w84). all statistical analyses were performed with spss 18.0j software (spss inc, chicago, il). clinical parameters in subjects with or without death were compared using the unpaired t test, and categorical parameters were compared with the test. next, we used cox regression analysis to examine the independent effects of diabetes or bmi on the risk of all - cause death. after adjusting for significant covariates, such as age, sex, current smoking status, and systolic bp values, the hazard ratios (hr) and 95% cis were calculated for all - cause death, cvd death, or cancer death in subjects with diabetes or leanness (obesity). finally, our population was subdivided into six categories according to bmi (leanness, normal bmi, and obesity) and the presence of diabetes, and the hr (95% ci) of all - cause deaths in each of the six categories was calculated. in that analysis, to examine whether the association among the six categories and all - cause death differed between middle - aged / younger individuals and older individuals, we used a cox regression analysis separately in subjects aged < 65 and 65 years. this analysis included significant covariates for adjusted variables, such as sex, current smoking status, systolic bp values, and pre - existing myocardial infarction, stroke, or cancer. we then performed additional adjustments for each of the following possible confounders : physical activity (n = 3,615), educational level (n = 3,601), and marital status (n = 3,616). finally, using a cox regression analysis, we examined whether there were any interactions between diabetes and bmi in the risk of all - cause death separately in those aged < 65 years and those 65 years. to synchronize the methods of data collection, we established a central committee composed of the chief medical officers from the participating districts. information about lifestyle and medical history was gathered by means of a written questionnaire. in some subjects, information about physical activity (n = 3,615 [99% ]), educational level (n = 3,601 [99% ]), and marital status (n = 3,616 [99% ]) were also obtained (supplementary data). systolic and diastolic bp were measured with a fully automated sphygmomanometer, bp203rv - ii (nippon colin, komaki, japan), which was placed on the right arm of a subject who had rested in a sitting position for 5 min before measurement. hypertension was defined as systolic bp / diastolic bp 140/90 mmhg or self - reported usage of antihypertensive medication. blood samples were drawn from the antecubital vein of seated subjects, with minimal tourniquet use (details are described in the supplementary data). total cholesterol and triglycerides were measured using an enzymatic method (wako ; interassay coefficient of variation [cv ], 1.5% for total cholesterol and 1.7% for triglyceride). hdl cholesterol was measured using the phosphotungstate precipitation method (wako ; interassay cv, 1.9%). blood glucose was measured via an enzymatic method (kanto chemistry ; interassay cv, 1.9%), and the value for hba1c was estimated as a national glycohemoglobin standardization program equivalent value calculated with the following formula (16) : hbalc (%) = hbalc (japan diabetes society) (%) + 0.4%. the japan society for the study of obesity defines obesity as a bmi 25 kg / m and leanness as a bmi < 18.5 kg / m (2,17) therefore, in the current study, we defined obesity as bmi 25 kg / m (mean 27.0 2.4 however, if we had defined leanness as bmi < 18.5 kg / m, only 160 subjects (4% of total patients) would qualify. thus, in the current study, we defined leanness as the lowest quartile of bmi (range 14.221.1 ; mean 19.5 1.2 kg / m ; n = 910). as a consequence, we defined normal bmi as bmi ranging from the second quartile of bmi to 25 kg / m (mean 22.7 1.0 kg / m ; n = 1,535). diabetes was defined in accordance with the american diabetes association guidelines (18) as a fasting glucose concentration of 126 mg / dl or higher, casual blood glucose concentration of 200 mg / dl or higher, hba1c of 6.5% or higher, or self - reported use of antihyperglycemic drugs. as described in previous reports (1315), mortality data from the date of entry to 31 december 2002 were collected from the cause - of - death register at public health centers in each community with the permission of the agency of general affairs and the ministry of health, labor, and welfare. the follow - up period was 10.2 2.1 years (37,278 person - years). information on the cause of death was coded for participants who died using icd-10 codes. causes of death were classified as follows : 1) cvd death : heart disease including sudden death (i21i23, i46, i48i50, q20q28), cvd (i60, i61, i63, i69), and other cvd (i71) ; 2) cancer death (c02, c10, c14c20, c22c26, c30, c34, c41, c50, c53, c54, c61, c64, c65, c71, c74, c76, c81c85, c90c93) ; and 3) other causes, such as infection and suicide (a41, b1519, d65, g12, g21, g93, j10j18, j43, j84, j96, k72, m62, n00n08, r57, r54, r64, s06, t58, x60x84, y85y87, w75w84). all statistical analyses were performed with spss 18.0j software (spss inc, chicago, il). clinical parameters in subjects with or without death were compared using the unpaired t test, and categorical parameters were compared with the test. next, we used cox regression analysis to examine the independent effects of diabetes or bmi on the risk of all - cause death. after adjusting for significant covariates, such as age, sex, current smoking status, and systolic bp values, the hazard ratios (hr) and 95% cis were calculated for all - cause death, cvd death, or cancer death in subjects with diabetes or leanness (obesity). finally, our population was subdivided into six categories according to bmi (leanness, normal bmi, and obesity) and the presence of diabetes, and the hr (95% ci) of all - cause deaths in each of the six categories was calculated. in that analysis, to examine whether the association among the six categories and all - cause death differed between middle - aged / younger individuals and older individuals, we used a cox regression analysis separately in subjects aged < 65 and 65 years. this analysis included significant covariates for adjusted variables, such as sex, current smoking status, systolic bp values, and pre - existing myocardial infarction, stroke, or cancer. we then performed additional adjustments for each of the following possible confounders : physical activity (n = 3,615), educational level (n = 3,601), and marital status (n = 3,616). finally, using a cox regression analysis, we examined whether there were any interactions between diabetes and bmi in the risk of all - cause death separately in those aged < 65 years and those 65 years. the mean sd age of the 3,641 subjects was 53.7 12.1 years, and 1,220 (33.5%) were men. at the time of study recruitment, there were 507 patients (13.9%) defined as having diabetes, 1,277 (35.1%) with hypertension, 58 (1.6%) with a pre - existing myocardial infarction or stroke, and 43 (1.2%) with a pre - existing cancer. during an average duration of 10.2 2.1 years (37,278 person - years), 240 deaths occurred (6.4 events/1,000 person - years), including 54 cvd deaths, 101 cancer deaths, and 85 other - cause deaths (e.g., infection, suicide, accident). the baseline clinical characteristics according to the incidence of all - cause death are reported in table 1. the prevalence of leanness or diabetes was higher in subjects with death than in those without death. the crude incidence rate of all - cause death was 13.0 events/1,000 person - years for diabetes and 5.4 events/1,000 person - years for nondiabetes. by comparison, the crude incidence rate per 1,000 person - years of all - cause death was 8.4 events in lean subjects, 5.2 events in subjects with normal bmi, and 6.5 events in obese subjects. the prevalence of cause - specific death subdivided by the presence of diabetes or bmi is reported in supplementary tables 1 and 2. kaplan - meier curves showing cumulative all - cause death according to the presence of diabetes or the classification of bmi are shown in fig. 1a and b. there was no significant difference in the rate of all - cause death among the subjects subdivided by the presence of diabetes or bmi in the first 5-year period, whereas the differences were significant for the second 5-year period (both p < 0.05 by log - rank test). baseline clinical characteristics of the study population according to the occurrence of death kaplan - meier curves show the cumulative incidence of all - cause death by the presence of diabetes vs. nondiabetes (a), or the classification of bmi by leanness or obesity vs. normal bmi (b). next, the hr (95% ci) of all - cause death associated with leanness (obesity) or diabetes was calculated using cox regression analysis (table 2). the hr (95% ci) of all - cause death in subjects with leanness, but not obesity, and diabetes were significant, even after adjustment for significant covariates. stratification with cause - specific deaths showed that leanness and obesity were both associated with an increased risk of cvd death, whereas diabetes was associated with an increased risk of cancer death. when we defined obesity as the highest quartile of bmi (25.3 kg / m ; mean 27.7 kg / m, n = 910) instead of bmi 25 kg / m, the risk of cvd death in obesity remained unchanged (data not shown). furthermore, when we examined the male - to - female differences in the risk of all - cause death as well as cancer death in diabetes, we found that this conclusion remained unchanged (supplementary tables 3 and 4). cox regression analysis for all - cause death, cvd death, and cancer death in the total population (n = 3,641) the increased risk of all - cause death in leanness or diabetes (table 2) did not change when the 66 subjects who died within 2 years of follow - up were excluded (data not shown). after exclusion of 58 subjects with a pre - existing myocardial infarction or stroke and 43 with cancer at baseline, associations of leanness or diabetes with all - cause death remained significant (data not shown). when we adjusted for various confounding factors, such as physical activity, educational level, and marital status in the associations between diabetes or leanness and all - cause death, the risk of diabetes or leanness remained unchanged (data not shown). among 2,866 subjects aged < 65 years, the risk of all - cause death conferred by diabetes, compared with nondiabetes with normal bmi (reference), increased when diabetes was accompanied by leanness or obesity (fig. in contrast, among the 775 subjects aged 65 years, the risk of all - cause death conferred by diabetes, compared with nondiabetes with normal bmi, increased when the diabetes was accompanied by leanness but not by obesity (fig. we repeated the analysis after excluding subjects who died within the initial 2 years of follow - up or those who had a pre - existing myocardial infarction, stroke, or cancer at baseline, and the conclusions remained unchanged (data not shown). furthermore, we performed additional adjustments for each of the following possible confounders in fig. the hr (95% ci) of all - cause deaths according to the presence of diabetes and bmi are shown separately in those aged < 65 years (a) and those 65 years (b). the analysis was adjusted for sex, current smoking status, systolic bp values, and pre - existing myocardial infarction, stroke, or cancer. p < 0.05 vs. reference group, p < 0.01 vs. reference group, and p < 0.001 vs. reference group. finally, using a cox regression analysis (including sex, current smoking status, systolic bp values, and pre - existing myocardial infarction, stroke, or cancer), we examined whether there was an interaction between diabetes and bmi in the risk of all - cause death separately in those aged < 65 years and those 65 years. as a result, we found a significant interaction between diabetes and leanness only in subjects aged < 65 years (p = 0.047). the mean sd age of the 3,641 subjects was 53.7 12.1 years, and 1,220 (33.5%) were men. at the time of study recruitment, there were 507 patients (13.9%) defined as having diabetes, 1,277 (35.1%) with hypertension, 58 (1.6%) with a pre - existing myocardial infarction or stroke, and 43 (1.2%) with a pre - existing cancer. during an average duration of 10.2 2.1 years (37,278 person - years), 240 deaths occurred (6.4 events/1,000 person - years), including 54 cvd deaths, 101 cancer deaths, and 85 other - cause deaths (e.g., infection, suicide, accident). the baseline clinical characteristics according to the incidence of all - cause death are reported in table 1. the prevalence of leanness or diabetes was higher in subjects with death than in those without death. the crude incidence rate of all - cause death was 13.0 events/1,000 person - years for diabetes and 5.4 events/1,000 person - years for nondiabetes. by comparison, the crude incidence rate per 1,000 person - years of all - cause death was 8.4 events in lean subjects, 5.2 events in subjects with normal bmi, and 6.5 events in obese subjects. the prevalence of cause - specific death subdivided by the presence of diabetes or bmi is reported in supplementary tables 1 and 2. kaplan - meier curves showing cumulative all - cause death according to the presence of diabetes or the classification of bmi are shown in fig. 1a and b. there was no significant difference in the rate of all - cause death among the subjects subdivided by the presence of diabetes or bmi in the first 5-year period, whereas the differences were significant for the second 5-year period (both p < 0.05 by log - rank test). baseline clinical characteristics of the study population according to the occurrence of death kaplan - meier curves show the cumulative incidence of all - cause death by the presence of diabetes vs. nondiabetes (a), or the classification of bmi by leanness or obesity vs. normal bmi (b). next, the hr (95% ci) of all - cause death associated with leanness (obesity) or diabetes was calculated using cox regression analysis (table 2). the hr (95% ci) of all - cause death in subjects with leanness, but not obesity, and diabetes were significant, even after adjustment for significant covariates. stratification with cause - specific deaths showed that leanness and obesity were both associated with an increased risk of cvd death, whereas diabetes was associated with an increased risk of cancer death. when we defined obesity as the highest quartile of bmi (25.3 kg / m ; mean 27.7 kg / m, n = 910) instead of bmi 25 kg / m, the risk of cvd death in obesity remained unchanged (data not shown). furthermore, when we examined the male - to - female differences in the risk of all - cause death as well as cancer death in diabetes, we found that this conclusion remained unchanged (supplementary tables 3 and 4). cox regression analysis for all - cause death, cvd death, and cancer death in the total population (n = 3,641) the increased risk of all - cause death in leanness or diabetes (table 2) did not change when the 66 subjects who died within 2 years of follow - up were excluded (data not shown). after exclusion of 58 subjects with a pre - existing myocardial infarction or stroke and 43 with cancer at baseline, associations of leanness or diabetes with all - cause death remained significant (data not shown). when we adjusted for various confounding factors, such as physical activity, educational level, and marital status in the associations between diabetes or leanness and all - cause death, the risk of diabetes or leanness remained unchanged (data not shown). among 2,866 subjects aged < 65 years, the risk of all - cause death conferred by diabetes, compared with nondiabetes with normal bmi (reference), increased when diabetes was accompanied by leanness or obesity (fig. in contrast, among the 775 subjects aged 65 years, the risk of all - cause death conferred by diabetes, compared with nondiabetes with normal bmi, increased when the diabetes was accompanied by leanness but not by obesity (fig. we repeated the analysis after excluding subjects who died within the initial 2 years of follow - up or those who had a pre - existing myocardial infarction, stroke, or cancer at baseline, and the conclusions remained unchanged (data not shown). furthermore, we performed additional adjustments for each of the following possible confounders in fig. the hr (95% ci) of all - cause deaths according to the presence of diabetes and bmi are shown separately in those aged < 65 years (a) and those 65 years (b). the analysis was adjusted for sex, current smoking status, systolic bp values, and pre - existing myocardial infarction, stroke, or cancer. p < 0.05 vs. reference group, p < 0.01 vs. reference group, and p < 0.001 vs. reference group. finally, using a cox regression analysis (including sex, current smoking status, systolic bp values, and pre - existing myocardial infarction, stroke, or cancer), we examined whether there was an interaction between diabetes and bmi in the risk of all - cause death separately in those aged < 65 years and those 65 years. as a result, we found a significant interaction between diabetes and leanness only in subjects aged < 65 years (p = 0.047). in the present 10-year prospective study in a rural japanese general population, we have demonstrated for the first time the associations between diabetes, bmi, and the risk of death. the main findings of the current study are that 1) leanness, but not obesity, and diabetes were independently associated with an increased risk of all - cause death ; 2) leanness and obesity were both associated with an increased risk of cvd death, whereas diabetes was associated with cancer death ; and 3) lean diabetic subjects had a substantially high risk of all - cause death regardless of age, whereas obese diabetic subjects showed increased risk only in those aged < 65 years. these findings provide opportunities for a careful evaluation of the risks accompanying diabetes that can be modified by bmi or age. most of the literature regarding diabetes and excess mortality has been derived from western populations (19,20), and there are few prospective studies on mortality and cause of death from diabetes in japan. the recent substantial increase in rates of diabetes in asian countries (2,3) highlights the need to better understand the mortality related to diabetes and to identify high - risk subjects in order to prevent excess mortality from diabetes within each asian country. in the current study, diabetes almost doubled the incidence of all - cause death compared with nondiabetes, and diabetes increased the risk of all - cause deaths by 65%, even after adjustment for significant covariates (table 2). contrary to previous reports from western populations (21,22), we observed that cvd mortality was not significantly greater in diabetes than nondiabetes. because the number of cvd death was small, this issue merits further analysis. nevertheless, a direct comparison between diabetes in japanese in caucasians showed that the cvd mortality is threefold higher in caucasians (23). moreover, a world health organization multinational study demonstrated that japanese cohorts showed a very low excess mortality from cvd death compared with western populations (24). these findings indicate that the association of diabetes with cvd mortality may differ between japanese and western populations ; further studies on a large cohort from a japanese general population will be required to clarify that issue. diabetes and cancer are common conditions, and their codiagnosis in the same individual is not infrequent (25). in a previous japanese cohort study, diabetes was associated with an increase in the risk of total cancer incidence of 27% in men and 21% in women (26). we also demonstrated that cancer death was higher in diabetes compared with nondiabetes without sex differences (supplementary tables 3 and 4). it is not easy to differentiate whether diabetes causes cancer or whether risk factors for diabetes, such as obesity and physical inactivity, are associated with cancer. however, our data showed that an increased risk of cancer mortality in diabetes remained unchanged even after adjustment for significant covariates, including obesity or physical activity. by site, emerging data support higher risks of death from cancers of the liver, pancreas, and colon among adults with diabetes, whereas the evidence with other malignancies is equivocal (2527). there were not enough cancer deaths in the current study to analyze the association between diabetes and cancer death by tumor site (supplementary tables 1 and 2). the biological plausibility of the associations between diabetes and cancer has tended to be site - specific but may partly be the result of diabetic metabolic and hormonal alternations as well as their underlying common biological mechanisms such as hyperinsulinemia, abnormal production of adipocytokines and growth factors, and epigenetic changes (25). because our data showed only cancer deaths, but not cancer incidences, we can not exclude the possibility that a diagnosis of diabetes or poor glycemic control can lead to poor outcomes among subjects who developed cancer during the follow - up period (28). further studies are warranted to quantify the specific impact of diabetes on cancer incidence versus cancer survival. we demonstrated that, compared with nondiabetes with normal bmi, lean diabetes showed a substantially higher risk of all - cause death in subjects aged < 65 years and also those aged 65 years. especially in subjects aged < 65 years, a significant interaction was found between diabetes and leanness in the risk of all - cause death. obese diabetes, by contrast, showed an elevated risk only in subjects aged < 65 years (fig. 2), a finding similar to the previous findings of nilsson. (29) that obese diabetes in older persons (all subjects were 75 years old) presented no mortality risk. this may be partly explained by a survival bias ; that is, the detrimental effects of obesity - related metabolic abnormalities are generally less prominent among older subjects than they are in middle - aged individuals (30,31). the reasons for pronounced mortality in lean diabetes in the current study are not clear. the pathophysiology of lean diabetes is heterogeneous, including impaired insulin secretion, increased endogenous glucose production, high inflammatory state, and higher perceived stress hormones (32,33). the contrasting general clinical phenotypes of lean diabetes compared with obese diabetes have recently been discussed (32,33), although we can not contribute to that discussion. incomplete control for confounding or reverse causation bias in an effort to control for reverse causation, we repeated the analysis after excluding subjects who died within the initial 2 years of follow - up and those who had a pre - existing myocardial infarction, stroke, or cancer at baseline ; the conclusions remained unchanged. however, that the leanness associated with higher mortality during the follow - up period was a result of occult diseases involving muscle or fat wasting can not be excluded. the strengths of the current study include 1) this is a population - based study in an asian (japanese) population where there are limited data available addressing the association between diabetes, leanness, and mortality ; and 2) complete follow - up was achieved in almost the entire cohort. a number of people from the original cohort (1315) were excluded (71%) ; thus, there is a potential bias in the study participants chosen for the study analysis. data on diabetes type, duration (i.e., no distinction between subjects with new - onset diabetes and those with longer - duration diabetes), disease severity, and the presence of diabetes complications at baseline would have been informative and would have extended the knowledge achieved in the current study. second, we could not separate diabetes into type 1 or type 2 diabetes, although the incidence of type 1 diabetes is extremely low (approximately 2 cases / year/100,000 individuals) in japan (34). lastly, information regarding bmi and glucose values was available only at baseline, making it impossible to evaluate the effects of changes during the follow - up period in either or both of these factors. in conclusion, among the japanese general population, a diagnosis of diabetes confers an increased risk of all - cause death. particular attention must be paid to the pronounced high mortality for lean diabetes. taken together with the evidence of a rapidly increasing number of diabetes cases in asia, the findings from the current study suggest that an integrated strategy combining preventive actions against diabetes, improvement of care for diabetes that includes early detection of diabetes complications as well as undiagnosed chronic disease, and multidisciplinary care programs is needed. most of the literature regarding diabetes and excess mortality has been derived from western populations (19,20), and there are few prospective studies on mortality and cause of death from diabetes in japan. the recent substantial increase in rates of diabetes in asian countries (2,3) highlights the need to better understand the mortality related to diabetes and to identify high - risk subjects in order to prevent excess mortality from diabetes within each asian country. in the current study, diabetes almost doubled the incidence of all - cause death compared with nondiabetes, and diabetes increased the risk of all - cause deaths by 65%, even after adjustment for significant covariates (table 2). contrary to previous reports from western populations (21,22), we observed that cvd mortality was not significantly greater in diabetes than nondiabetes. because the number of cvd death was small, this issue merits further analysis. nevertheless, a direct comparison between diabetes in japanese in caucasians showed that the cvd mortality is threefold higher in caucasians (23). moreover, a world health organization multinational study demonstrated that japanese cohorts showed a very low excess mortality from cvd death compared with western populations (24). these findings indicate that the association of diabetes with cvd mortality may differ between japanese and western populations ; further studies on a large cohort from a japanese general population will be required to clarify that issue. diabetes and cancer are common conditions, and their codiagnosis in the same individual is not infrequent (25). in a previous japanese cohort study, diabetes was associated with an increase in the risk of total cancer incidence of 27% in men and 21% in women (26). we also demonstrated that cancer death was higher in diabetes compared with nondiabetes without sex differences (supplementary tables 3 and 4). it is not easy to differentiate whether diabetes causes cancer or whether risk factors for diabetes, such as obesity and physical inactivity, are associated with cancer. however, our data showed that an increased risk of cancer mortality in diabetes remained unchanged even after adjustment for significant covariates, including obesity or physical activity. by site, emerging data support higher risks of death from cancers of the liver, pancreas, and colon among adults with diabetes, whereas the evidence with other malignancies is equivocal (2527). there were not enough cancer deaths in the current study to analyze the association between diabetes and cancer death by tumor site (supplementary tables 1 and 2). the biological plausibility of the associations between diabetes and cancer has tended to be site - specific but may partly be the result of diabetic metabolic and hormonal alternations as well as their underlying common biological mechanisms such as hyperinsulinemia, abnormal production of adipocytokines and growth factors, and epigenetic changes (25). because our data showed only cancer deaths, but not cancer incidences, we can not exclude the possibility that a diagnosis of diabetes or poor glycemic control can lead to poor outcomes among subjects who developed cancer during the follow - up period (28). further studies are warranted to quantify the specific impact of diabetes on cancer incidence versus cancer survival. a recent meta - analysis from western populations demonstrated the obesity paradox in diabetes and the importance of assessing nonobese (bmi < 25 kg / m) diabetes as being high - mortality cases (9). we demonstrated that, compared with nondiabetes with normal bmi, lean diabetes showed a substantially higher risk of all - cause death in subjects aged < 65 years and also those aged 65 years. especially in subjects aged < 65 years, a significant interaction was found between diabetes and leanness in the risk of all - cause death. obese diabetes, by contrast, showed an elevated risk only in subjects aged < 65 years (fig. 2), a finding similar to the previous findings of nilsson. (29) that obese diabetes in older persons (all subjects were 75 years old) presented no mortality risk. this may be partly explained by a survival bias ; that is, the detrimental effects of obesity - related metabolic abnormalities are generally less prominent among older subjects than they are in middle - aged individuals (30,31). the reasons for pronounced mortality in lean diabetes in the current study are not clear. the pathophysiology of lean diabetes is heterogeneous, including impaired insulin secretion, increased endogenous glucose production, high inflammatory state, and higher perceived stress hormones (32,33). the contrasting general clinical phenotypes of lean diabetes compared with obese diabetes have recently been discussed (32,33), although we can not contribute to that discussion. incomplete control for confounding or reverse causation bias in an effort to control for reverse causation, we repeated the analysis after excluding subjects who died within the initial 2 years of follow - up and those who had a pre - existing myocardial infarction, stroke, or cancer at baseline ; the conclusions remained unchanged. however, that the leanness associated with higher mortality during the follow - up period was a result of occult diseases involving muscle or fat wasting can not be excluded. the strengths of the current study include 1) this is a population - based study in an asian (japanese) population where there are limited data available addressing the association between diabetes, leanness, and mortality ; and 2) complete follow - up was achieved in almost the entire cohort. a number of people from the original cohort (1315) were excluded (71%) ; thus, there is a potential bias in the study participants chosen for the study analysis. data on diabetes type, duration (i.e., no distinction between subjects with new - onset diabetes and those with longer - duration diabetes), disease severity, and the presence of diabetes complications at baseline would have been informative and would have extended the knowledge achieved in the current study. second, we could not separate diabetes into type 1 or type 2 diabetes, although the incidence of type 1 diabetes is extremely low (approximately 2 cases / year/100,000 individuals) in japan (34). lastly, information regarding bmi and glucose values was available only at baseline, making it impossible to evaluate the effects of changes during the follow - up period in either or both of these factors. in conclusion, among the japanese general population particular attention must be paid to the pronounced high mortality for lean diabetes. taken together with the evidence of a rapidly increasing number of diabetes cases in asia, the findings from the current study suggest that an integrated strategy combining preventive actions against diabetes, improvement of care for diabetes that includes early detection of diabetes complications as well as undiagnosed chronic disease, and multidisciplinary care programs is needed. | objectiveto examine the bmi - stratified associations between diabetes and the risks of all - cause death, cardiovascular disease (cvd) death, and cancer death.research design and methodsusing a prospective study with 12 rural japanese general populations (n = 3,641, mean age, 53.7 years ; 33.5% men), we examined the associations between diabetes and the risk of all - cause death, cvd death, and cancer death. we also examined the effects of bmi and age on such associations.resultsduring an average duration of 10.2 years (37,278 person - years), 240 deaths occurred (54 deaths from cvd, 101 from cancer, and 85 from other causes). cox regression analysis showed leanness (defined as the lowest quartile of entire bmi ; mean, 19.5 kg / m2), but not obesity (bmi 25 kg / m2), and diabetes were independently associated with an increased risk of all - cause death (hazard ratio [hr ] 1.70 and 1.65, respectively ; both p < 0.01.). stratification with cause - specific deaths showed that leanness and obesity were associated with cvd death (hr 3.77 and 2.94, respectively), whereas diabetes was associated with cancer death (hr 1.87 ; all p < 0.05). the increased risk of all - cause death in diabetes was substantially higher in lean subjects aged < 65 years (hr 3.4) or those aged 65 years (hr 4.2), whereas the risk in obese diabetes patients was significant only in subjects aged < 65 years (hr 2.32 ; all p < 0.05).conclusionsamong the japanese general population, diabetes confers an increased risk of all - cause death. particular attention must be paid to the pronounced high mortality in diabetes accompanied with leanness, regardless of age. |
approximately 26% of american adults are suffering from a diagnosable mental disease, and nearly half (45%) of them meet criteria for two or more disorders strongly related to comorbidity. major depressive disorder (mdd), one of the most common mental disorders, affects almost 15 million american adults (about 7% of the population), and women report more depressive symptoms than men. furthermore, mdd is the leading cause of disability in the united states for persons between the ages of 15 and 44, resulting in almost half of all lost productivity that translates into a cost burden of $ 44 billion per year. in addition, the world health organization 's global burden of disease study measured lost years of healthy life in the developed world, regardless of whether they were lost to premature death or disability for various diseases, concluding that disability burden caused by mdd ranks second only to cardiovascular disease. while 3545% of depressed patients receiving fda - approved antidepressants experience complete relief from their symptoms, 5565% have inadequate response and/or side effects, such as sexual dysfunction, insomnia, weight gain, restlessness, and memory lapses, among others. moreover, antidepressants have also been found to have serious side effects, such as suicide, violence, psychosis, and abnormal bleeding [7, 8 ]. thus, many patients end up on revolving medication trials, switching repeatedly from one drug to another or combining drugs to maximize their effects. in addition, a literature review found that among patients with mdd placebo was as effective as antidepressants. interestingly, according to several studies, modifiable factors, such as optimizing nutritional status, may help to improve the symptoms of depression. in one study, 12 g / day of inositol resulted in positive therapeutic improvements similar to common antidepressant drugs, but without untoward side effects. ernst found that ginkgo biloba was effective at improving depressed mood, anxiety, memory, concentration, and fatigue. s - adenosyl methionine (same) was compared to oral doses of imipramine in a double - blind study for 14 days. significant improvements were observed with same by the end of the first week, and at the end of the protocol 66% of the same patients had a clinically significant improvement in depressive symptoms compared to 22% of the imipramine patients. another study that included 23 elderly patients with type 2 diabetes and hypomagnesemia showed that magnesium chloride was as effective as 50 mg / day of imipramine in improving depressive symptoms. given these positive findings, a low - cost, safe alternative to medication can be considered for persons suffering from mdd. thus, our study will extend the evaluative process of a vitamin b complex supplement 's efficacy and safety in improving depression, anxiety, and quality of life in a sample of adults diagnosed with depression through a randomized, double - blind, placebo - controlled clinical trial. with the overall high prevalence of dietary supplement use, the multifaceted problems associated with depression, including the untoward effects of standard treatment, and the growing number of readily available alternative and complementary remedies in the united states, the efficacy and safety of these substances demand more randomized clinical studies. therefore, only scientifically valid results from well - controlled trials can help to evaluate and support claims of effectiveness of all treatments, including natural products. the study was conducted with the approval of the university of miami institutional review board for human subjects research, and each subject signed informed consent and hipaa forms before enrolling in the study. potential participants (n = 120) were identified through referrals from clinical offices and centers at the university of miami miller school of medicine and from local community centers from miami - dade county from march 2010 to october 2011. thirty - six participants failed the screening inclusion / exclusion criteria, 24 participants were eligible for the study, but never enrolled, and 60 eligible participants were enrolled in the study at baseline. potential study candidates were identified as individuals who expressed an interest in a study assessing the efficacy of a dietary supplement on depression, anxiety, and quality of life. subjects were enrolled if they were (a) 18 years of age and older ; (b) currently diagnosed with mdd or a related depressive disorder as classified by the dsm - iv - tr ; (c) english speaking ; (d) had an elevated level of homocysteine (> 10 mol / l) at screening as a marker of inflammation ; (e) interested in participating in a novel nutritional supplement program ; and (f) willing to follow recommendations, including discontinuing all dietary supplements (e.g., multivitamin and mineral formula and vitamin b complex) for depression 2 weeks before starting and during the entire intervention period. exclusion criteria consisted of (a) current enrollment in another research trial for depression treatment ; (b) inability to consent to the study ; or (c) pregnancy in women. potential study subjects were prescreened for the inclusion and exclusion criteria and given a brief introduction to the nature and purpose of the study. all otherwise eligible subjects had a sample of venous blood drawn to measure homocysteine, a sulfurated amino acid derived from methionine, with higher levels directly related to depression. the blood sample was collected in an edta tube and delivered to the laboratory for processing within 2 hours of collection. following completion of the baseline assessment, participants were randomly assigned to one of two conditions (a) max stress b (a whole - food dietary supplement) or (b) placebo. assignment of subjects into one of the two treatment groups was accomplished with a computer - generated table of random permutations, designed to balance the number of subjects in each group. the table was arranged in advance, and the predetermined list of treatments served to prepare the numbered supplement containers (used in order) and the envelopes to be opened in the case of emergency. all subjects and investigators were blind to the treatment condition. only the staff at premier research labs (the manufacturer of max stress b) knew the assignment of treatment condition. each participant completed a basic sociodemographics and medical history questionnaire, including current medications, at baseline. they were also asked to note any changes in type or amount of medications during the course of the study. criteria used to select the assessment instruments included (a) appropriateness for the population ; (b) ease of administration and scoring ; (c) experience administering these measures ; and (d) employment of measures involving a multimethod (i.e., self - report and physical measures) approach to enhance the validity of the overall assessment. the beck depression inventory - ii (bdi) and beck anxiety inventory (bai) were administered at baseline and 30- and 60-day followups to assess changes in depressive and anxiety symptoms, respectively, over the course of the intervention. both the bdi and bai consist of 21 items and are scored 0 to 63, where higher levels indicate progressive levels of depression (09 : minimal, 1018 : mild, 1929 : moderate, and 3063 : severe) and anxiety (07 : minimal, 815 : mild, 1625 : moderate, and 2663 : severe). the secondary outcome, administered at baseline and 30- and 60-day followups, included change in general health - related quality of life according to the medical outcomes study short form 36 (sf-36). the sf-36 provides psychometrically based physical and mental health summary measures and as such is sensitive to subtle changes in relatively healthy persons, including those due to illness or injury. in addition, the sf-36 is reliable, valid, and provides a t score for each scale or domain ranging from 0 to 100 with higher scores representing better perceived quality of life. as a control variable, physical activity was assessed at baseline and 30- and 60-day followups by the stanford 7-day physical activity recall. this instrument has been validated for use in community - based settings, and it assesses the amount (number of hours) of moderate, physically challenging, and very physically challenging activities over the past 7 days. this assessment tool provides useful estimates of habitual physical activity for research and highly correlates with daily self - report of physical activity [20, 21 ]. for the 60-day intervention period, participants who enrolled in the study received (a) max stress b (a whole nutrient natural source extract from probiotic colonies that contains vitamins b1, b2, b3, b5, b6, and b12, and folate, paba, biotin, inositol, purified water, and certified organic alcohol) or (b) placebo (an oil / water emulsion with food coloring similar to the test product). subjects were instructed to consume 1 vial (equivalent to 1/2 teaspoon) of product in at least 12 ounces of water over the course of each day. they were not advised to modify eating or physical activity habits or nondepression prescription medication use. subjects were also instructed not to consume other nutritional supplements containing any of the vitamin b complex nutrients, same, inositol, paba, or folate for two weeks prior to having the baseline assessment and until the conclusion of the 60-day intervention period. analysis of variance and chi - square were utilized to determine the presence of differences in background contextual variables by study arm assignment. we utilized linear mixed modeling (lmm) to assess the fixed effect of time by randomization (max stress b versus placebo) on changes in our outcome variables from baseline to 60-day followup. if the type iii test of the fixed effect of time by randomization was significant, then we used pairwise comparisons to determine the unique differences in effects over time by study arm between baseline and followup at 30 and 60 days for depression, anxiety, and quality of life variables. lmm with heterogeneous compound symmetry covariance allowed us to account for subject attrition, intercorrelated responses between time points, and nonconstant variability. during the entire study period, no subjects reported adverse events or complications from the test products. table 1 presents the sociodemographic variables by study arm assignment for age, gender, race / ethnicity, education, and marital status. the sample (n = 60) comprised of 68% males (n = 41) and 32% females (n = 19) with a mean age of 51 years (sd = 7.8 ; r = 22,68). the racial / ethnic distributions of the subjects were as follows : 63% black, non - hispanic (n = 38), 20% hispanic (n = 12), and 16.7% white, non - hispanic (n = 10). table 2 shows the most commonly prevalent history of diseases and disorders among this sample, including hypertension, arthritis, hepatitis, low back pain / herniated disc, and sleep apnea. table 3 displays the most prevalent current prescription medications and over - the - counter remedies, including antianxiety, antidepressant, antiviral, antihypertensive, and insomnia and aspirin, tylenol, and vitamin / mineral dietary supplements. no proportions were significantly different between the study groups. regarding physical activity, study groups were statistically similar on the number of times exercised at strenuous, moderate, and mild exertion levels in the previous 7 days at each assessment. figure 1 shows the mean values for the bdi and bai over the course of the intervention for both study groups. for the bdi, the fixed effects for time (f[2,83.3 ] = 21.7, p < 0.01) and randomization (f[1,57.6 ] = 4.0, p = 0.05) post hoc comparisons revealed that the bdi significantly decreased from baseline to 30 days (mean difference = 6.8 ; se = 1.2 ; 95% ci : 3.8, 9.7 ; p < 0.01) and 60 days (mean difference = 7.7 ; se = 1.3 ; 95% ci : 4.5, 10.9 ; p < 0.01) for the total sample. for the max stress b group, the bdi significantly decreased from baseline to 30 days (mean difference = 5.8 ; se = 1.7 ; 95% ci : 1.7, 10.0 ; p < 0.01) and 60 days (mean difference = 7.5 ; se = 1.8 ; 95% ci : 3.1, 12.0 ; p < 0.001). for the placebo group, the bdi significantly decreased from baseline to 30 days (mean difference = 7.7 ; se = 1.7 ; 95% ci : 3.4, 11.9 ; p < 0.001) and 60 days (mean difference = 7.9 ; se = 1.9 ; 95% ci : 3.2, 12.5 ; p < 0.001). for the bai, the fixed effect for time (f[2,93.4 ] = 3.8, p < 0.05) was significant, but the effects for randomization and time by randomization were nonsignificant. post hoc comparisons revealed that the bai significantly decreased from baseline to 60 days (mean difference = 3.8 ; se = 1.4 ; 95% ci : 0.4, 7.2 ; p < 0.05) for the total sample. the bai showed a positive trend, but was statistically not significant, from baseline to 60 days (mean difference = 4.2 ; se = 1.9 ; 95% ci : 0.5, 8.8 ; p = 0.10) for the max stress b group, whereas the placebo group 's score stayed flat over the course of the intervention (mean difference = 3.4 ; se = 2.0 ; 95% ci : 1.4, 8.3 ; p = 0.25). table 4 shows the descriptive values for the sf-36, including the scores for physical functioning, role - physical, general health, vitality, social functioning, role - emotional, mental health, and bodily pain. for role - physical, general health, and bodily pain the fixed effects for time, randomization, and time by randomization were nonsignificant. for physical functioning, the time by randomization effect was not significant (f[2,87.9 ] = 3.0, p = 0.06), and post hoc comparisons revealed that the 60-day score was significantly higher than the baseline (mean difference = 11.6 ; se = 4.6 ; 95% ci : 0.3, 22.9 ; p < 0.05) for the placebo group. for vitality, the fixed effect for time (f[2,78.9 ] = 5.6, p < 0.01) was significant, but the effects for randomization and time by randomization were nonsignificant. post hoc comparisons revealed that the score significantly increased from baseline to 30 days (mean difference = 6.3 ; se = 2.5 ; 95% ci : 0.3, 12.3 ; p < 0.05) and 60 days (mean difference = 8.3 ; se = 2.7 ; 95% ci : 1.7, 14.8 ; p < 0.01) for the total sample. for social functioning, the fixed effect for time by randomization (f[2,85.1 ] = 2.5, p = 0.09) was not significant, and the effect for time (f[2,85.1 ] = 5.9, p < 0.01) was significant, but the effect for randomization was nonsignificant. post hoc comparisons revealed that the placebo group score was significantly higher than the max stress b group (mean difference = 16.6 ; se = 6.6 ; 95% ci : 3.3, 29.9 ; p < 0.05) at 30 days. for the placebo group, the score at 30 days (mean difference = 14.8 ; se = 4.5 ; 95% ci : 3.9, 25.7 ; p < 0.01) and 60 days (mean difference = 11.3 ; se = 4.7 ; 95% ci : 0.1, 22.8 ; p = 0.05) significantly improved compared to baseline. for role - emotional, the fixed effect for time (f[2,77.4 ] = 5.3, p < 0.01) was significant, but the effects for randomization and time by randomization were nonsignificant. post hoc comparisons revealed that the score significantly increased from baseline to 30 days (mean difference = 10.3 ; se = 3.6 ; 95% ci : 1.4, 19.2 ; p < 0.05) and 60 days (mean difference = 10.3 ; se = 3.9 ; 95% ci : 0.7, 19.8 ; p < 0.05) for the total sample. for mental health, the fixed effects for time by randomization (f[2,92.1 ] = 3.6, p < 0.05) and time (f[2,92.1 ] = 13.6, p < 0.01) were significant, but the effect for randomization was nonsignificant. post hoc comparisons revealed that the placebo group score was significantly higher than the max stress b group (mean difference = 13.9 ; se = 6.2 ; 95% ci : 1.5, 26.2 ; p < 0.05) at 30 days. for the max stress b group, the scores at 30 days (mean difference = 9.5 ; se = 3.5 ; 95% ci : 0.9, 18.2 ; p < 0.05) and 60 days (mean difference = 15.8 ; se = 3.4 ; 95% ci : 7.6, 24.0 ; p < 0.01) were significantly improved compared to baseline. for the placebo group, the score at 30 days (mean difference = 13.0 ; se = 3.6 ; 95% ci : 4.2, 21.8 ; p < 0.01) significantly improved, but not at 60 days. in the current study, we have demonstrated moderate short - term (60 days) improvements in depression, anxiety, and overall mental health with the max stress b product, which contains several whole - food nutrients. globally, the bdi and bai are valid and reliable assessments used to detect many symptoms of depression and anxiety, respectively [17, 18 ], and both study groups demonstrated improved scores from baseline on both assessment tools. the max stress b group showed greater improvement on the bai, while the placebo group demonstrated greater improvement on the bdi (25% versus 22% and 34% versus 39%, respectively). however, the max stress b group achieved a more continuous decrease throughout the protocol, while the placebo group had less or no improvement from 30 to 60 days. considering the positive trend demonstrated by the max stress b group, our study thus, our findings are modestly similar to those of others showing that dietary supplements have the ability to improve certain aspects of mood [1115 ]. in addition to some mood benefits, the max stress b arm showed positive effects on the mental health scale according to the sf-36. our findings are consistent with another study that showed improvements in mood (depression and anxiety) along with memory, concentration, and fatigue in response to consuming ginkgo biloba. another recent study found that ginkgo biloba was beneficial on scores of neuropsychiatric impairment, including apathy and depression / dysphoria, compared to placebo in a sample of adults with dementia. an additional study showed some improvement in ratings of depression and cognition after taking ginkgo biloba in a sample of adults who had alzheimer 's. thus, our findings appear to support other research that max stress b, similar to other nutrients, has the ability to improve subjective ratings of mental health. in summary, we have showed that max stress b offers utility for improving the overall mental health quality of life of adults with mdd or another depressive disorder with no side effects. furthermore, future trials should include longer interventions to more firmly determine the effect of max stress b on mental health and mood of adults suffering from depressive disorders. we enrolled english speaking individuals only, so our results may not be generalizable to non - english speaking persons of different racial / ethnic backgrounds. our findings may be restricted by the length of the intervention, given that mood disorder symptom changes may take longer than 60 days to occur. we did not assess dietary intake ; thus, we were not able to control for possible influences that variable may have had on our final results. however, we did assess physical activity levels, which were found to be unrelated to the outcome variables. furthermore, we did not restrict or change the use of medications by our participants, such as the use of steroids, antidepressants, or anti - inflammatory agents, given the ethical considerations associated with such decision. the findings of our study are also potentially limited by a small sample size in each study arm. a larger sample size could result in even more significant findings for mood symptoms and quality of life. assessing the changes in homocysteine and depression and anxiety scores could have provided useful information and would be of interest in future trials. depression is a significant problem that is increasing in prevalence. in fact, approximately 15 million adults have mdd, and the prevalence of mdd and its associated financial costs are a significant drain on an already overburdened united states health system and are getting worse. this disease shows signs of spiraling out of control, as options for prevention or treatment are limited. thus, any safe intervention that demonstrates promise for either sustaining mood or improving the condition of persons with mdd or another depressive disorder is urgently needed. the formula used in the current study the max stress b formula showed modest improvements in mood and mental health according to the bdi, bai, and sf-36, making our findings consistent with the prior studies. thus, our study shows that a high quality, whole - food dietary supplement may offer an opportunity for adults with depression to improve mood symptoms and quality of life. | depression, the most common type of mental illness, is the second leading cause of disability and is increasing among americans. the effect of improved nutrition, particularly with dietary supplements, on depression may provide an alternative to standard medical treatment. some studies have shown that certain nutrients (e.g., inositol and s - adenosyl methionine) are effective at improving depressed mood, although the results are not unequivocal. the current study was a randomized, double - blind, placebo - controlled trial to evaluate the efficacy of a vitamin b complex nutritional supplement (max stress b) for improving depressive and anxiety symptoms according to the beck depression and anxiety inventories (bdi and bai) in 60 adults diagnosed with major depression or other forms of depressive disorders. secondary outcomes included quality of life according to the sf-36. participants were assessed at baseline and 30- and 60-day followups. max stress b showed significant and more continuous improvements in depressive and anxiety symptoms, compared to placebo. additionally, max stress b showed significant improvement on the mental health scale of the sf-36 compared to placebo. thus, we showed modest utility of max stress b to improve mood symptoms and mental health quality of life in adults with depression. |
reed 's syndrome refers to the onset of uterine leiomyomas with cutaneous leiomyomas, the latter occurring segmentally or affecting a particular dermatome, that are classified into type 1 and 2. type 1 is caused by a novel postzygotic segmental mutation ; type 2 reflects an additional postzygotic loss of heterozygosity of the gene locus responsible for cutaneous leiomyomatosis in an initially heterozygous embryo. loss of heterozygosity is a genetic process when a heterozygous cell becomes homozygous or hemizygous by loosing the corresponding wild - type allele. this phenomenon can be regarded as a precondition for tumor growth in type 2 cases, the segmental manifestation is more distinctive with additional disseminated disease because of a germline mutation with heterozygosity of all somatic cells outside the strongly affected area. a subset of individuals with reed 's syndrome is predisposed to develop papillary renal cell carcinoma. herein, we report such rare occurrence of familial myomatosis cutis et uteri with type 2 segmental variety. a 50-year - old female presented to us with history of developing multiple brown painful lesions which appeared initially over her left leg. they extended to appear insidiously over trunk, chest, and upper limbs about 25 years back which extended to appear insidiously despite excision of the left leg lesion. she also underwent hysterectomy, owing to menorrhagia and multiple uterine fibromas, 25 years ago. dermatological examination revealed multiple tender, hyperpigmented papulonodules over the left lower limb, trunk, right side of the chest, and bilateral upper limbs [figures 1a, b and 2a, b ]. a clinical suspicion of leiomyomas was confirmed by histopathological findings of a circumscribed tumor in the dermis composed of bundles of smooth muscles cells arranged in an interlacing and whorled pattern, having abundant eosinophilic cytoplasm and elongated nuclei with blunt ends. deep red color with masson 's trichome confirmed smooth muscle fibers [figure 3a, b ]. gynecological consultation for her daughter 's complaints of menorrhagia revealed uterine fibroids. a diagnosis of familial myomatosis cutis et uteri, segmental type 2 was made. (a) unilateral distribution of leiomyomas on the left leg, (b) multiple papulonodules on the extensor aspect of the left arm (a) multiple papulonodules distributed over the right side of chest, (b) multiple leiomyomas distributed over the trunk (a) histopathology shows eosinohillic smooth muscles arranged in interlacing and whorled pattern (h and e, 40), (b) histopathology showing red color stained smooth muscle fibers (masson 's trichome, 40) leiomyoma, a rare benign tumor of smooth muscle derived from arrector pili muscle, media of blood vessels, smooth muscle of scrotum, labia majora, and nipples, can be of pilar, genital (dartoic), and angioleiomyomic type. pilar leiomyoma originating from the arrector pili is the most common type of cutaneous leiomyoma and usually occurs in early adult life. it classically presents as a collection of pink, red or dusky brown firm nodules of varying size. the gene that predisposes to multiple pilar leiomyomas has been mapped to chromosome 1q 42.3-q 43. multiple cutaneous leiomyomas, with inherited predisposition, are linked to uterine leiomyoma (reed 's syndrome), and increased incidence of renal cell carcinoma. type 1 reflects heterozygosity for the underlying mutation, with a clinical picture similar to that in a nonmosaic phenotype. leads to homo- or hemizygosity, with a pronounced segmental manifestation of lesions in the affected segment. though the exact molecular etiopathogenesis of multiple cutaneous leiomyomas is not known, recent studies have demonstrated the involvement of a classical tumor suppressor gene encoding fumarate hydratase, in the pathogenesis of multiple leiomyomas. the lesions are often sensitive to touch, cold, emotional stress, or spontaneous pain. it could be attributed to the local pressure exerted by the tumor on cutaneous nerves. the excitation of these muscles occurs via the sympathetic nervous system resulting in contraction with the influx of calcium ions. hence, nifedipine, a calcium channel blocker, has a role in relieving pain associated with cutaneous leiomyoma. this case of familial cutaneous myomatosis et uteri is reported for its rare occurrence and is rarer for being segmental type 2 variety. | reed 's syndrome or familial myomatosis cutis et uteri, an autosomal dominant inherited condition with incomplete penetrance, is characterized by multiple cutaneous and uterine leiomyomas.[1 ] uterine leiomyomas usually commence earlier compared to that in the general population and cutaneous leiomyomas may precede, follow or occur concurrently. few patients may have associated renal cell carcinoma. herein we report a case of a 50-year - old female with multiple, painful cutaneous leiomyomas and who had undergone hysterectomy owing to large uterine fibroids. her 18-year - old daughter also has uterine fibroids. |
dna double - strand breaks are extremely toxic dna lesions that arise from a variety of sources, including ionizing radiation, radiomimetic drugs [2, 3 ], oxidative stress [4, 5 ], abortive or inhibited topoisomerase reactions, and immunological processes such as v(d)j and class - switch recombination. thus, dsb repair is a critical process to which mammalian cells have devoted enormous resources, creating a complex network of repair systems that are intricately linked with cell cycle control and survival / death pathways. remarkably, molecular mechanisms of dsb repair in mammalian cells almost completely eluded researchers for decades, until the implication of ku autoantigen in 1994 unleashed a cascade of investigations by which the major players and primary mechanistic details of dsb rejoining were rather rapidly defined. much of the seminal work elucidating these repair systems has taken advantage of defined dsb substrates, either constructed in vitro or formed in cells by site - specific nucleases. these defined dsbs typically have canonical 5-phosphate and 3-hydroxyl termini suitable for further processing by exonucleases, polymerases, and ligases. thus, this experimental approach, while extremely powerful, bypasses an important step in repair, namely, the cleanup of the chemically modified termini and/or damaged bases that accompany most dsbs formed in a natural or clinical environment. several enzymes have been described that are capable of resolving various end modifications, and their specificity and cofactor requirements have been determined in some detail. a limited number of studies have been directed toward distinguishing which of these enzymes are actually used for repair in cells, and much work remains to be done in this area. other cellular studies, however, suggest that resolution of damaged ends, especially those with complex or multiple modifications, can be a critical and in some cases rate - limiting step in repair. this paper will attempt to summarize current knowledge of the molecular mechanisms for resolution of damaged dsb ends in mammalian cells and the biological consequences of that processing. studies based on other organisms such as yeast are included only insofar as they provide insight into questions for which no comparable mammalian data are available. dsbs induced by ionizing radiation stem from attack by free radicals, primarily the hydroxyl radical, on deoxyribose, with formation of carbon - centered free radicals on any of the five deoxyribose carbons potentially leading to strand cleavage. significant free radical - induced blocked termini include nucleoside 5-aldehyde on the 5-side, and phosphoglycolate (pg), phosphoglycoaldehyde, formyl phosphate, and 3-keto-2-deoxynucleotide on the 3-side [1, 11, 12 ] (figure 1). most of these moieties, as well as most of the free radical - generated oxidized abasic sites, are unstable and break down spontaneously to leave breaks with 3- and 5-phosphates. a notable exception is the 3-pg, whose stability against further degradation even under harsh conditions has rendered it the probe of choice for studies of 3-end - processing. however, the 2-oxidized abasic site is considerably more stable than other abasic lesions, and both this lesion and the 5-aldehyde can readily be isolated intact from treated dnas. the relative frequencies of the various modified termini are presumably similar for single versus double strand breaks, but there are few quantitative data available on this point. an early study utilizing end - labeled dna suggested that approximately half of all radiation - induced breaks bore 3-pg termini, with 3-phosphates comprising most of the remainder. however, a more recent measurement by mass spectrometry suggested a lower 3-pg incidence of approximately 10% of total sugar oxidation products. considerable attention has been devoted recently to clustered damage induced by spurs of closely spaced ionizations that surround tracks of the secondary electrons dislodged by -rays [25, 26 ]. such lesions presumably contain random mixtures of strand breaks, abasic sites, and any of the myriad forms of oxidative base damage, including 8-oxoguanine (8-oxog) and thymine glycol [1, 27 ]. monte carlo calculations of radiation tracks predict that a substantial portion of damage sites will harbor multiple lesions, including dsbs with accompanying nearby base damage [28, 29 ]. the primary empirical evidence for clustered lesions is the generation of additional dsbs in irradiated dna or cells by posttreatment with glycosylases and abasic endonucleases that together cleave sites of base damage [4, 30 ]. while these data clearly confirm that complex lesions do occur, biochemical studies with purified glycosylases and defined synthetic substrates indicate that nearby breaks and gaps can block recognition and removal of damaged bases [3133 ], suggesting that glycosylases require a relatively intact duplex dna structure on which to act. thus, base damages near dna ends, even if not sequestered by dsb repair proteins, are likely to be poor substrates for repair glycosylases and would instead have to be removed by dsb - specific mechanisms such as end trimming. similar to radiation, the radiomimetic natural products bleomycin, neocarzinostatin (zinostatin), and calicheamicin induce dsbs by free radical mechanisms, but the damage is largely restricted to the deoxyribose sugar moiety and to a few specific carbons therein [2, 3 ]. the zinostatin- and calicheamicin - induced dsbs are similar in chemical structure but are formed on a 2-base (zinostatin) and 3-base (calicheamicin) 3-stagger between breaks in opposite strands. for either compound, one dsb end has a 5-aldehyde and mixture of 3-phosphate and 3-pg termini on a 1-base (zinostatin) or 2-base (calicheamicin) 3-overhang. the opposite dsb end has both 5- and 3-phosphate termini, on a 2-base (zinostatin) or 3-base (calicheamicin) 3-overhang. bleomycin induces dsbs with predominantly blunt ends or 1-base 5-overhangs, with predominantly 3-pg termini, owing to its specifically targeting oxidation of the c-4 position, from which 3-pgs derive. for diffusible oxidants such as h2o2 while the chemistry of dna cleavage is similar to that seen with radiation, attributable mostly to oxidative fragmentation of deoxyribose by attack of hydroxyl radicals, there are marked sequence preferences for cleavage, owing to preferential fe binding at certain sites on dna. presence of at least 25% 3-pg termini was reported for strand breaks in dna from h2o2-treated cells, and this fraction would likely be similar for ssbs and dsbs. however, the initial ratio of ssbs to dsbs is much higher for h2o2 (~500 : 1) than for radiation (~25 : 1). thus, most dsbs in h2o2-treated cells result from collision of ssbs with replication forks. dsbs in nonreplicating g1 cells are much less frequent but they do occur, possibly as a result of local repetitive redox cycling of dna - bound fe or by collision of ssbs with transcription bubbles and subsequent oxidative or endonucleolytic cleavage of the exposed single - stranded dna. dna topoisomerases i and ii (top1 and top2) relax dna by inducing transient ssbs (top1) and dsbs (top2) wherein the topoisomerase is covalently linked through a tyrosine to dna 3-phosphate (top1) or 5-phosphate (top2) termini [42, 43 ]. normally the breaks are rapidly rejoined by the topoisomerase with concomitant dissolution of the tyrosine - dna covalent bond, but reversal can be prevented by inhibition or inactivation of the enzyme, or by oxidative damage to the dna. in the case of top2 (figure 2), the irreversible dsb will then have 3-hydroxyl termini, and 4-base 5-overhangs terminated in a tyrosyl - linked topoisomerase. top1-mediated dsbs arise primarily by collision of replication forks with ssbs, resulting in a so - called one - ended these dsbs would likely have normal 3-hydroxyl and 5-hydroxyl termini and blunt ends if formed from a ssb in the leading template strand, or 5-phosphate and 3-top2-linked termini, if formed from a ssb in the lagging template strand, although neither of these structures has been verified empirically (figure 3). moreover, dsbs can also be formed by a replication - independent but transcription dependent process, probably involving collision of a transcription complex with a ssb [6, 44 ]. these dsbs are correlated with formation of r - loops (locally denatured dna segments with one strand partially annealed to nascent mrna) in the wake of arrested transcription complexes. however, the mechanism by which r - loops promote dsbs is currently unknown, and thus the structure of these dsbs is difficult to predict. it is possible that the breaks in both dna strands are nucleolytic and occur some distance from the initiating top1-mediated ssb. particularly when considering the repair of damage induced by topoisomerase inhibitors, it is important to distinguish between irreversible breaks and cleavable complexes. the latter are merely intermediates in dna relaxation that are stabilized by the inhibitor [45, 46 ]. while the dna strands in cleavable complexes are indeed broken and are detected as breaks upon detergent lysis of the cells, they are rapidly religated by the topoisomerase if instead the inhibitor is simply removed. when replication forks or transcription complexes collide with cleavable complexes that are persistent due to inhibitors, oxidative dna damage, or topoisomerase inactivation, proteasomes are recruited to degrade the topoisomerase [4750 ], resulting in an irreversible break with a topoisomerase fragment linked to one dna terminus (figure 2), which forms the actual substrate for the end processing enzymes discussed in the following sections. consideration of the resolution of damaged dsb ends must take into account the two distinctly different mechanisms by which dsbs are repaired in mammalian cells. in nhej, 5- and 3-termini are in general minimally processed to yield ends that can be annealed or juxtaposed, then patched and ligated (figure 4). thus, at least one strand must have a 3-hydroxyl terminus suitable for polymerase - mediated extension and ligation on one end and a 5-phosphate on the other end. given the relatively high tolerance of the xrcc4/dna ligase iv (x4l4) complex for imperfectly matched ends (see section 5), the other strand could in principle bear unligatable termini (although probably not large adducts), which could be resolved after ligation of the first strand. however, at least in some cases, enzymes capable of removing damaged termini on either recessed or protruding 5- or 3-single strands would be required. such terminal processing could occur at several points in the nhej pathway (figure 5), for example, even before ku binding, or after synapsis and dna - pk autophosphorylation. moreover, processing of blocked termini on the second strand could occur after ligation of the first strand and dissociation of the nhej repair complex. hrr, on the other hand, involves extensive exonucleolytic 5-resection, followed by invasion of a homologous sister duplex by the exposed 3-overhang. in this case, removal of any 5-blocks, at both ends of the break, is essential at a very early step in repair (figure 5). the strand invasion step would likely proceed despite small chemical modifications of the 3-overhang, although any blocked 3-termini would have to be removed prior to the extension step. tdp1 was isolated biochemically as a yeast enzyme that removed a protein fragment of top1 from the 3-end of dna. the gene was identified by random mutagenesis, and the corresponding human gene was soon cloned by homology. in vitro, human tdp1 removes tyrosyl - linked peptides as well as simple tyrosyl moieties from 3-termini of either ssbs or dsbs, leaving a 3-phosphate that can then be removed by pnkp (see section 4.3). tdp1 is also capable of removing other 3-blocks such as 3-pgs and cleaved abasic sites, although less efficiently than 3-tyrosyl linkages ; for example, human tdp1 removes 3-phosphotyrosyl moieties about 100 times more efficiently than 3-pgs. the active site of the enzyme contains a putative binding channel for single - stranded dna, suggesting that double - stranded substrates become partially denatured prior to phosphotyrosyl cleavage. as predicted from such a model, tdp1 acts more efficiently on single - strand than on double - strand substrates and more efficiently on single - strand 3-overhangs than on 3-recessed ends of dsbs. the extremely rare human genetic disease spinocerebellar ataxia with axonal neuropathy (scan1) is associated with an h493r mutation in tdp1 that reduces the rate of hydrolysis of tyrosyl - dna bonds by about 25-fold, and also dramatically increases the lifetime of a transient intermediate wherein tdp1 is covalently linked to the dna 3-terminus. despite the leakiness and complexity of this mutation, these cells provide a model of tdp1 deficiency for assessing its role in repair of various dna lesions. tdp1-deficient mice, murine embryonic fibroblasts (mefs) [6264 ], and dt-40 chicken erythrocytes have also been generated. there is little doubt that tdp1 is instrumental in removing 3-linked top1 fragments from ssbs, as ssbs are more persistent in scan1 cells than in normal cells after treatment with top1 inhibitors. moreover, tdp1 binds to dna ligase iii, which carries out the ligation step in ssb repair. conversely, there is no evidence of tdp1 binding to any nhej or hrr proteins nor any evidence that tdp1 is recruited to dsb ends by other dsb repair factors. in fact, in vitro nhej proteins inhibit tdp1 activity toward dna ends, probably by restricting access to the termini. as judged by h2ax focus - formation assays, there is no detectable effect of tdp1 deficiency on repair of radiation - induced dna dsbs in either scan1 cells or mefs. the slight sensitivity of scan1 cells to radiation is apparently attributable to a defect in repair of ssbs. tdp1 mefs and dt-40 cells as well as scan1 cells are sensitive to the top1 inhibitor camptothecin, but this sensitivity is probably likewise mostly due to a defect in ssb repair, and it is unclear whether tdp1 is involved in top1 removal from the dsbs. nevertheless, tdp1 mice and tdp1 dt-40 cells are both sensitive to bleomycin, while tdp1-knockdown hela cells are slightly sensitive to calicheamicin. moreover, following treatment with calicheamicin, scan1 cells show more chromosome aberrations, particularly dicentric chromosomes, than normal cells. in extracts of scan1 cells and tdp1 mefs, pg termini on 3-overhangs of dsbs are highly persistent, with no detectable processing for several hours, while in extracts from normal cells, substantial processing can be seen within minutes. thus, despite the fact that tdp1 's activity toward pg termini is about 100 times weaker than toward its canonical 3-phosphotyrosyl substrate, all these results suggest a major role for tdp1 in resolution of 3-pg termini of dsbs, on both blunt ends and 3-overhangs. with calicheamicin in particular, it is unlikely that the observed sensitivities result from effects on ssb repair, as nearly all lesions induced by calicheamicin are bistranded. neither yeast tdp1 nor human tdp1 has any detectable activity toward a simple 5-phosphotyrosyl oligonucleotide. however, a more realistic substrate consisting of an oligonucleotide with phosphotyrosyl - linked peptide, derived from a top2 cleavable complex, is cleaved by yeast tdp1 and less efficiently by human tdp1 (j. l. nitiss, university of illinois college of pharmacy, personal communication). however, tdp1 dt-40 cells are sensitive to top2 inhibitors, while overexpression of tdp1 in 293 cells reduces the level of dna damage seen after top2 inhibitor treatment. apparent species - specific differences may reflect the differences in the efficiency of alternative repair pathways for these lesions. tdp2, which has no homology to tdp1, has robust phosphodiesterase activity toward 5-tyrosyl dna ends and much weaker activity toward 3-tyrosyl ends. tdp2 mefs and dt-40 cells show significant sensitivity to top2 inhibitors [74, 75 ]. thus, tdp2 apparently plays a major role in resolution of top2-linked dsbs (figure 2), although increased persistence of top2-linked dna in tdp2-deficient cells has yet to be directly demonstrated. ctip is a critical factor in channeling dsbs to either the nhej or the hrr pathway. in late s and g2 phase, ctip phosphorylation at s327 by cdk2 promotes brca1 recruitment and the initiation of 5-resection, which in turn precludes nhej and commits a dsb to repair by hrr [7678 ]. however, ctip also has modulatory effects on nhej even in g1, where it promotes certain microhomology - mediated end joining (mmej) events. sae2, the s. cerevisiae homologue of ctip, harbors endonuclease activity that acts on overhangs near hairpins [79, 80 ], and it has been proposed that this activity initiates 5-resection for hrr by endonucleolytically releasing an oligonucleotide (1040 bp) from the 5-end. such sae2-dependent cleavage can most clearly be seen in the processing of spo11-linked breaks generated during meiosis, and similar cleavage can be seen in mouse testes, presumably by either an endonucleolytic activity of ctip or a ctip - dependent activity of mre11. an initial report of mre11-dependent ctip nuclease activity in vitro has yet to be further elucidated. human fibroblasts deficient in either ctip or mre11 show a profound deficiency in repair of dsbs induced by the top2 inhibitor etoposide in g1 phase, as judged by h2ax focus formation. similar results were seen when ctip was knocked down by sirna in either fibroblasts or hela cells. inasmuch as the repair was shown to be ligase iv - dependent, these results suggest that both ctip and the mre11/rad50/nbs1 (mrn) complex are required for nhej of top2-linked dsbs, most likely reflecting a role in endonucleolytic removal of the top2 peptide from the 5-ends of the dsbs (figure 2). remarkably, in these studies there was virtually no repair in ctip - knockdown fibroblasts even as long as 6 hr after treatment. it seems surprising that tdp2 apparently could not substitute, even partially, for ctip in resolving these blocked dsb ends. however, dt-40 cells harboring an unphosphorylatable s332a ctip allele are viable and proficient in homologous recombination but are sensitive to both top1 and top2 inhibitors. since the s332a mutation abrogates ctip binding to brca1, these results suggest that brca1 and ctip are both required for endonucleolytic release of both top1 and top2 fragments from 3- and 5-termini, respectively, of topoisomerase - mediated dna breaks, although again increased persistence of topoisomerase - linked dna was not explicitly demonstrated. curiously, while ctip(s322a)/tdp1 dt-40 cells were much more sensitive than either single mutant to camptothecin, the two mutations were epistatic in conferring sensitivity to etoposide. these results suggest that ctip and tdp1 are essential factors in a single pathway for resolution of top2 dsbs, a conclusion that is somewhat difficult to rationalize in terms of their known biochemical activities. moreover, the inference that this dsb repair requires ctip phosphorylation at s332 (equivalent to human s327) and ctip binding to brca1 is difficult to reconcile with the human cell studies, wherein cells expressing an s327a mutant of ctip had wild - type proficiency in g1 repair of etoposide - induced dsbs. pnkp removes phosphates from 3-dna ends and phosphorylates 5-ends, using atp as a cofactor [86, 87 ], but has no other known activities toward other modified termini, including 3-pgs. pnkp shows no strict dependence on dna secondary structure, acting on simple oligomers, as well as nicks, gaps, and dsb ends. pnkp binding to xrcc1 and xrcc4 suggests recruitment to repair complexes for ssb and dsb repair, respectively, although recruitment to ssbs does not require and may precede xrcc1 recruitment. other than apurinic / apyrimidinic (ap) lyases, human cells contain two known ap endonucleases, ape1 and ape2, that cleave the phosphodiester bond between the 5-phosphate of the ap site and the preceding nucleotide, leaving a 3-hydroxyl end. both enzymes are homologous to e. coli exonuclease iii and have similar activities of ap endonuclease, 35 exonuclease, and a phosphodiesterase activity that removes 3-pg and 3-phosphate termini [9294 ]. the canonical and by far the most efficient activity of ape1 is the cleavage of ap sites in duplex dna. ape1 also removes 3-pgs and other 3-blocks from dna ends, although its activity (measured as kcat / km) toward 3-pgs at internal nicks, recessed 3-ends, and blunt ends is 100, 500, and 1800 times lower, respectively, than its abasic endonuclease activity. thus, the substrate preferences of ape1 are complementary to those of tdp1, which acts more efficiently on overhangs. studies of ape1 function have been complicated by the fact that it is essential for survival. ape1-deficient mice and mefs are inviable, and conditional ape1 cells die within days of ape1 deletion. however, ape1 knockdown renders tk6 lymphoblastoid cells and hct116 colorectal carcinoma cells more resistant to radiation, but more sensitive to the radiomimetic agent bleomycin. to explain this paradoxical result, it was proposed that bleomycin sensitivity reflects a defect in removal of the predominant 3-pg moieties at ends of (mostly blunt - ended) bleomycin - induced dsbs, while radioresistance may result from fewer complex lesions such as strand breaks with closely opposed strand breaks being converted to toxic dsbs when ape1 is knocked down. thus, these results suggest that ape1 's phosphodiesterase activity on 3-pg dsb ends is biologically significant, despite its inefficiency. an early report of association between ape1 and the core nhej protein ku has been neither confirmed nor refuted. ape1 can also remove 3-phosphotyrosyl moieties from a recessed 3-end, but even less efficiently than 3-pgs. however, it has robust 35 exonuclease activity toward mismatched 3-terminal bases, as well as 3-pg removal activity [93, 94, 101 ]. both activities are significantly stimulated by proliferating cell nuclear antigen (pcna), with which ape2 colocalizes at nuclear foci in cells following exposure to oxidative stress. thus, based on its known activities and its homology to ape1, ape2 is a candidate enzyme for resolving 3-pgs and other 3-blocks, although its detailed substrate requirements and the kinetic parameters for various substrates have not been rigorously defined. artemis was isolated as the factor mutated in a subset of human severe combined immune deficiency patients with accompanying radiosensitivity (rs - scid). rs - scid is also known as athabascan scid (scid - a), owing to a single mutation detected with relatively high incidence in athabascan amerindians. artemis has intrinsic 53 exonuclease activity (recently suggested to be a contaminant), but upon complexation with dna - pk it acquires an endonuclease activity that opens dna hairpins, which are formed as intermediates in v(d)j recombination. this same activity trims both 3- and 5-overhangs of dna dsb ends, usually removing the 5-overhang entirely while shortening 3-overhangs to 4 - 5 bases (figure 6(a)). however, upon extended incubation, these short 3-overhangs are further shortened to 2 - 3 bases. although only dna - pkcs is strictly required for artemis endonuclease activity, ku enhances activity, especially for less favorable substrates, probably by improving dna end binding. artemis is capable of trimming 3-pg - terminated overhangs, thus providing a 3-hydroxyl terminus for patching and ligation of dsbs, and on shorter overhangs (35 bases) the pg terminus stimulates trimming. artemis also coordinately trims both dna strands at blunt ends, whether terminated in a 3-pg or a 3-hydroxyl (figure 6(b)). this process is much slower than the trimming of overhangs and proceeds via endonucleolytic removal of several bases from the 5-terminal strand, followed by trimming of the resulting 3-overhang. whereas the 53 exonucleolytic activity of artemis requires a 5-phosphate terminus, this endonucleolytic trimming does not. thus, based on its biochemical properties, artemis could resolve a 3-pg, and probably any other small 3-modification, in almost any context, including the 3-overhanging pgs of zinostatin- and calicheamicin - induced dsbs, as well as blunt - ended bleomycin - induced dsbs. consistent with a significant role in dsb repair, rs - scid patient - derived artemis - deficient fibroblasts show increased sensitivity to zinostatin, bleomycin, and ionizing radiation, as well as a defect in dsb repair following treatment with these agents. moreover, both hypersensitivity and dsb repair deficiency can be rescued by stable complementation with wild - type artemis expressed from lentivirus, but not with endonuclease - deficient mutant of artemis. these results imply that radiosensitivity in artemis - deficient cells reflects a defect in dna processing, rather than the cell signaling functions of artemis. in principle, artemis could trim even the most extensively damaged dna ends, for example, a radiation - induced dsb with multiple base damages near the end, and trimming might continue until an undamaged dna segment is exposed. base damage could promote single strandedness near the dna end and thus promote artemis - mediated cleavage, although these same structural modifications could have the opposite effect of interfering with substrate recognition. however, implication of artemis in trimming of modified dna ends for dsb repair is complicated by the finding that the defect in repair is confined to a small fraction of the total dsbs, typically 1020% in the case of radiation or zinostatin [108, 109 ]. according to some studies, in artemis - deficient cells, these breaks are never repaired, even after several days, while the other ~90% of dsbs are repaired as quickly as in normal cells. the repair - resistant breaks appear to be primarily those in heterochromatin, and their repair also requires atm, the mre11/rad50/nbs1 (mrn) complex, and 53bp1. a model has been proposed wherein both atm and 53bp1 are required primarily to phosphorylate the heterochromatin protein kap-1, resulting in decondensation of heterochromatin to allow access to dsb repair factors [109, 111 ]. since artemis and atm are epistatic for repair of these slowly rejoined breaks, it may be inferred that artemis, like atm, is required only for repair of breaks in heterochromatin, although this has not been explicitly shown. inasmuch as it is unlikely that the chemistry of the dsbs, especially the relatively well - defined breaks induced by zinostatin and bleomycin [2, 3 ], is substantially different between heterochromatic and euchromatic breaks, it is difficult to explain why artemis - mediated trimming of the dsb ends would be required only when the dsb is in heterochromatin. it is possible that the dsb repair mechanisms for persistent breaks such as those in heterochromatin are sufficiently different from those for rapidly repaired breaks and that enzymes which act on damaged ends are somehow excluded from acting on the more persistent dsbs. alternatively, an intriguing study of dsbs induced at a putative partially heterochromatic dna locus by the rare - cutting endonuclease i - scei suggested that, rather than trimming dna ends, artemis excises an entire dsb - containing nucleosome, thus allowing rejoining of the more accessible linker regions on either side of that nucleosome and deletion of the dna within the nucleosome. with respect to a role for artemis in repairing top2-linked dsbs, there are conflicting data on the effect of artemis deficiency on sensitivity to top2 inhibitors. homologous knockout of artemis conferred significant (~2-fold) etoposide sensitivity to nalm-6 pre - b cells, while knockout in hct116 colorectal carcinoma cells had no effect on sensitivity. rs - scid patient - derived, artemis - deficient fibroblasts showed no defect in rejoining of etoposide - induced dsbs, although in separate study, similar patient - derived cell lines showed slight sensitivity to etoposide. a 3-phosphotyrosyl - terminated 3-overhang was cleaved, albeit rather slowly, by artemis in presence of dna - pk, but dna ends bearing 5- or 3-phosphotyrosyl - linked protein fragments (i.e., structural models of topoisomerase - mediated dsbs) have apparently not been tested as artemis substrates. metnase, also called setmar, has both protein methyltransferase and endonuclease activities and was discovered in a search for human proteins homologous to bacterial transposases. although the nuclease activity of metnase does not open dna hairpins and does not require any protein cofactors, its specificities are otherwise remarkably similar to those of artemis. it is inactive toward intact double - stranded dna, but it trims both 5- and 3-overhangs of dsbs, as well as flaps and y - structures mimicking frayed dsb ends. it appears to require a free 5- or 3-dna terminus for entry, as it does not cleave single - stranded loops flanked at both ends by double - stranded regions. its methyltransferase activity promotes nhej, at least in part by methylating histone h3 at lys36. however, its activity in stimulating and improving the fidelity of end joining of transfected plasmid substrates is dependent on its nuclease activity. thus, given its biochemical specificity, its binding to xrcc4, and its implication in nhej, metnase is a candidate for trimming diverse types of damage to dna ends, thereby creating substrates more amenable to patching and ligation. mammalian cells contain a variety of 35 and 53 exonucleases that could in principle serve to remove terminal modifications, similar to exonuclease iii in e. coli. however, relatively few mammalian exonucleases have been tested for activity toward terminally modified substrates and those that have shown little such activity. the major 35 exonuclease activity in mammalian cell extracts is dnase iii, also called trex1. exonucleolytic digestion of dna by dnase iii is completely blocked by a 3-pg terminus, in either the presence or absence of ku or by a 3-phosphotyrosyl terminus. its crystal structure revealed a tight nucleotide binding pocket that would be unlikely to either accommodate the extra bulk of a terminal pg or recognize the pg itself as a terminal nucleotide. the werner syndrome - associated protein wrn1, which binds to both ku and xrcc4 (suggesting some role in nhej), likewise has no activity toward either 3-pg or 3-phosphotyrosyl substrates. other mammalian 35 exonucleases, including rad9 and mre11 (which is strongly implicated in both nhej and hrr), do not appear to have been tested for activity toward modified termini. mammalian 53 exonucleases include apollo (snm1b, dclre1b), artemis (snm1c, dclre1b, discussed above), aprataxin, exonuclease 1 (exo1), fanconi - associated nuclease 1 (fan1), flap endonuclease 1 (fen1), and the aprataxin - and - pnkp - like factor (aplf, palf) [126128 ]. aprataxin has been implicated in resolution of abortive 5-adenylated ligase intermediates for both ssb repair and nhej. in general, the 53 exonuclease function of the other enzymes either requires or strongly prefers a 5-phosphate. apollo, artemis (discussed above), aprataxin, exonuclease i, and palf all have single - strand endonuclease activity as well that could in principle resolve either 3- or 5-terminal blocks. however, except for artemis and aprataxin, there is only indirect evidence that resolution of damaged termini is a biologically relevant function of these enzymes in cells. palf binds to xrcc4 and can trim 3-overhangs to promote end joining in a defined in vitro nhej system based on purified recombinant proteins. fen-1 has been implicated in resolution of flap structures for nhej in yeast, but there is no comparable evidence in mammalian cells. the mechanism by which the 5-aldehyde termini of zinostatin- and calicheamicin - induced dsbs are resolved is essentially unknown. one possibility is release of a 5-terminal oligonucleotide by ctip (see section 4.2). alternatively, as 5-aldehydes are formed in only one strand of these dsbs, they could be removed by displacement synthesis following religation of the break in the complementary strand (see section 5). threads onto dna ends, making tight contact with the dna grooves. once bound to a dna end, ku recruits other nhej factors and ku also has a potent lyase activity that serves to cleave abasic sites near dna ends. this lyase activity also removes deoxyribose-5-phosphate moieties such as would be present at one 5-terminus of a dsb formed by ape1-mediated cleavage of an ap site near a closely opposed strand break. although lyase activity might be expected for any protein whose basic amine residues are in tight contact with dna grooves, the lyase activity of ku shows specificities which suggest that it may be specifically adapted to promote efficient and accurate nhej. for example, it is more active toward ap sites on 5-overhangs, where it would generate ligatable 5-phosphates than on 3-overhangs where a nonligatable cleaved sugar moiety would be left blocking the 3-terminus. in addition, ap sites which are sufficiently distant from a terminus such that ligation can still occur despite their presence are relatively resistant to ku 's lyase activity. in other words, ku appears to cleave ap sites preferentially in situations where doing so would facilitate end joining or increase its accuracy, whereas cleavage is suppressed when it would lead to unnecessary deletion of terminal nucleotides prior to religation. like all dna ligases, the nhej - associated dna ligase iv has an absolute requirement for 5-phosphate and 3-hydroyxl termini. however, in its usual tight complex with xrcc4, ligase iv has considerable tolerance for ends that are not perfectly matched and overhangs that are not perfectly annealed. for example, short, partially complementary overhangs can be annealed and ligated despite the presence of single - stranded flaps, mismatched bases, and missing nucleotides in the presumed annealed segment (figure 7). moreover, the efficiency of ligation of imperfectly matched ends is increased markedly by the presence of xlf [20, 136, 137 ], a scaffold protein that forms filaments of alternating homodimers with xrcc4. in vitro, the combination of x4l4, ku, dna - pkcs, and xlf can join two ends with completely mismatched overhangs, albeit only in one strand (figure 7(b)), and can also join a protruding 5-overhang to a blunt end (not shown). the gap - filling nhej - associated dna polymerase displays a similar tolerance for imperfectly annealed template / primers [138, 139 ]. while ligases and polymerases could in principle have higher stringency in the context of a full repair complex than when acting alone on a dna substrate in vitro, end joining experiments in both extracts and intact cells confirm that ligation of mismatched overhangs can occur. for example, in chinese hamster ovary (cho) cells, end joining of two - ataa 3-overhangs of i - scei - induced dsbs apparently proceeds predominantly by annealing the two terminal taa trinucleotides to each other (despite the internal aa mismatch), followed by single - base gap filling and ligation (figure 7(c)). further studies in human cell extracts supplemented with an extremely error - prone mutant form of polymerase likewise show that repair patches containing multiple mismatches can be ligated during nhej (figure 7(d)). by analogy, it is likely that similar patching and ligation can occur even on ends that contain some degree of base damage and other modifications at or very near dna termini. a few studies using defined substrates that incorporate such damage support this proposal. for example, similar to the -ataa overhangs mentioned above, a substrate bearing at an -acg 3-overhang on one end and an -ac(8-oxog) overhang at the other, can be annealed at the terminal dinucleotides, patched and ligated (figure 7(e)). in this cell extract - based nhej model, the 8-oxog - containing strand clearly undergoes polymerase -dependent single - base extension and ligation without prior removal of the damaged base. likewise, the nonplanar base thymine glycol suppressed patching and ligation of partially complementary -cta overhangs by at least 90%. this same in vitro system was able to join two ends, one with a pg - terminated -acg 3-overhang and one with an identical but hydroxyl - terminated overhang (figure 7(f)). in this case, the hydroxyl - terminated strand was often patched and ligated, without any processing of the 3-pg - terminated complementary strand. with this substrate, the second, 3-pg - terminated break could likely be repaired at a later time by the less error - prone base excision and ssb repair pathways. similar postend - joining repair of residual damage could also occur for the dsb substrates containing abasic sites and damaged bases, presumably in a relatively error - free manner. thus, in all these cases, tolerance for residual damage in end joining could actually increase fidelity, because a less tolerant system might require endonucleolytic trimming of these damaged ends, resulting in deletion of terminal nucleotides in the repaired products. in a different extract - based end joining assay, either 8-oxog or a base mismatch at the penultimate base pair of a blunt - ended dsb was found to strongly inhibit end joining, while an abasic site the same position blocked joining entirely. although the specific substrates used were not identical, this experimental system appears to show somewhat less tolerance for nearby mismatches, abasic sites, and base damage than those mentioned above. nevertheless, end joining products that retained damaged bases or base mismatches were detected in this system as well. overall, mammalian cells are seen to harbor multiple redundant pathways and enzymes for resolution of damaged dsb ends, with diverse specificities and various degrees of sequence conservation. much remains to be determined regarding which mechanisms predominate under particular circumstances and how the various pathways are prioritized in the cell. experimentally, a major obstacle to addressing these issues is the lack of assays capable of tracking dsb end processing in intact cells. technologies to induce terminally modified dsbs at a defined time and at specific sites would be very helpful in this regard. while chemical techniques such as mass spectrometry are still far too insensitive to be used to follow dsb end processing, continuing improvements and adaptations may yet make such direct chemical analysis tractable. with respect to therapeutic implications, the most obvious application would be to identify inhibitors that would block end processing and thereby enhance the antitumor activity of dsb - inducing cancer chemotherapeutic agents. given the redundancy of end processing pathways outlined above, resolution of damaged ends might seem an unlikely target for inhibition. nevertheless, despite apparent redundancy, some of the single mutants, such as the tdp2 dt-40 cells and mefs [74, 75 ], do show significant sensitivity to certain types of dsbs. given the general tendency of cancer cells to lose proficiency in one or more repair systems, there is reason to hope that at least for select classes of tumors, such sensitization might be induced selectively in tumor cells, while having less effect on fully repair - competent normal cells. | most dna double - strand breaks (dsbs) formed in a natural environment have chemical modifications at or near the ends that preclude direct religation and require removal or other processing so that rejoining can proceed. free radical - mediated dsbs typically bear unligatable 3-phosphate or 3-phosphoglycolate termini and often have oxidized bases and/or abasic sites near the break. topoisomerase - mediated dsbs are blocked by covalently bound peptide fragments of the topoisomerase. enzymes capable of resolving damaged ends include polynucleotide kinase / phosphatase, which restores missing 5-phosphates and removes 3-phosphates ; tyrosyl - dna phosphodiesterases i and ii (tdp1 and tdp2), which remove peptide fragments of topoisomerases i and ii, respectively ; and the artemis and metnase endonucleases, which can trim damaged overhangs of diverse structure. tdp1 as well as ape1 can remove 3-phosphoglycolates and other 3 blocks, while ctip appears to provide an alternative pathway for topoisomerase ii fragment removal. ku, a core dsb joining protein, can cleave abasic sites near dna ends. the downstream processes of patching and ligation are tolerant of residual damage and can sometimes proceed without complete damage removal. despite these redundant pathways for resolution, damaged ends appear to be a significant barrier to rejoining, and their resolution may be a rate - limiting step in repair of some dsbs. |
a 65-year - old man with a medical history of stage iii nasopharyngeal cancer, end - stage renal disease treated with dialysis, hyperthyroidism, type 2 diabetes mellitus, hypertension, atrial fibrillation, and secondary hyperparathyroidism presented at the oral medicine clinic of the college of dentistry, university of florida for dental evaluation prior to the start of radiotherapy. intraoral radiographs were taken using a cs2000 intraoral x - ray system (carestream dental llc, atlanta, ga, usa) with # 2 soredex optime (soredex, charlotte, nc, usa) photo - stimulable phosphor (psp) sensors at 70 kvp, 7 ma, and a 0.142-second exposure time, using a standard bitewing technique. a panoramic image was obtained using an orthopantomograph op100 d digital panoramic x - ray unit (instrumentarium dental, tuusula, finland) with exposure factors of 70 kvp with 12 ma for 17.6 seconds. the panoramic and bitewing radiographs revealed multiple tortuous vascular calcifications in the soft tissue of the neck and cheek bilaterally, with a dense " rail track " pattern of linear calcifications within the facial artery. based upon the radiographic presentation and the patient 's known medical history, multiple radiopaque entities were also noted in the soft tissue of the neck, more on the left than on the right, consistent with a diagnosis of carotid atherosclerosis (figs. 1 and 2). positron emission tomography / computed tomography (pet / ct) scans were performed using a philips gemini gxl 16 pet / ct system with 5 mm thickness. the acquired data were reviewed by a medical radiologist, and " pipe - stem " calcifications were observed in the bilateral, lingual, and facial arteries (fig. calcifications may occur in several locations in the cardiovascular system, including the intima and media of vessels. intimal arterial calcification is associated with atherosclerosis, and vascular plaques form within the intima of the involved vessel.3 however, in mnckeberg arteriosclerosis, the calcific deposits are located entirely within the medial layer of the arterial wall and both the internal and external elastic membranes are spared.712 in a recent study, the prevalence of mnckeberg arteriosclerosis in the population was found to be 13.3% for males and 6.9% for females, and it is a well - recognized age - related phenomenon.1112 medial artery calcification can lead to vascular stiffness, resulting in increased vascular resistance, reduced compliance of the artery, and an inability to properly vasodilate in the setting of increased stress.13 medial calcinosis contributes to significant adverse cardiovascular outcomes in patients with chronic kidney disease and diabetes, where higher levels of medial artery calcification are a risk factor for amputation.14 the affected artery may not demonstrate evidence of a pulse. the exact pathogenic mechanism of medial calcinosis is not well understood. however, degenerative processes leading to the apoptosis or necrosis of medial smooth muscle cells and osteogenic processes leading to formation of bone - like structures are two distinct pathologic mechanisms that have been suggested for mnckeberg arteriosclerosis.15 meema.8 have suggested the possibility that two clinically and histologically different types of medial calcifications may exist. the first type is a benign, slowly progressive, essentially asymptomatic form with thin medial calcifications and little or no narrowing of the arterial lumen. in contrast, the second type is defined as a malignant, rapidly progressive form, in which massive and extensive medial calcifications may displace the internal elastica toward the lumen, causing luminal narrowing.78 mnckeberg initially described medial calcinosis as primarily affecting the arteries of the lower limbs, and occasionally affecting the peripheral arteries of the upper extremities. however, the process rarely affects the intraabdominal arteries, with the exception of the renal and splenic arteries.7 some reports in the literature have described mnckeberg arteriosclerosis. in 1977, lachman.7 described the involvement of coronary, peripheral, and visceral arteries with mnckeberg calcification. a case of mnckeberg arteriosclerosis involving the aorta, pelvic, and lower limb arteries was reported by lanzer in 1998.16 the nonvascular involvement of soft tissue (pharynx and larynx) with mnckeberg sclerosis was reported by couri.6 in this report, we described a diabetic patient with end - stage renal disease on dialysis, who had advanced and previously undiagnosed mnckeberg medial calcinosis of the facial and lingual arteries. knowledge of the radiographic appearance of this calcification is clinically useful in developing a differential diagnosis. the proper interpretation of radiographic images presupposes a thorough knowledge of the anatomy, distribution, number, size, and shape of the calcifications. the calcified vessel appears as a parallel pair of thin, radiopaque lines that may have a straight course or a tortuous path, showing a pattern of blood vessels that looks like railroad tracks.511 carotid artery calcifications and phleboliths are calcifications that can be seen in the same location on a panoramic radiograph. carotid artery calcifications radiographically appear as curvilinear irregular parallel radiopacities in the soft tissues of the neck at or below the third and fourth cervical vertebrae, and inferior and lateral to the hyoid bone.1718 phleboliths can be seen on a panoramic radiograph as round or oval in shape with a homogeneously radiopaque center, giving phleboliths a " target " appearance.19 mnckeberg sclerosis is listed among the primary diseases of vessels that can be visualized on panoramic radiographs. to the best of our knowledge, our report describes the first known case of medial calcification in the facial artery on the panoramic radiograph of a diabetic patient with end - stage renal disease. soft tissue calcifications in the maxillofacial area are relatively common and can occur as the result of physiologic or pathologic mineralization, and generally correspond to radiographic findings in routine examinations, such as panoramic radiographs. a comprehensive review and thorough interpretation of all conventional and routine dental radiographs, especially beyond the region of interest, is necessary, and dental practitioners should be aware of the various calcified structures seen on panoramic radiographs, especially those associated with systemic diseases. a proper knowledge of radiographic features, however subtle they may be, assists the clinician in following up and further managing the patient, including appropriate referrals. | mnckeberg sclerosis is a disease of unknown etiology, characterized by dystrophic calcification within the arterial tunica media of the lower extremities leading to reduced arterial compliance. medial calcinosis does not obstruct the lumina of the arteries, and therefore does not lead to symptoms or signs of limb or organ ischemia. mnckeberg sclerosis most commonly occurs in aged and diabetic individuals and in patients on dialysis. mnckeberg arteriosclerosis is frequently observed in the visceral arteries, and it can occur in the head and neck region as well. this report describes a remarkable case of mnckeberg arteriosclerosis in the head and neck region as detected on dental imaging studies. to the best of our knowledge, this is the first case that has been reported in which this condition presented in the facial vasculature. the aim of this report was to define the radiographic characteristics of mnckeberg arteriosclerosis in an effort to assist health care providers in diagnosing and managing this condition. |
cerebral palsy includes a group of nonprogressive movement disorders due to brain lesions or abnormalities in early development. its prevalence of 2 per 1000 newborns overall rises to 77 per 1000 preterms born at below 28 0/7 weeks of gestation [2, 3 ]. a major cause is cystic periventricular leukomalacia (cpvl) comprising necrosis and subsequent cyst formation of the periventricular white matter : 60100% of children with cpvl develop cerebral palsy [46 ]. although the etiology and pathogenesis of cpvl remain unelucidated, several perinatal risk factors appear involved. chorioamnionitis is thought to provoke a fetal inflammatory response syndrome associated with increased fetal cytokines that may lead to neonatal brain injury. several studies indicate that the cytokines can themselves damage white matter without bacteremia being required [815 ]. an important predictor of chorioamnionitis is preterm prelabor rupture of membranes (pprom). chorioamnionitis is quite common and often subclinical : fever and inflammatory marker elevation are rare in the early stages, making diagnosis difficult. against this background the risks of prematurity from immediate delivery have to be balanced against those of ascending intrauterine infection and its probable consequences. moreover subclinical chorioamnionitis is believed to cause pprom. at a gestational age below 34 0/7 weeks, half the gynecologists in australia and new zealand preferred to induce labor, while the other half chose conservative management. a cochrane review from 2010 found no evidence about which strategy is favorable. despite a lack of randomized studies new british and german guidelines advise active management before 34 weeks and active management between 34 and 36 weeks. zurich university hospital has hitherto favored conservative management, delaying delivery until clinically mandatory, on the grounds that the higher mortality and morbidity of newborns at lower gestational age are proven whereas the effect of increasing cpvl risk by prolonging pregnancy remains unknown. the more limited objectives of the present study were to identify the risk factors for pvl in the conservative pprom management setting and determine whether prolonging gestation outweighs the risk of cpvl due to chorioamnionitis. the study population comprised all babies with cpvl born in zurich university hospital 's obstetric department between 1993 and 2008. cranial ultrasound was obtained in infants with gestational age below 32 0/7 weeks or birth weight below 1500 g at days 1, 3, and 7 of life and repeated weekly until hospital discharge. all 6440 infants born between 2005 and 2007 and not affected by pvl served as controls. during the study period women with premature contractions received tocolytic drugs (hexoprenaline only until 2001, nifedipine or hexoprenaline from 2002 to 2008) for 48 hours to allow lung maturation with 24 mg of betamethasone. urinary tract infection or bacterial vaginosis was treated with antibiotics (co - amoxiclav or clindamycin). steroids were repeated every 10 days until 2002. since then, all women with threatened preterm delivery have received a single course of steroids. after pprom co - amoxiclav was used until 2001 when it was changed to erythromycin, chorioamnionitis was monitored using blood tests (including leukocytes and c - reactive protein (crp) 12 hourly for the first 48 hours), maternal temperature, and fetal heart rate. clinical chorioamnionitis (3 of following markers : leukocytes > 20,000/l, crp > 40 mg / dl, maternal temperature > 38c, maternal tachycardia > 100 bpm, and fetal tachycardia > 160 bpm) was treated with antibiotics (co - amoxiclav) and prompt delivery. if the diagnosis was uncertain, delivery was deferred until chorioamnionitis became clinically obvious or delivery could be delayed no longer for other reasons. diagnosis was based on placental histology, positive amniotic fluid cultures sampled at cesarean section, or clinical parameters. babies born below 25 0/7 weeks were excluded in both groups because in most instances neonatal care was restricted to comfort care. the risk factors recorded in both groups were chorioamnionitis, pprom - delivery interval, gestational age at delivery, birth weight, gender, race, and parity. all statistical analyses were performed with stata 10 statistics / data analysis software (stata corporation, college station, tx) using pearson 's test for comparisons of frequencies and wilcoxon 's rank - sum test for group comparisons. subsequent to univariate analysis, multivariate logistic regression was performed to test the impact of factors such as chorioamnionitis, gestational age, birth weight, gender, and pprom - delivery interval on the incidence of cpvl. the results were used to calculate the risks of developing cpvl at different fetal weights and pprom - delivery intervals. given that the analysis was of anonymized data, the study was exempt from local institutional review board approval. follow - up data of study preterms below 32 0/7 weeks were extracted from the prospective national database of the swiss neonatal network & follow - up group. according to the recommendations of our research ethics committee between 1993 and 2008, 32,276 infants were born at zurich university hospital, including 6027 (18.7%) preterms (below 37 0/7 weeks) ; over the same period 32 cases of cpvl were recorded, representing an overall prevalence of 0.99 (tables 1 and 2). cpvl prevalence among preterm infants was 5.3. all 32 infants with pvl were delivered preterm and all but one before 34 0/7 weeks. cpvl risk decreased exponentially with increasing birth weight (figure 1) and increasing gestational age. males were 3 times more affected than females (male : female ratio 24 : 8). all birth weights in newborns with cpvl were less than 2500 g (table 1). the individual pprom - delivery intervals of all pvl cases are shown in figure 2. of the 32 infants, five (16%) died within the first 6 weeks after birth. of the 27 surviving infants, three (11%) were lost to follow - up (parental refusal), while 24 (89%) were neurodevelopmentally assessed at a median (range) age of 3.8 years (2.010.2 years) : one (4%) was normal, nine (38%) were moderately disabled, defined as cerebral palsy grade 20,000/l, crp > 40 mg / dl, maternal temperature > 38c, maternal tachycardia > 100 bpm, and fetal tachycardia > 160 bpm. thus, 18/32 cases (56%) were classified as having been complicated by chorioamnionitis. two cases were assigned to the nonchorioamnionitis group despite the absence of placental histology and a number of clinical parameters. review of the placental histology and clinical parameters reduced these to 71 cases of histological chorioamnionitis and two cases of clinical chorioamnionitis in the absence of placental histology. preliminary logistic regression revealed significant associations between cpvl and chorioamnionitis, male sex, and birth weight. chorioamnionitis had the highest impact on cpvl risk (or 35.9, 95% ci 12.6102.7). however, because a prenatal diagnosis of chorioamnionitis is often not possible, logistic regression was performed, replacing chorioamnionitis by the pprom - delivery interval. this revealed significant impacts on cpvl by sex (p = 0.008), pprom - delivery interval > 48 hours (p 24 hours (p = 0.002) and gestational age (p < 0.001). no significant influence of ethnicity (p = 0.49), fetal weight (p = 0.37), or parity (p = 0.79) was observed. according to logistic regression analyses tabulation of estimated cpvl incidence at varying pprom - delivery intervals and birth weights for boys and girls (table 4), assuming fetal weight gain of 200 g / week, revealed a slight rise in the first 48 hours, followed by a significantly lower risk after the first and second week of prolongation of pregnancy. increasing fetal weight during pprom latency had a far stronger protective effect despite a prolonged pprom - delivery interval being a risk factor for cpvl. this accounts for the high prevalence of preterm deliveries compared to the national average (19% versus 9%). the prevalence of cpvl in our study group (0.99) appears lower than the few reports in the literature. hamrick. reported an incidence of 1.8% at uc san francisco in 1992, falling to 0.2% in 2002 ; the incidence of cpvl in children weighing < 1500 g decreased from 2.9% to 0.5% over the same period. the difference may be partly due to zurich 's conservative management of newborns below 25 0/7 weeks of gestation (restriction to comfort care in the majority of cases). we may also have missed some cases of late cpvl diagnosis in children born after 32 0/7 weeks (there were no instances of late diagnosis of brain lesions in preterms included in long - term follow - up). given our small sample size, we could only extrapolate cpvl incidence for birth weights < 1000 g (figure 1). the vermont oxford network reported approximately 3% cpvl at birth weights 7511500 g ; risk was highest (6%) at birth weights < 751 g. our data confirm the reported exponential decrease in cpvl incidence with increasing birth weight [28, 30 ]. they also confirm a similar exponential decrease with advancing gestational age independently of birth weight. again, our data at below 26 0/7 weeks are not comparable to other centers due to our conservative management of newborns around 25 0/7 weeks. our data support the dependency of cpvl risk on the pprom - delivery interval [8, 31 ], but not on either low parity or prom [8, 10, 31 ]. the finding of a 4 : 1 male / female ratio confirms previous reports [8, 15, 31 ] but remains unelucidated. we also confirmed the several reports of a significant association between cpvl and chorioamnionitis [8, 9, 11, 14, 15 ]. the fact that only one cpvl baby had a positive blood culture within 3 days of birth supports the hypothesis that fetal inflammatory response syndrome is perfectly capable of causing brain damage even in the absence of bacteremia [3234 ]. apart from being a risk factor for cpvl, chorioamnionitis is well - recognized as correlating with neonatal morbidity and mortality [3234 ]. unfortunately, these risk factors can not resolve our strategic dilemma of immediate versus delayed delivery for lowering short - term mortality and long - term sequelae. chorioamnionitis is a major complication of pprom but probably even more often the cause of pprom. the increase in cpvl risk during the first 48 hours after pprom and the substantial decrease thereafter at varying birth weights and pprom - delivery intervals (table 4) can be interpreted in several ways. for example, the initial increase may relate to the use of antenatal steroids to induce lung maturation. steroids could facilitate the spread of bacterial infection ; they could also modulate the fetal inflammatory cytokines thought to cause brain damage. corroborative evidence is that the incidence of cpvl in our group decreased from 1.3 on repeated steroid courses to 0.7 on single - course steroids (although we must admit to having concomitantly switched to erythromycin and introduced nifedipine tocolysis). this study could not add any information about the impact of the different changed interventions to the lower cpvl incidence 20022008. another explanation is that the inverted u - shaped risk for cpvl with respect to the pprom - delivery interval results from superposing two distinct groups of women : one with pprom due to chorioamnionitis and the other with pprom from a noninfectious cause. thus, women still pregnant after one week of pprom are more likely to have a noninfectious cause of fluid leakage. as a consequence, delaying delivery in these cases would lower cpvl risk by allowing birth weight to increase. at the same time, by increasing gestational age and birth weight, this strategy would substantially decrease all other complications of prematurity such as cerebral hemorrhage or lung pathology. an increased risk for neurodevelopmental impairment in the first 48 to 72 hours after pprom was also observed in a large french cohort with 1884 infants born at 2432 weeks of gestational age. given this strong association between cpvl and chorioamnionitis, it is absolutely essential to diagnose intrauterine infection as early as possible. this points once again to the urgent need for a tool that reliably diagnoses chorioamnionitis. in the absence of such a tool because cpvl affects only a small proportion of newborns, with other problems of prematurity playing a much larger role, it seems not unreasonable to pursue a conservative strategy until prospective randomized trials provide a definitive answer or, at the least, until we have a reliable test for the early diagnosis of chorioamnionitis. perinatal morbidity is strongly correlated with prematurity and latency does not appear to worsen outcome in pprom. the fact that chorioamnionitis and the pprom - delivery interval seem to have a high impact on the risk of developing cpvl in our study could have important consequences for the future management of pprom. the strengths of this study are that all infants and mothers were monitored in the same department and that all preterms below 32 0/7 weeks received long - term follow - up, where possible (the actual follow - up rate was 89%). only in midstudy were there any relevant changes in the management of pprom (antibiotics, tocolytic, and steroid courses). we had particularly few birth weights below 1000 g, partly because the swiss society of neonatology recommends restricting neonatal management to comfort care at gestational ages below 24 0/7 weeks. between 24 0/7 and 25 6/7 weeks of gestation the decision to undertake intensive care is individual and influenced by prenatal factors such as birth weight, gender, antenatal steroid use, intrauterine growth restriction, chorioamnionitis, fetal malformation, multiple gestation, and clinical condition immediately after delivery (asphyxia, heart rate, activity, and response to initial resuscitation). intervention continues in the neonatal intensive care unit with the primary goal of survival with an acceptable quality of life. other factors accounting for small sample size in any study of this kind include the fact that the true number of newborns with cpvl can be difficult to evaluate due to early postnatal death, especially when gestational age is very low. cpvl can often only be diagnosed weeks after birth because it takes time for the periventricular cysts to become visible on ultrasound. failure of underreporting when making a late diagnosis of cpvl is another source. even if a prolonged pprom - delivery interval may briefly increase the risk of cpvl, we believe that conservative management makes sense in the absence of clinical chorioamnionitis. higher infant weight at delivery compensates for the impact of pprom latency on neonatal outcome provided that the pregnancy can be prolonged by more than 48 hours. | objective. to identify the risk factors for cystic periventricular leukomalacia (cpvl) and their implications for deciding between immediate delivery and conservative management of preterm prelabor rupture of the membranes (pprom). methods. the following risk factors were compared between cpvl infants and 6440 controls : chorioamnionitis, sex, gestational age (ga), birth weight, pprom, and pprom - delivery interval. factor impact on cpvl risk and clinical decision - making was determined by multivariate logistic regression. results. overall cpvl prevalence (n = 32) was 0.99/1000 births. all cpvl infants but one were born 48 hours (or 9.0 ; 95%-ci 4.120.0), male gender (or 3.2 ; 95%-ci 1.47.3). ga was not a risk factor if birth weight was included. risk decreased with increasing fetal weight despite a prolonged pprom - delivery interval. conclusion. pprom - delivery interval is the single most important prenatally available risk factor for the development of cpvl. immediate delivery favors babies with chorioamnionitis but disfavors those with non infectious pprom. in the absence of clinical chorioamnionitis fetal weight gain may offset the inflammatory risk of cpvl caused by a prolonged pprom - delivery interval. |
the belgian pharmacologist bacq discovered the existence of toxins in nemertines during the mid- 1930s while searching for invertebrate neurotransmitters. an aqueous homogenate of a small intertidal hoplonemertine species (amphiporus lactifloreus), like acetylcholine (ach), contracted isolated frog skeletal muscle and stimulated the cat cervical autonomic ganglion. unlike ach the cholinergic activity of the homogenate was stable in highly alkaline solution and was soluble in organic solvents under basic conditions. king showed that amphiporine acted as an organic base and attempted its further purification by crystallization with standard alkaloidal precipitants, however a crystalline salt was not obtained. while over a 1,000 species have been described, the actual number of species in this inconspicuous phylum is likely to be several times this figure. being soft - bodied and relatively vulnerable to predators, they contain integumentary toxins which serve as chemical defenses against predators. the phylum is roughly divided into two large groups, the enoplans (hoplonemertines) bearing a mineralized proboscis stylet and the anoplans (paleo- and heteronemertines) lacking a stylet. the relatively small size of most nemertines makes them more difficult to collect than many aquatic animals. another problem is species identification, as the external morphologies of some species may be so similar that the preparation of fixed and stained tissue sections for histological examination may be necessary for unequivocal identification. nonetheless, the phylum undoubtedly represents an unusually rich source of alkaloid, peptide and protein toxins, most of them still awaiting investigation. approximately thirty years elapsed after bacq s discovery before another study of nemertine toxins was reported. extracts of most species were found to be toxic to crustaceans, but only those of hoplonemertines displayed nicotinic agonist properties and contained pyridyl alkaloids. since this article focuses on the alkaloids, those interested in the peptide toxins should consult a recent review. this first nemertine alkaloid to be isolated and identified, anabaseine, occurs in relatively large concentrations in the intertidal pacific nemertine paranemertes peregrina. the peregrine (wandering) designation refers to the relatively unique foraging behavior of this moderately large (> 15 cm) species : it glides along the exposed surface of mud flats at low tide searching for annelid worms in full view of potential predators such as seagulls, raccoons and other large predators. several thousand worms were collected and an alkaloid fraction was obtained from the ethanolic extract, much as described by king. because more than a gram of alkaloid was isolated, it was possible to obtain a homogeneous picrate salt, even though in relatively small yield. after conversion back to the free base, nuclear magnetic resonance and mass spectrometric analyses indicated that the alkaloid was anabaseine, a previously synthesized compound that had not been reported as a natural product. this was corroborated by comparison of the chemical and toxicological properties of natural and synthetic samples. anabaseine is chemically similar to the tobacco alkaloid, anabasine, but possesses an imine double bond in the otherwise saturated piperidine ring (fig. 2). imine - enamine tautomerism constrains the -carbon to lie within the same plane as the -carbon and the imine nitrogen. this electronic conjugation strongly favors the two rings of anabaseine being approximately co - planar with respect to each other. this contrasts with nicotine and anabasine, whose respective pyrrolidine and piperidine rings are oriented approximately at right angles in their preferred conformations. anabaseine was first prepared as an intermediate in the synthesis of anabasine by two austrian tobacco chemists. a mixed aldol - like condensation reaction between nicotinic acid ethyl ester and n - benzoyl piperidone yielded the expected diketone, which rearranged in the presence of concentrated hydrochloride acid at high temperature to anabaseine hydrochloride. conversion of the salt to the free base, extraction of the free base with organic solvent and purification by distillation, the method reported by spath and mamoli or column chromatography generally provided anabaseine in relatively low yields. 251) obtained in this manner exists as the ammonium - ketone form and contains one molecule of water. while stable as the dried salt, aqueous solutions of anabaseine hydrochloride should be refrigerated when not in use and replaced after several weeks. the cationic forms of anabaseine are quite soluble in protic solvents such as water, methanol and ethanol, but the more lipophilic free base is best dissolved in non - aqueous solvents such as alcohols, acetone, or ethyl acetate. although anabaseine appears to be chemically simple, it actually occurs in several different forms under physiological conditions. at neutral ph there are three forms in roughly equal concentrations : the unprotonated cyclic imine, the monocationic cyclic iminium and the monocationic ammonium - ketone (fig. this multiplicity complicated our initial attempts to determine which forms interact with achrs based upon the ph dependence of anabaseine potency, so stable analogs of each form were prepared so that the pharmacological properties of the different forms could be inferred. 2,3-bipyridyl, which can be prepared by oxidation of anabaseine or anabasine, is predicted to possess a chemical conformation similar to the cyclic imine, so it was selected as an analog of the unprotonated form, while 2-(3,4,5,6-tetrahydropyrimidinyl)-3-pyridine (pthp) was selected as a stable permanently ionized analog of the cyclic iminium form. two stable analogs of the open - chain ammonium - ketone form were prepared by di- or tri - methylation of the ammonium group. amongst the various stable analogs, only pthp potently stimulated skeletal muscle and brain nicotinic acetylcholine receptor (achr)-expressing cells (kem., in preparation). thus, we conclude that only the cyclic iminium form of anabaseine is active on these mammalian achrs. achrs are a family of receptors that belong to the cys - loop ligand - gated ion channel superfamily that includes types a and c -amminobutyric acid, glycine, and type 3 serotonin (5-hydroxytryptamine ; 5-ht) receptors (reviewed in). in the peripheral nervous system, achrs can be subdivided into muscle - type, that have the stoichiometry 121 (embryonic or torpedo) or 121 (adult), and ganglionic achrs (e.g., 34). in the central nervous system (cns), achrs are of two main subclasses : receptors that bind the competitive antagonist -bungarotoxin (-btx) with high affinity but the agonist nicotine with low affinity (e.g., 7-containing receptors), and achrs that bind nicotine with high affinity but -btx with low affinity (e.g., 42-containing receptors). the physiological and pharmacological effects of anabaseine on a variety of vertebrate achrs were previously reported. like nicotine, anabaseine stimulates all achrs to some degree and thus must be classified as a non - selective nicotinic agonist. however, it preferentially stimulates the same achrs (e.g., skeletal muscle and brain 7 subtypes) that display high affinities for the snake toxin -btx. in contrast, nicotine preferentially and almost fully stimulates 42 (brain) and 34 (predominantly autonomic) receptors. anabaseine is a full agonist at the 7 receptor but only a very weak (low efficacy) agonist at the 42 subtype. the maximal effect of nicotine on the latter receptor is much greater than its maximal effect on the 7 receptor. since nicotine also binds to 42 receptors at much lower (about 100-fold) concentrations than to the 7 receptor, its in vivo effects at smoking concentrations are most likely mediated through 2 subunit - containing receptors. anabaseine stimulates pc12 cell and guinea pig ileum achrs thought to contain 34 (and probably other) autonomic receptors. a more recent study of anabaseine action on rat achrs expressed in xenopus oocytes indicates that anabaseine is a rather weak partial agonist on the 34 receptor subtype. the whole animal (mouse) toxicity of anabaseine is very similar to that of nicotine and is significantly higher than for anabasine. in contrast, anabaseine has very weak partial agonist activity at this receptor and probably exerts its toxicity by causing peripheral neuromuscular block and respiratory arrest. because of its high toxicity and relative lack of receptor selectivity, few in vivo studies have been carried out with anabaseine. the significantly higher potency of nicotine relative to anabaseine in causing prostration is consistent with the notion that 42 receptors primarily mediate this characteristic behavior. marine annelids which are the usual prey for paranemertes are paralyzed, as are crustaceans and insects. nicotinic cholinergic receptors primarily reside on central neurons in arthropods, but are also found in their cardiac pacemaker ganglion. 2,3-bipyridyl, a largely unionized analog of anabaseine, is even more active than anabaseine in paralyzing crustaceans. while it does not cause paralysis, nemertelline (a tetrapyridyl found in amphiporus angulatus), like anabaseine and 2,3-bipyridyl, stimulates an unusual receptor in the stomatogastric muscle of the crayfish which is apparently a chloride channel. at present this is the only known action of this alkaloid, which is the most abundant pyridine in this species of amphiporus. a variety of pyridine compounds including anabaseine and 2,3-bipyridyl stimulate chemoreceptor present in sensory neurons present at the surfaces of crayfish and lobster walking legs that influence feeding behavior. anabaseine and 2,3-bipyridyl were found to be two of the most active compounds in stimulating similar pyridine receptors on spiny lobster sensory nerves. the nemertine alkaloids, by acting upon these chemoreceptors, may act as repellants against certain predators. some of these compounds are also able to inhibit the settlement of barnacle larvae to marine surfaces and thus might be useful antifouling additives to marine paints. this first nemertine alkaloid to be isolated and identified, anabaseine, occurs in relatively large concentrations in the intertidal pacific nemertine paranemertes peregrina. the peregrine (wandering) designation refers to the relatively unique foraging behavior of this moderately large (> 15 cm) species : it glides along the exposed surface of mud flats at low tide searching for annelid worms in full view of potential predators such as seagulls, raccoons and other large predators. several thousand worms were collected and an alkaloid fraction was obtained from the ethanolic extract, much as described by king. because more than a gram of alkaloid was isolated, it was possible to obtain a homogeneous picrate salt, even though in relatively small yield. after conversion back to the free base, nuclear magnetic resonance and mass spectrometric analyses indicated that the alkaloid was anabaseine, a previously synthesized compound that had not been reported as a natural product. this was corroborated by comparison of the chemical and toxicological properties of natural and synthetic samples. anabaseine is chemically similar to the tobacco alkaloid, anabasine, but possesses an imine double bond in the otherwise saturated piperidine ring (fig. 2). imine - enamine tautomerism constrains the -carbon to lie within the same plane as the -carbon and the imine nitrogen. this electronic conjugation strongly favors the two rings of anabaseine being approximately co - planar with respect to each other. this contrasts with nicotine and anabasine, whose respective pyrrolidine and piperidine rings are oriented approximately at right angles in their preferred conformations. anabaseine was first prepared as an intermediate in the synthesis of anabasine by two austrian tobacco chemists. a mixed aldol - like condensation reaction between nicotinic acid ethyl ester and n - benzoyl piperidone yielded the expected diketone, which rearranged in the presence of concentrated hydrochloride acid at high temperature to anabaseine hydrochloride. conversion of the salt to the free base, extraction of the free base with organic solvent and purification by distillation, the method reported by spath and mamoli or column chromatography generally provided anabaseine in relatively low yields. 251) obtained in this manner exists as the ammonium - ketone form and contains one molecule of water. while stable as the dried salt, aqueous solutions of anabaseine hydrochloride should be refrigerated when not in use and replaced after several weeks. the cationic forms of anabaseine are quite soluble in protic solvents such as water, methanol and ethanol, but the more lipophilic free base is best dissolved in non - aqueous solvents such as alcohols, acetone, or ethyl acetate. although anabaseine appears to be chemically simple, it actually occurs in several different forms under physiological conditions. at neutral ph there are three forms in roughly equal concentrations : the unprotonated cyclic imine, the monocationic cyclic iminium and the monocationic ammonium - ketone (fig. this multiplicity complicated our initial attempts to determine which forms interact with achrs based upon the ph dependence of anabaseine potency, so stable analogs of each form were prepared so that the pharmacological properties of the different forms could be inferred. 2,3-bipyridyl, which can be prepared by oxidation of anabaseine or anabasine, is predicted to possess a chemical conformation similar to the cyclic imine, so it was selected as an analog of the unprotonated form, while 2-(3,4,5,6-tetrahydropyrimidinyl)-3-pyridine (pthp) was selected as a stable permanently ionized analog of the cyclic iminium form. two stable analogs of the open - chain ammonium - ketone form were prepared by di- or tri - methylation of the ammonium group. amongst the various stable analogs, only pthp potently stimulated skeletal muscle and brain nicotinic acetylcholine receptor (achr)-expressing cells (kem., in preparation). thus, we conclude that only the cyclic iminium form of anabaseine is active on these mammalian achrs. achrs are a family of receptors that belong to the cys - loop ligand - gated ion channel superfamily that includes types a and c -amminobutyric acid, glycine, and type 3 serotonin (5-hydroxytryptamine ; 5-ht) receptors (reviewed in). in the peripheral nervous system, achrs can be subdivided into muscle - type, that have the stoichiometry 121 (embryonic or torpedo) or 121 (adult), and ganglionic achrs (e.g., 34). in the central nervous system (cns), achrs are of two main subclasses : receptors that bind the competitive antagonist -bungarotoxin (-btx) with high affinity but the agonist nicotine with low affinity (e.g., 7-containing receptors), and achrs that bind nicotine with high affinity but -btx with low affinity (e.g., 42-containing receptors). the physiological and pharmacological effects of anabaseine on a variety of vertebrate achrs were previously reported. like nicotine, anabaseine stimulates all achrs to some degree and thus must be classified as a non - selective nicotinic agonist. however, it preferentially stimulates the same achrs (e.g., skeletal muscle and brain 7 subtypes) that display high affinities for the snake toxin -btx. in contrast, nicotine preferentially and almost fully stimulates 42 (brain) and 34 (predominantly autonomic) receptors. anabaseine is a full agonist at the 7 receptor but only a very weak (low efficacy) agonist at the 42 subtype. the maximal effect of nicotine on the latter receptor is much greater than its maximal effect on the 7 receptor. since nicotine also binds to 42 receptors at much lower (about 100-fold) concentrations than to the 7 receptor, its in vivo effects at smoking concentrations are most likely mediated through 2 subunit - containing receptors. anabaseine stimulates pc12 cell and guinea pig ileum achrs thought to contain 34 (and probably other) autonomic receptors. a more recent study of anabaseine action on rat achrs expressed in xenopus oocytes indicates that anabaseine is a rather weak partial agonist on the 34 receptor subtype. the whole animal (mouse) toxicity of anabaseine is very similar to that of nicotine and is significantly higher than for anabasine. in contrast, anabaseine has very weak partial agonist activity at this receptor and probably exerts its toxicity by causing peripheral neuromuscular block and respiratory arrest. because of its high toxicity and relative lack of receptor selectivity, few in vivo studies have been carried out with anabaseine. the significantly higher potency of nicotine relative to anabaseine in causing prostration is consistent with the notion that 42 receptors primarily mediate this characteristic behavior. marine annelids which are the usual prey for paranemertes are paralyzed, as are crustaceans and insects. nicotinic cholinergic receptors primarily reside on central neurons in arthropods, but are also found in their cardiac pacemaker ganglion. 2,3-bipyridyl, a largely unionized analog of anabaseine, is even more active than anabaseine in paralyzing crustaceans. while it does not cause paralysis, nemertelline (a tetrapyridyl found in amphiporus angulatus), like anabaseine and 2,3-bipyridyl, stimulates an unusual receptor in the stomatogastric muscle of the crayfish which is apparently a chloride channel. at present this is the only known action of this alkaloid, which is the most abundant pyridine in this species of amphiporus. a variety of pyridine compounds including anabaseine and 2,3-bipyridyl stimulate chemoreceptor present in sensory neurons present at the surfaces of crayfish and lobster walking legs that influence feeding behavior. anabaseine and 2,3-bipyridyl were found to be two of the most active compounds in stimulating similar pyridine receptors on spiny lobster sensory nerves. the nemertine alkaloids, by acting upon these chemoreceptors, may act as repellants against certain predators. some of these compounds are also able to inhibit the settlement of barnacle larvae to marine surfaces and thus might be useful antifouling additives to marine paints. while anabaseine is a broad spectrum nicotinic agonist, a large variety (> 200) of substituted anabaseines that have been synthesized over the past two decades displayed selective agonistic effects on the 7 achr. the 3-arylidene - anabaseines are of special potential therapeutic interest because they have been shown to possess neuroprotective as well as cognition enhancing properties. here we shall only consider 3-(2,4-dimethoxybenzylidene)-anabaseine (dmxba), whose pharmaceutical code name gts-21 refers to its origination as the 21 compound generated in a joint project by gainesville (university of florida) and tokushima (taiho pharmaceuticals) scientists. dmxba was the first nicotinic agonist reported to selectively stimulate 7 achrs ; it was also the first 7 agonist to enter clinical tests for possible treatment of cognition problems such as occur in schizophrenia, parkinsonism and alzheimer s disease (ad). dmxba is prepared by reaction of 2,4-dimethoxybenzaldehyde with anabaseine in acidic alcohol at approximately 70c, in a manner similar to the preparation of 3-(4-dimethylaminobenzylidene)- anabaseine. the resulting product can be precipitated and recrystallized using less polar solvents. whereas the two rings of anabaseine have been shown to be electronically conjugated and thus nearly coplanar, all three rings of dmxba are predicted to lie in different planes. unlike anabaseine, 3-arylidene - anabaseines do not readily hydrolyze to open - chain forms at physiological phs like anabaseine. in principle, these compounds can adopt two possible conformations with respect to the vinyl portion of the arylidene ring, namely e- or z. by nmr we have shown that only the e form occurs in aqueous solution when the synthetic dmxba dihydrochloride is dissolved in water. only after intense or maintained light exposure does the e to z conversion become significant (kem. thus, the synthetic compound solid and stock solutions of the compound must be stored in containers that exclude light and stock. while photosensitivity of dmxba was observed in the laboratory, when plasma samples from animal and human tests were prepared in an unlighted fume hood and subsequently determined by hplc with a photodiode array detector, no z - isomeric product was observed. dmxba is a lipophilic compound which readily passes across biological membranes including the gastrointestinal wall and the blood - brain barrier and reaches peak concentrations in the blood and brain within a very short time. it is o - demethylated primarily at the p - position of the benzylidene ring, but demethylation at the o - methoxy group also occurs to a much lesser extent. while the resulting hydroxy metabolites are actually more efficacious at the 7 receptor in vitro, their peak brain concentrations are much less than for dmxba. based on the crystal structure of acetylcholine - binding protein (achbp), monomer, homodimer, and homopentamer models of the 7 achr were derived. since the agonist binding sites are located at the subunit interface (reviewed in), a detailed analysis about the interface, as well as its interaction with the hepes molecule that has been observed in the achbp crystal, was performed. furthermore, a ligand - binding pocket was defined providing useful information for conducting various mutagenesis studies to get clues for drug design. although computer - predicted protein structures are still not as accurate as x - ray structures, the three modeled structures can at least serve as a basis for designing new ligands. theoretical and molecular modeling studies were done to better understand the details of how dmxba and its two primary o - demethylated metabolites (2oh- and 4oh - mba) might bind to 7 achrs. figure 4 depicts a model of the binding pocket of 2oh - mba at the extracellular domain of the 7 achr. there was rather good accordance of the calculated preferred energies with the observed binding affinities. van der waals repulsions made the dominated contribution to the predicted binding energy for the receptor. dmxba and its metabolites seemed larger than the optimal size for fitting into the agonist binding site. thus, one possible approach to improving the effectiveness of benzylidene - anabaseine binding might be to reduce the molecular volume while retaining the active groups. we are optimistic that molecular modeling, in combination with experimental studies on model proteins such as achbp, may provide a useful basis for rational design of nicotinic drug candidates for treating neurodegenerative and possibly other diseases. while dmxba selectively stimulates 7 achrs, at significantly higher concentrations it also is an antagonist of 42 achrs and related type 3 5-ht receptors. at even higher concentrations dmxba also is a weak antagonist at other achrs. in at least some achr subtypes, dmxba and its metabolites for instance, it has been found that dmxba at micromolar concentrations [inhibition constant (ki) = 13 1 m ] displaces the binding of [h]thienylcyclohexylpiperidine ([h]tcp) within the channel of the neuromuscular - type achr. while the 4-hydroxy metabolite also displayed this inhibitory binding, it occurred at a higher concentration (ki = 48 5 m). schild - type analyses of these experiments indicated that these competitions are mediated by a steric mechanism. thus, considering that [h]tcp is a structural and functional analog of the dissociative general anesthetic and potent noncompetitive antagonist phencyclidine (pcp), we suggest that the anabaseine analog binding site overlaps the pcp locus in the desensitized ion channel. in this regard, photoaffinity labeling studies using [h]ethidium diazide, which binds with high affinity to the pcp locus, helped to determine the structural components of this site in the desensitized state (reviewed in). the results indicated that residues leu at position 9 (e.g., the leucine ring) and ser at position 10 from the 1-m2 transmembrane segment as well as other unknown amino acids located in the m1 and m2 transmembrane segments from the subunit are structurally involved in the pcp binding site. in the resting state (in the presence of -btx), anabaseine analogs modulate either [h]tcp, [h]tetracaine, or [c]amobarbital binding to the torpedo achr in an allosteric fashion. these results indicate that the anabaseine analog binding site overlaps neither the pcp, the tetracaine, nor the amobarbital binding domain in the resting ion channel. we suggested that the pcp binding site in the resting state is located more extracellularly than that in the desensitized state, probably close to the mouth of the external vestibule (probably after position 13 and closer to position 20), whereas the barbiturate locus is located practically in the middle of the resting ion channel (between position 9 and 13) (reviewed in). in addition, the tetracaine binding domain bridges both the pcp and the amobarbital loci in the resting ion channel (probably between position 5 and 20). interestingly, anabaseine analogs enhance [h]tcp binding to the torpedo achr when the receptor is in the resting but activatable state (in the absence of -btx). we consider that this enhancement is due to an anabaseine analog - induced achr desensitization process. this hypothesis was supported by the fact that anabaseine analogs also increase the binding of the agonist [h]cytisine to the resting but activatable achr. in this regard, achr desensitization seems to be another mechanism by which anabaseine analogs produce the noncompetitive inhibition of achrs, which in turn, might account for the partial agonistic effect of these compounds in 7 achrs. considering this new experimental evidence it is plausible that the maximal channel activation observed in conventional voltage - clamp electrophysiological recordings might be influenced by the propensity of anabaseine analogs for causing channel block and/or desensitization as well as the probability of the bound agonist to trigger the conformational changes associated with moving from a resting (closed but activatable) to an activated (open) channel state, to finally a desensitized (closed) conformation. what makes dmxba of considerable scientific as well as potential clinical interest is its selective stimulation of 7 achrs. the physiological function of this receptor had been very difficult to investigate in the past due to its propensity to rapidly desensitize when high concentrations of agonist are applied. later, after cloning and expression in cultured cells, it was found to be physiologically active as a ligand - gated ion channel with unusually high permeability for calcium ions. 7 achrs occur at presynaptic as well as on postsynaptic sites at densities that are sometimes as high as that of glutamate receptors. by causing an influx of calcium ions even at normal membrane resting potentials, when most voltage - gated calcium channels are closed, these achrs are able to stimulate a variety of second messenger systems responsive to elevations in intracellular calcium, including nitric oxide synthesis. that dmxba enhances performance in cognitive tasks indicates that 7 achrs play a significant role in learning and memory. that dmxba and other nicotinic agonists primarily exert their cognition - enhancing actions through achr stimulation rather than desensitization follows from the finding that their pro - cognitive effects are inhibited by administering nicotinic antagonists like mecamylamine, -btx and methyllycaconitine. dmxba and nicotine both enhance long - term potentiation (ltp) in the hippocampus. one hypothesis is that stimulation of 7 receptors by released ach or choline (an endogenous weak agonist) enhances the action of synaptically liberated glutamate on nearby n - methyl - d - aspartate (nmda)-type glutamate receptors, as the depolarization resulting from 7 channel opening would eliminate the resting block of nmda receptors by intracellular magnesium ions. the dmxba enhancement displays less acute tolerance (i.e., reduced response with successive applications) than does the nicotine effect. since the auditory gating effects of both compounds in mice are prevented by prior administration of -btx, 7 receptors are the dominant achrs mediating this action. schizophrenics suffer from a relative inability to filter or gate repetitive sensory stimuli, particularly auditory and visual stimuli. this gating defect probably contributes to the negative symptoms of the disease, which are not well treated by neuroleptic drugs which in general are dopamine receptor antagonists. a recent recommendation by an expert panel of psychiatrists recommended development of new therapies for treating the cognitive problems associated with this disease, since they are particularly problematic in preventing schizophrenics from holding jobs and functioning in society. the university of colorado and university of florida labs have recently collaborated on a phase 1 test of dmxba in schizophrenics. the results of this study, both regarding safety and initial assessments of efficacy, encourage further tests. it has also been shown that the deleterious effects of cocaine on auditory gating can be counteracted by dmxba. thus, 7 nicotinic agonists may also be useful in treating psychoses resulting from use of these stimulants. two decades ago a drastic decrease in achrs was first reported in alzheimer s patients and some parkinson s patients. this finding stimulated considerable academic and pharmaceutical interest in the development of nicotinic agonists that could selectively stimulate the remaining brain achrs involved in cognitive and other critical mental functions. at that time it was already apparent that cholinesterase inhibitors and non - selective muscarinic agonists were relatively weak therapeutic agents for counteracting the neurodegeneration and dementia associated with ad. dmxba is a relatively unique drug candidate which readily enters the brain and acts as an 7 achr partial agonist. its effects upon cognitive behavior have been investigated by many laboratories using a variety of mammalian species (reviewed in). nucleus basalis - lesioned rats or aging rats, mice and rabbits were often used to simulate a cholinergic deficit. initially it was observed that the compound enhanced passive avoidance performance in rats and active avoidance in mice and acquisition of conditioned eye - blink reflex in aging rabbits. memory of more intricate learning tasks such as water and radial maze performance by rats and delayed matching by monkeys was also enhanced, suggesting that the compound may be able to enhance cognition in aging humans, particularly ad patients. the latter paper is noteworthy in providing compelling evidence that single doses of relatively short plasma half - life (hours) nicotinic agonists are capable of enhancing cognition for relatively long periods of time (days). generally, cognition enhancement is more readily demonstrated under conditions where cognitive function is deficient, as in chemically lesioned or aging animals. in the case of dmxba performance on several cognitive tasks thus, 7 nicotinic agonists may be useful in treating deficits in cognition, regardless of age. one advantage of targeting 7 receptors for therapeutic enhancement of cognition, instead of 42 receptors, is that modulation of the former receptor does not seem to affect activities associated with nicotine dependence, namely hyperlocomotion, nicotine discrimination and nicotine self - administration, whereas the latter receptor is thought to be a major mediator of the euphoric and anxiolytic effects of nicotine. the actions of subcutaneously administered anabaseine and dmxba upon the brain levels of ach and several biogenic amines have been investigated using cerebral (frontoparietal location) microdialysis methods. anabaseine, like nicotine and other 42 agonists, elevated ach levels (table 2). however, an equimolar (3.6 mol / kg) dose of dmxba did not affect ach levels at this cortical site or within the hippocampus. both anabaseine and dmxba elevated dopamine and norepinephrine levels, but did not significantly affect serotonin levels. however, when mecamylamine, a noncompetitive achr antagonist, was administered thirty minutes before administration of either compound, significant increases in ach and 5-ht levels were observed. explanations for these extraordinary mecamylamine effects are not yet at hand ; one possible interpretation would be that at the mecamylamine dose administered, the more sensitive 42 receptors expressed on inhibitory (gabaergic) neurons innervating the basalis (cholinergic) and raphe (serotonergic) nuclei were preferentially inhibited, leaving the excitatory effects of anabaseine and dmxba on the most resistant achrs (7) to be expressed without opposition from these inhibitory effects. further investigation of the effects of 7 agonists upon brain neurotransmitter levels and their sensitivity to block by achr antagonists is clearly warranted. several laboratories have recently reported that -amyloid (a) binds to 7 receptors at very low concentrations. the peptide inhibits -btx binding to its ach - binding site and has been reported to activate or inhibit this achr. it was hypothesized that 7 receptors are a target of a action on brain neurons. the initially reported selective inhibition of 7 receptors was followed by reports from other laboratories that non-7 achrs are also affected by similarly low concentrations of the peptide. if the 7 achr were a major target for a in generating ad, then one would predict that neurons bearing high concentrations of this receptor would be particularly susceptible and this would lead to a decrease in brain 7 receptors as the disease progresses. however, the loss of 7 receptors in ad brains is much smaller than the loss of 42 achrs. 7 receptor levels in transgenic mice overexpressing a may be reduced although this may occur very early in life. further investigations of the interaction of a with achrs are needed to determine whether 7 and/or other achrs are directly involved in mediating neuronal destruction in ad. besides affecting cognitive functions, dmxba and other nicotinic agonists also display neuroprotective properties such as inhibiton of the excitotoxic effects of a and high concentrations of ethanol. in a stroke model, at very high concentrations (> 10 m), approximately 50 times higher than would occur under clinical conditions (200 nm), rapid addition of this compound to cultured neurons dmxba was also excitotoxic. if the compound was allowed to reach the cells gradually these high concentrations were not toxic. the neuroprotective effects of dmxba were inhibited by reducing extracellular and intracellular calcium levels, and thus seem to be a consequence of calcium influx into the neuron. these are interesting times for the investigation of achrs and their roles in health and disease. besides the brain achr targets discussed above, several peripherally expressed achrs may also be useful therapeutic targets for treating other disease states. examples which readily come to mind are acute inflammation and controlling the growth (angiogenesis) of new blood vessels. in these two examples considerable evidence we hope to have convinced the reader that naturally occurring toxins acting on achrs, besides being useful probes for particular nicotinic receptors, can also serve as molecular models for the design of nicotinic agonists and antagonists of possible therapeutic utility. | nemertines are a phylum of carnivorous marine worms that possess a variety of alkaloidal, peptidic or proteinaceous toxins that serve as chemical defenses against potential predators. the hoplonemertines additionally envenomate their prey with a mixture of proboscis alkaloids delivered with the help of a calcareous stylet that punctures the skin of the victim. anabaseine, the first of these alkaloids to be identified, stimulates a wide variety of animal nicotinic acetylcholine receptors (achrs), especially the neuromuscular [e.g., 121 (embryogenic) or 121 (adult) ] and 7 achrs that are inhibited by the snake peptide -bungarotoxin. a synthetic derivative, 3-(2,4-dimethoxybenzylidene)-anabaseine (dmxba ; also called gts-21), improves memory in experimental animals and humans and is currently in clinical trials to determine whether it can ameliorate cognitive problems associated with schizophrenia. here we summarize present knowledge concerning the chemistry and mechanisms of action of these two substances (anabaseine and dmxba) on achrs, especially those found in the mammalian brain. |
recent work has demonstrated the importance of the abdominal muscles in ensuring sufficient spine stability to prevent buckling and enhance function2, 3. the curl - up, performed as an abdominal exercise, has been shown to produce reasonable levels of activity in the rectus abdominis (ra) muscle while minimizing the resultant spine load and has been incorporated into several low back fitness programs4. the use of unstable surfaces underneath the subject for stability training of the injured low back is becoming more popular4. swiss balls have been incorporated into strength - training regimens and can reportedly be used to more effectively train the musculoskeletal system. performing strength exercises on swiss balls has been advocated based on the belief that a labile surface will provide a greater challenge to the trunk musculature, increase the dynamic balance of the user, and possibly train users to stabilize their spines to prevent and treat injury5. studies have been conducted to document the loads imposed on the spine during various abdominal exercises6, but the effect of unstable surfaces has not been examined. we believe that there is a clinical need to understand the effects of using unstable surfaces to challenge the muscular system during the curl - up exercise. recent evidence regarding the conservative management of low back pain suggests that the restoration of neuromuscular control in the transverse abdominis (tra), together with minimal contraction of other superficial oblique, internal, and external abdominal muscles, is essential for effective treatment during the early stages of rehabilitation7, 8. previous studies have demonstrated that performance of the abdominal hollowing exercise in particular is far more effective than performance of general core - stabilizing techniques in improving the cross - sectional area of the tra9, 10. the abdominal hollowing exercise is designed to emphasize deep local muscle activity while minimizing the activity of the more superficial global muscles. although many studies on the effect of the abdominal hollowing exercise in rehabilitation programs have been performed, none have examined the combined effect of the abdominal hollowing exercise and curl - up exercise on an unstable surface. therefore, the purpose of this study was to investigate the effects of the abdominal hollowing exercise on trunk muscle activity during the curl - up exercise on an unstable surface by measuring electromyography (emg) activity. fourteen healthy subjects (nine males, five females) volunteered to participate in this study (table 1table 1.summary of anthropometric characteristics and neck disability index of the study participantscharacteristiccontrol group(n = 13)mnp group(n = 14)gender (n, male)66age (years), mean (sd)20.6 1.620.6 1.5height (cm), mean (sd)167. 6.9168.0 8.0weight (kg), mean (sd)60.7 10.761.0 12.4neck disability index (ndi) (%), mean (sd)3.3 2.616.9 7.1mnp : mild neck pain ; sd : standard deviation. all subjects were in good health and reported no history of neurological and/or respiratory diseases. none of them had undergone institutional care for any pathological back conditions during the preceding year. significant difference between the two groups (p < 0.05) emg data were collected using a biopac mp150wsw data acquisition system (biopac systems, inc., surface electrode pairs were oriented along the line of action of the underlying muscle fibers on the right side : ra, centered 2 cm lateral and caudal to the umbilicus ; tra, centered 2 cm cephalad to the pubic bone, just lateral to the midline, and parallel to the superior pubic ramus ; external oblique (eo), centered over the tip of the eighth rib diagonally ; and internal oblique (io), centered 2 cm proximal to the midpoint from the anterior superior iliac spine (asis) to the symphysis pubis diagonally11. the maximum voluntary isometric contraction (mvic) of each muscle was measured using the maneuver suggested by kendall.12 for normalization. the subjects were instructed on how to perform the curl - up and abdominal hollowing exercises by the investigator. all subjects were instructed to place their hands on their abdomen for tactile feedback during both exercises. for the abdominal hollowing, the subjects were instructed to draw the lower part of the abdomen up and in toward the spine without moving the trunk or pelvis. a pressure biofeedback unit (chattanooga group, inc., tn, usa) was used to ensure that the subjects were performing the exercises correctly. a pressure cuff unit was placed under their lumbar spine and inflated to 40 mmhg before the exercises were performed13. when the subject performed the hollowing correctly, the pressure either stayed at 40 mmhg or decreased, and the subjects felt their abdomen hollow. the subjects held the trunk and pelvis in that position for 5 s while continuing to breathe normally1. the first task was to perform a traditional curl - up exercise on a flat floor with the hips and knees flexed to 90 and legs supported on a platform. the subject s hands were placed by the sides of their body, and only the head and shoulders were elevated from the flat floor. the next two tasks varied based on the type of unstable surface. for the second task, the subjects assumed the supine position on a flat floor with the hips and knees flexed to 90 and legs supported on a gym ball. ball inflation was checked between subjects to ensure that the diameter remained at 70 cm prior to each test. for the third task, the subjects assumed the supine position on a flat floor with the hips and knees flexed to 90 and legs supported on a gym ball. only the head and shoulders were then elevated from the flat floor with performance of the abdominal hollowing exercise during the curl - up exercise. the subjects held each contraction for 5 s and then returned to the resting position. each subject had 1 min of rest between each trial and 5 min of rest between the tasks. repeated - measures anova with post hoc bonferroni correction was used to determine the trunk muscle activation during the three exercises (p < 0.05). the mean emg amplitudes of the abdominal muscles during the three curl - up exercises are presented in table 2table 2.mean relative muscle activity (% mvic) and standard deviation of the various abdominal muscles during three different curl - up exercisesmusclesstable surfaceunstable surfaceunstable surfacewith abdominalhollowing exerciserectus abdominis41.6 14.649.3 15.026.2 10.5external oblique43.6 18.648.2 12.849.8 19.8internal oblique42.1 18.047.2 16.662.6 21.2transverse abdominis19.2 7.438.4 13.556.5 20.4p < 0.05. the emg activities of the ra and eo trunk muscles during the curl - up exercise were significantly higher on an unstable surface than on a stable surface. the emg activity of the ra was lower when combined with the abdominal hollowing exercise on an unstable surface than that when performing the curl - up exercise on both a stable and unstable surface. the emg activities of the tra and io were higher when combined with the abdominal hollowing exercise on an unstable surface than those during the curl - up exercise on both a stable and unstable surface. the present study showed that the combination of the abdominal hollowing exercise and curl - up exercise on an unstable surface was associated with higher emg activities of the tra and io and lower emg activity of the ra than during the curl - up exercise on both stable and unstable surfaces. the emg activities of the ra and eo were significantly higher on an unstable surface than on a stable surface during the curl - up exercise. several studies have suggested that the swiss ball may be helpful in increasing trunk muscle activity for stabilization of the lumbar spine during rehabilitation exercises5. during the curl - up exercise on the swiss ball, the ra and eo activity may undergo more physical demand, as evidenced by the fact that the swiss ball created more perturbations in our study. contraction of superficial trunk muscles such as the ra and eo without local muscle contraction may lead to compressive loading and shearing forces during the curl - up exercise, which may induce stress on the inner tissues during the entire range of motion and serve as a major source of spinal pain. although the curl - up exercise on an unstable surface induced higher emg activities of the ra and eo, a reverse effect may be seen in individuals with spinal instability. thus, clinicians who apply an unstable surface to increase the trunk muscle activity must consider the provocation that patients endure. in this study, the emg activities of the tra and io were higher during the combination of the abdominal hollowing exercise and curl - up exercise on an unstable surface. conversely, the emg activity of the ra decreased during the combination of the abdominal hollowing exercise and curl - up exercise on an unstable surface. the abdominal hollowing exercise was developed for neuromuscular retraining and kinesthetic awareness of the tra14. our results showed that abdominal hollowing exercise resulted in higher emg activity of local muscles than of global muscles. the local muscle system involves the tra and io, which provide dynamic stability to each spinal segment15. the findings of the present study suggest that the abdominal hollowing exercise as a therapeutic option may be used to preferentially load certain local muscle groups during the curl - up exercise on an unstable surface, and that these effects may be favorably produced in individuals with low back pain. first, our results can not be generalized to other populations because all the subjects who participated in the study were healthy young individuals. second, surface emg was used to monitor muscle activity, leaving the possibility of crosstalk from adjacent muscles. this study provides empirical evidence that the curl - up exercise in combination with the abdominal hollowing exercise is more useful for enhancing abdominal muscle activity than is the curl - up exercise on an unstable surface. this finding offers clinical insight into the additive effect of the abdominal hollowing exercise in selectively stimulating local muscles and suggests that it may be used as an alternative core stabilization technique for the management of patients with low back pain. these findings should be considered when selecting rehabilitation exercises for neuromuscular retraining of the abdominal muscles. | [purpose ] the purpose of this study was to investigate the effects of the abdominal hollowing exercise on trunk muscle activity during the curl - up exercise on an unstable surface by measuring electromyography (emg) activity. [subjects ] fourteen young healthy adults (nine male, five female) voluntarily participated in this study. [methods ] each subject was asked to perform a curl - up exercise on two supporting surfaces (stable and unstable surfaces) combined with the abdominal hollowing exercise on an unstable surface. the muscle activities of the rectus abdominis (ra), external oblique (eo), internal oblique (io), and transverse abdominis (tra) were measured using surface emg during performance of the curl - up exercise. [results ] the emg activity of the ra and eo was significantly higher on an unstable surface than on a stable surface during the curl - up exercise. the emg activities of the tra and io were greater in combination with the abdominal hollowing exercise on an unstable surface than during the curl - up exercise on both a stable and unstable surface. [conclusion ] these findings suggest that the local trunk muscle activity during the curl - up exercise is more strongly affected by combination with the abdominal hollowing exercise than by performance on an unstable supporting surface. |
to report the ultrasound biomicroscopy (ubm) and surgical findings in a subject with a syndrome of ectopia lentis, spontaneous filtering blebs, and craniofacial dysmorphism (traboulsi syndrome). case report, using a 40-mhz ubm wide - field anterior segment scan and anterior segment optical coherence tomography (oct). a 16-year - old orphan girl presented with visual loss to the level of 6/60 (20/200) bilaterally. ubm and anterior segment oct revealed chronic apposition of the iris to the cornea with angle closure, delineation of the bleb tract and rarefaction of the zonules. the girl had abnormal facial features (a beaked nose and long face) with normal chromosomal studies, negative fluorescent in situ hybridization study for velocardiofacial syndrome and an absence of signs suggesting marfan syndrome. under general anesthesia, attempts at deepening the anterior chamber with sodium hyaluronate 3% led to a spontaneous dislocation of the lens into the anterior chamber, facilitating its aspiration. best corrected visual acuity did not improve from the preoperative level beyond 6/60 (20/200) because of central retrocorneal fibrosis. early surgical removal of the lens is necessary in this syndrome to avoid irreversible corneal and trabecular meshwork damage in chronic apposition of the iris to the cornea. ubm can help in the delineation of the bleb tract and document resolution of angle closure after surgery. inadvertent bleb may form as a result of a fistula which allows aqueous to flow from the anterior chamber into the subconjunctival space. most conjunctival blebs follow episodes of scleritis, accidental penetrating injury or ocular surgical procedures (cataract surgery, scleral fixated intraocular lens implantation, scleral tunnel lensectomy, and cyclophotocoagulation). spontaneous filtering blebs are rare and have been observed with few systemic disorders (scleroderma), ocular abnormalities (terrien 's marginal degeneration and axenfeld syndrome), or with systemic conditions, such as familial craniofacial dysmorphism with spontaneous bleb formation [6, 7 ] known in lebanon as traboulsi syndrome. we present the anterior segment imaging and treatment in a case that provides new insights into the pathophysiology of traboulsi syndrome. 1) has been complaining of bilateral visual loss for several years. both uncorrected and corrected (with 7.0 dpt) she had a central superficial corneal opacification as well as central retrocorneal nodular thickening bilaterally. the posterior pole could be visualized in the left eye with difficulty using a 90-dpt lens and revealing a cup - to - disc ratio of 0.1. using ultrasonography, the axial length measured 19.41 mm in the right eye and 20.12 mm in the left eye. the cornea was visualized using anterior optical coherence tomography (oct) (visante oct ; carl zeiss meditec inc., near apposition of the cornea to the iris was clearly demonstrated with angle closure (fig. few zonules were delineated. besides the apposition of the iris to the cornea with angle closure, both rarefaction of the zonules and bleb tracts were more clearly imaged by ultrasound biomicroscopy (ubm) using a 40-mhz ubm probe (eye cubed ellex ; ellex innovative imaging inc.,, usa). under general anesthesia, attempts at deepening the anterior chamber with sodium hyaluronate 3% led to spontaneous dislocation of the lens into the anterior chamber (fig. 2). deepening of the angle was somewhat more evident on ubm than anterior segment oct after lens removal (fig. 3), but the bleb height decreased clinically and by ubm (comparing to the fellow eye) after surgery (fig. 5). best corrected visual acuity did not improve from the preoperative level beyond 6/60 (20/200) partly from the central retrocorneal scar (fig. the patient declined a second eye surgery to remove the subluxated lens after 4 years of follow - up. 1). she had some features suggestive of velocardiofacial syndrome like a prominent broad nose. genetic consultation ruled out both homocystinuria (normal serum amino acid quantitation) and digeorge syndrome (no deletion in the region 22q11.2 by fluorescent in situ hybridization), and confirmed a normal karyotype. traboulsi and colleagues described 6 members of a family with a syndrome of mild facial dysmorphism, subluxation of the crystalline lenses, variable degrees of angle closure with iridocorneal adhesions, patchy iris atrophy, and scleral thinning. three non - operated eyes of two patients had spontaneous filtering blebs that presented as avascular cystic elevations of the superior conjunctiva. systemic work - up of all patients was negative for evidence of diseases known to be associated with dislocated lenses. furthermore, traboulsi and colleagues, subsequently reported 4 members of a lebanese druze family with the syndrome of lens dislocation, spontaneous filtering blebs, scleral thinning, anterior segment abnormalities and a distinctive facial appearance not compatible with marfan syndrome (negative echocardiogram), but with autosomal recessive inheritance. the absence of scleral thinning in the current case may relate to the short eye status hence a thickened sclera. the formation of spontaneous filtering blebs in reported cases of traboulsi syndrome [6, 7 ] may relate to the scleral thinning. scleral thinning occurs in progressive myopia as in the family described by dagi and walton. a posterior channel of communication was apparent on ubm between the bleb and ciliary body similar to the findings in a traumatic bleb as described by khouri.. the current syndrome is different from the syndrome described by dagi and walton who described the clinical features of an atypical presentation of ectopia lentis consisting of primary anterior axial lens subluxation in childhood, associated progressive myopia, and complicating angle - closure glaucoma but without bleb formation. this clinical triad consists of rapidly increasing myopia, normal axial length, and progressive axial subluxation. prophylactic lensectomy provides a safe and sight - saving treatment that arrests and, at times, reverses the progression of angle - closure glaucoma. ectopia lentis with anterior axial subluxation and progressive myopia can be a sight - threatening condition when significant forward mobilization of the lens results in synechial angle - closure glaucoma. recognition of this clinical entity can expedite diagnosis and prevent irreversible loss of vision secondary to glaucoma. because the primary mechanism of angle closure is angle crowding, peripheral iridotomy does little to control the rise in intraocular pressure. according to dagi and walton, lensectomy appears to represent the most definitive treatment in this syndrome ; and lensectomy was ultimately required to halt progressive angle closure resulting from relentless axial subluxation. similarly, peripheral iridotomy may be of little value in our case and our therapy is to restore the angle anatomy by lens removal. we hypothesize that uveal effusion in a nanophthalmic or small eye led to angle apposition with anterior segment ischemia resulting in zonular damage and secondary lens subluxation. alternative explanations include : (1) primary cleft with secondary forward iris movement and corneal iris apposition and (2) a primary collagen synthesis problem with subsequent scleral melt, bleb formation, and secondary forward iris movement. weakening, stretching, or breakage of the zonules frees the lens to migrate and sublux. ectopia lentis occurs in marfan syndrome as well as in a large number of other inheritable disorders (homocystinuria, weill - marchesani syndrome, ectopia pupillae, microspherophakia, mandibulofacial dysostosis, etc.). it can occur as an isolated abnormality, secondary to ocular trauma or syphilis myopia in the presence of ectopia lentis in marfan syndrome may be due to axial elongation of the eye. in addition, two lenticular mechanisms may also contribute to the myopia, and we suspect they are its cause in the present case : an anterior shift of the lens - iris diaphragm moving the focal point of the eye very anteriorly, as well as an antero - posterior thickening of the lens (spherophakia - like). also, peters anomaly is a possibility in the differential diagnosis of central opacity of the cornea with retrocorneal fibrous tissue and with iridocorneal synechiae. peters anomaly is characterized by central corneal opacity (leukoma), thinning of the posterior aspect of the cornea, iridocorneal adhesions, and keratolenticular adhesion or cataract. the presence of lens abnormalities in peters anomaly is more frequently associated with systemic anomalies such as a cleft lip and palate, short stature, broad hands and feet, and variable mental delay. we presented additional insight into the traboulsi syndrome of facial dysmorphism with spontaneous conjunctival blebs using anterior segment oct and ubm technologies. early lensectomy seems indicated to prevent irreversible corneal scarring and angle damage from chronic apposition of the iris to the cornea. | purposeto report the ultrasound biomicroscopy (ubm) and surgical findings in a subject with a syndrome of ectopia lentis, spontaneous filtering blebs, and craniofacial dysmorphism (traboulsi syndrome).methodscase report, using a 40-mhz ubm wide - field anterior segment scan and anterior segment optical coherence tomography (oct).resultsa 16-year - old orphan girl presented with visual loss to the level of 6/60 (20/200) bilaterally. she had a central corneal opacification with retrocorneal fibrosis. the anterior chamber was flat with a very poorly dilating pupil. the lens was central in location. perilimbal conjunctival blebs were bilateral with an intraocular pressure of 8 mm hg. ubm and anterior segment oct revealed chronic apposition of the iris to the cornea with angle closure, delineation of the bleb tract and rarefaction of the zonules. the girl had abnormal facial features (a beaked nose and long face) with normal chromosomal studies, negative fluorescent in situ hybridization study for velocardiofacial syndrome and an absence of signs suggesting marfan syndrome. under general anesthesia, attempts at deepening the anterior chamber with sodium hyaluronate 3% led to a spontaneous dislocation of the lens into the anterior chamber, facilitating its aspiration. deepening of the angle was found after lens removal. retrocorneal fibrosis persisted after surgery, but the bleb height decreased. best corrected visual acuity did not improve from the preoperative level beyond 6/60 (20/200) because of central retrocorneal fibrosis.conclusionsearly surgical removal of the lens is necessary in this syndrome to avoid irreversible corneal and trabecular meshwork damage in chronic apposition of the iris to the cornea. ubm can help in the delineation of the bleb tract and document resolution of angle closure after surgery. |
normally, maintenance of the intestinal epithelium involves a dynamic process in which intestinal stem cells (iscs) located at the crypt base undergo differentiation, proliferation, and migration along the crypt villus axis. at the end of this journey, intestinal epithelial cells (iecs) these processes appear to be dysregulated in the inflamed mucosa of ibd patients, which exhibit hyperplasia, goblet cell depletion, and enhanced apoptosis. more severe damage may manifest as either total epithelial destruction and ulceration, or dysplasia and progressive tumorigenesis. because iecs and intestinal leukocytes both express prrs, downstream signaling has the potential to affect epithelial homeostasis at several levels. a landmark study using a mouse colitis model in which dextran sodium sulfate (dss) acutely damages the intestinal epithelium demonstrated that myd88 mice (deficient in toll - like receptor [tlr ], interleukin 1 receptor [il-1r ], and il-18r signaling) develop significantly enhanced disease compared with wild - type mice (rakoff - nahoum., 2004). in this context, innate signals are clearly protective and promote epithelial integrity via several mechanisms. these include the induction of antiapoptotic and cytoprotective factors and the promotion of tight junction formation (rakoff - nahoum., 2004 ; moreover, microbial sensing by prrs drives the recruitment of stromal and myeloid cells to the isc niche, thereby facilitating regeneration of the epithelium after injury (pull., 2005 ; brown., 2007). in addition to facilitating epithelial renewal, prr activation may limit bacterial translocation by inducing the production of antimicrobial peptides, such as defensins and regiii, by iecs these functions are partially mediated by activation of nf-b and pi3-akt dependent pathways in iecs (for review see cario, 2008). for example, mice whose iecs lack nemo (nemo, a component of the ib kinase required for nf-b activation, develop spontaneous intestinal inflammation (nenci., 2007). recent studies also support a role for nod - like receptor (nlr)mediated inflammasome activation in maintaining epithelial integrity (allen., 2010 ; dupaul - chicoine., 2010 ; zaki., 2010) nlrp3, pycard, and casp-1 mice, which lack the inflammasome signaling components nlrp3, asc, and caspase-1, respectively, all exhibit enhanced dss - induced colitis compared with wild - type controls (allen., 2010 ; dupaul - chicoine., 2010 ; zaki., 2010 these findings are consistent with a protective role for nlr signaling in this model, analogous to that of myd88 signaling. indeed, because microbial stimuli and host danger signals, such as uric acid, extracellular atp, and reactive oxygen species, synergize to activate the inflammasome (franchi., 2009), acute epithelial damage in the microbe - rich gut may create a potent niche for such regulation. however, the question of whether inflammasome activation in epithelial or hematopoietic cells confers this protection has produced mixed findings (allen., 2010 ; dupaul - chicoine., 2010 ; zaki., 2010). a major effector function of inflammasome - mediated activation of caspase-1 is the processing of pro il-1 and pro tellingly, il1r, il18r, and il18 mice exhibit enhanced dss - induced colitis compared with wild - type controls (takagi., 2003 ; lebeis., 2009 ; 2010), and recombinant il-18 can attenuate disease (dupaul - chicoine., 2010 ; zaki., crucially, because myd88 is a signaling adaptor shared by il-1r, il-18r, and tlrs, its indirect activation by nlrs and/or direct activation by tlrs may be a key event in the restoration of epithelial function. nonetheless, the role of il-1 and il-18 in intestinal inflammation remains contentious, given that earlier studies support a pathogenic role of these cytokines (for review see siegmund, 2002). a comparable and perhaps more physiological model where tlr or il-1r family signals limit colitis is that induced by the attaching and effacing bacterium citrobacter rodentium. indeed, increased numbers of mucosally associated adherent - invasive escherichia coli have been reported in cd and uc patients (rolhion and darfeuille - michaud, 2007). infected myd88 mice exhibit delayed neutrophil recruitment and enhanced bacterial outgrowth in the intestinal mucosa with pronounced ulceration, bleeding, and mortality (lebeis., 2007 ; gibson., 2008a). in this context, dysregulated epithelial restitution and repair is compounded by pathogen outgrowth and further immune activation. for example, tlr4 mice develop protective immunity to c. rodentium, but develop less severe intestinal pathology than infected wild - type mice (khan., 2006). this pathogenic role of tlr4 contrasts with that of tlr2 and il-1r, which both limit c. rodentium induced inflammation (gibson. these broadly protective roles for gut tlr and inflammasome signals in acute intestinal inflammation contrast starkly with pathogenic roles played by these signals in the il10or helicobacter hepaticus induced chronic colitis models. myd88 signaling promotes spontaneous colitis in both il10mice and lysm mice ; the latter mice are rendered hyporesponsive to il-10 by a myeloid cell specific deletion of stat3 (kobayashi., 2003 ; rakoff - nahoum., moreover, we have recently shown that colitis induced by h. hepaticus colonization of rag mice requires myd88 expression by hematopoietic cells (asquith., 2010). dependent activation of myeloid cells in the presence of a triggering microbiota can precipitate chronic intestinal inflammation. a key difference in these chronic colitis models is that damage to the epithelium is likely secondary to the microbiota - driven inflammatory response mediated by tissue - resident leukocytes. this notion is supported by the spontaneous colitis that occurs in nemo mice, in which tumor necrosis factor produced by myd88-activated myeloid cells is thought to drive epithelial apoptosis (nenci., 2007). in contrast, acute epithelial disruption by dss or c. rodentium infection may be a primary pathophysiological mechanism, with gross disruption of the epithelium allowing myd88-independent activation by the intestinal microbiota. in this acute disease setting, the epithelial repair functions of myd88 play a protective role, promoting restoration of epithelial barrier function and a return to homoeostasis. it will be of interest to extend studies of inflammasome function to chronic colitis models with distinct pathophysiological mechanisms. intestinal tumorigenesis is intimately linked to intestinal inflammation, and by extension to microbial sensing by prrs. intestinal tumors may originate from dysplastic epithelial or intestinal stem cells, and are infiltrated by several cell types, including lymphoid and myeloid cells (terzic., 2010). evidently, as in models of colitis, in models of cac, prr signals may modulate the neoplastic and infiltrating leukocyte populations distinctly, complicating the dissection of their role in cac pathophysiology. several previous studies have indicated that signals mediated by the tlr / il-1r family promote intestinal tumorigenesis. for example, tlr4 is up - regulated in both human and mouse neoplastic lesions of the inflamed intestine and promotes cac in mice treated with the chemical procarcinogen azoxymethane (aom) followed by dss (fukata., 2007). in addition, the size and frequency of adenocarcinomas in apc mice, which express a mutant version of the tumor suppressor gene apc, are greater than in apcmyd88 mice (rakoff - nahoum and medzhitov, 2007). moreover, il10 mice treated with aom develop myd88-dependent cac (uronis., 2009). in contrast to the tumorigenic role of tlr4 signals in the dss + aom model, the nlrp3 inflammasome and myd88 signals prevent cac in this setting. however, whereas epithelial tlr4 signals promote tumorigenesis (fukata., 2009), hematopoietic nlrp3 activation may be required for protection (allen., 2010). alternatively, myd88-independent tlr4 signaling through the adaptor trif may play a role, and merits further investigation. when treated with dss, myd88 (fukata. 2004), nlrp3, and casp-1 mice exhibit enhanced epithelial apoptosis and diminished or comparable epithelial proliferation relative to dss - treated wild - type mice (zaki., 2010). rather counterintuitively, myd88, nlrp3, and casp-1 mice show enhanced tumorigenesis compared with wild - type mice if also treated with aom (allen., 2010 ; salcedo., (2010) is that myd88 mice treated with aom + dss exhibit down - regulation of multiple dna repair genes and have a higher frequency of mutations in the oncoprotein -catenin, specifically in the domain required to target the protein for degradation. this indicates that failed repair of dna damage in myd88 mice may render epithelial cells prone to deregulation of the cell cycle and tumor progression, despite their increased propensity for cell death. expression of dna repair genes (salcedo., 2010), suggesting that this is a downstream effect of enhanced inflammation in these mice. excessive tlr / il-1r driven signaling can itself drive proinflammatory responses that fuel tumorigenesis, as shown by the myd88-dependent development of cac in il10 mice treated with aom (uronis., moreover, the removal of negative regulators of tlr / il-1r family and inflammasome signaling, such as tir8 or caspase-12, respectively, also leads to increased colitis and cac in the dss + aom model (dupaul - chicoine. in addition to excessive production of inflammatory cytokines in these mice, hyperproliferative epithelial repair responses may also drive tumorigenesis. prr signals may also modulate tumorigenesis independently of their pathogenic and protective roles in inflammation. this is exemplified by apcmice, which model familial adenomatous polyposis patients with mutations in the apc gene who develop intestinal polyps and colorectal cancer early in life. tlr - myd88 signals promote tumorigenesis in these mice, partially through stabilization of oncoprotein c - myc, which induces epithelial hyperproliferation and inhibits apoptosis (lee., 2010). in this case, the presence of a genetic lesion such as apc mutations may divert the homeostatic function of prr signaling toward tumorigenesis. functional analyses of prr pathways in intestinal homeostasis have revealed a complex picture with evidence for both protective and pathogenic roles. optimally, prr - signaling should be maintained at a homeostatic level at which tissue repair and host defense are preserved (fig. a key challenge now is to translate these diverse functions of prr in different animal models to determine which pathophysiological mechanisms underpin distinct forms of ibd and cac. for example, ablation of prr - driven leukocyte activation may limit pathogenesis in some settings, but may prove futile, and even exacerbate symptoms, if barrier dysfunction is the primary disease mechanism. careful dissection of prr - signaling networks in different cellular compartments and in acute and chronic models of intestinal inflammation is an important first step in designing rational therapeutic strategies designed to restore prr - mediated intestinal homeostasis in ibd. prr signals may be required to restore barrier function after epithelial insult and for protective immunity against pathogens ; impairment of these processes caused by insufficient prr signaling may result in pathogen outgrowth and, indirectly, excessive subsequent inflammation (left). excessive prr - driven repair or inflammatory responses (right) may also threaten homeostasis, e.g., through dysregulated epithelial proliferation leading to tumorigenesis and overexuberant pathogenic inflammatory responses to the intestinal microbiota. | inflammatory bowel disease (ibd) is characterized by dysregulated immune responses to the intestinal microbiota, and by chronic intestinal inflammation. several recent studies demonstrate the importance of innate microbial recognition by immune and nonimmune cells in the gut. paradoxically, either diminished or exacerbated innate immune signaling may trigger the breakdown of intestinal homeostasis, leading to ibd and colitis - associated cancer (cac). this dichotomy may reflect divergent functional roles for immune sensing in intestinal epithelial cells and leukocytes, which may vary with distinct disease mechanisms. |
although proinflammatory effects of acute hyperglycemia in inflammatory conditions and septic patients have been extensively investigated in clinical as well as experimental settings, there are still controversies about its relevance and mechanisms [1, 2 ].. found no beneficial effect of treating acute hyperglycemia in the clinical setting of sepsis. however, in this context it was difficult to dissect between effects directly attributable to insulin or thereby induced hypoglycemia. similarly, hyperglycemia - related proinflammatory effects should be separated into those directly evoked by glucose and those induced by secondary hyperglycemia (stress, inflammation). in case of acute hyperglycemia caused by critical illness, altered secretion of counterregulatory hormones and excessive release of proinflammatory cytokines is observed as well as suppression of the innate immune system [4, 5 ]. these consequences of impaired leukocyte function are in part comparable to the pathophysiology observed in patients with diabetes mellitus. despite tremendous research on proinflammatory conditions related to hyperglycemia direct effects of glucose on leukocyte recruitment during inflammation leukocyte recruitment into inflamed tissue follows a well - defined cascade of events beginning with the capture of free flowing leukocytes to the vessel wall followed by leukocyte rolling along and adhesion to the inflamed endothelial layer [9, 10 ]. during rolling, leukocytes get into intimate contact with the endothelial surface, which allows endothelial bound chemokines (i.e., cxcl1) to interact with their specific chemokine receptors (i.e., cxcr2 as the main chemokine receptor on neutrophil leukocytes) on the leukocyte surface. this triggers the activation of 2-integrins (i.e., lfa1 and mac1) which leads to firm leukocyte arrest on the endothelium. in addition, integrin - dependent signaling events induce cytoskeletal rearrangements and cell polarization, modifications necessary in helping to prepare the attached leukocyte to spread and crawl in search for its wayout of the vasculature into tissue [1013 ]. after crawling along the vessel wall little is known about how glucose might interfere with the cascade of leukocyte recruitment in vivo. insulin secretion promotes neutrophil chemotaxis under physiological conditions ; acute hyperglycemia leads to insulin resistance in and reduced respiratory burst of neutrophil leukocytes. insulin resistance is seen in different tissues including muscle cells and the substrate producing organ liver. hepatic insulin resistance leads to an exaggerated release and an increased blood concentration of not only glucose, but also of amino acids and of free fatty acids. paradoxically, for example, fatty acids are metabolized to a smaller extent in patients with critical illness, leading to a progressively increasing vicious circle of not extractable circulating metabolic substrates. although experimental design (route and doses of glucose, observed tissue) varied between existing studies, they revealed increased rolling, adhesion and transmigration of leukocytes after glucose application, an effect which was at least in part reversible after insulin injection. since changes of osmolarity were repeatedly discussed to be causative of alteration of leukocyte recruitment, azcutia. carried out experiments not only with the biological active d - glucose (that is referred to as glucose in all existing experimental settings as well as in our investigations), but also with its synthetically existing enantiomer l - glucose. osmotic changes were not causative in this setting, as l - glucose failed to alter leukocyte recruitment. since there are no intravital microscopic studies investigating the immediate effects of glucose on leukocyte recruitment in vivo, we aimed to observe leukocyte recruitment in response to intravenous glucose application in different inflammatory mouse models by intravital microscopy. based on our experimental results we found that in contrast to tnf stimulation leukocyte adhesion and transmigration can be additionally stimulated by glucose, which seems to be dependent on cxcl1-triggered interaction of icam1 and lfa1 during trauma - induced inflammation. icam1 mice were generated as described earlier and backcrossed for at least seven generations into the c57bl/6 background. lfa1 and c57bl/6 wildtype (wt) mice were provided by charles river (sulzfeld, germany). all mice were maintained as breeding colonies at the central animal facility of the university of heidelberg, germany. the animal experiments were approved by the animal care and use committee of the regierungsprsidium karlsruhe, germany. in certain experiments, recombinant murine tnf (r&d systems, minneapolis, usa) was injected intrascrotally at a dose of 500 ng per mouse 3 hours before intravital microscopy. in some experiments, recombinant murine chemokine cxcl1 (keratinocyte - derived chemokine kc ; peprotech, london, uk) was injected systemically at a dose of 600 ng per mouse. blocking antibodies against murine mac1 (tib128, clone m1/70, rat igg2b) and murine lfa1 (tib217, clone m17/4, rat igg2a) were obtained from american type culture collection (atcc, manasses, usa) and systemically administered with a dose of 100 g per mouse. bordetella pertussis ptx was purchased from sigma - aldrich (taufkirchen, germany) and administered in certain experiments at a dose of 4 g per mouse 3 h prior to preparation in the trauma model. mice were prepared for intravital microscopy as reported recently. briefly, after intraperitoneal (i.p.) injection of ketamine (125 mg / kg body weight, ketalar ; parke - davis, morris plains, usa) and xylazine (12.5 mg / kg body weight ; phoenix scientific, inc. joseph, usa) mice were placed on a heating pad to maintain body temperature. intravital microscopy was conducted on an upright microscope (leica ; wetzlar, germany) with a saline immersion objective (sw40/0.75 numerical aperture, zeiss, jena, germany). mice were intubated, and the left carotid artery was cannulated for blood sampling and the right jugular vein for glucose and systemic mab administration. d - glucose (200 mg / ml) was administered in doses of 0,25 g, 0,5 g, and 1 g / kg body weight. the injection of the equivalent volume of normal saline (nacl 0,9%) served as control agent. additional osmotic controls were performed with the biologically inactive l - glucose, purchased from sigma - aldrich (taufkirchen, germany). refers to d - glucose that was used in all other experiments that were carried out. blood glucose measurements were carried out throughout the experiment using accuchek (roche diagnostics, mannheim, germany) and additionally verified by comparing the results in sodium fluoride samples in the central laboratory of the university hospital heidelberg (analysezentrum, heidelberg, germany). after longitudinal incision and spreading of the muscle over a cover glass, the epididymis and testis were mobilized and pinned aside leading to full microscopic access to the cremaster muscle microcirculation. cremaster muscle venules were recorded via ccd camera (cf8/1, kappa, gleichen, germany) on a panasonic s - vhs recorder. postcapillary venules under observation were recorded before and during glucose administration and ranged from 20 to 40 m in diameter. systemic blood samples (10 l) were taken and assessed for white blood cell count before and after experiment. systemic blood samples (10 l) were taken after each mab injection and stained with trks solution 1 : 10 (merck, darmstadt, germany). in some experiments, recombinant murine chemokine kc (keratinocyte - derived chemokine ; peprotech, london, uk) was injected systemically at a dose of 600 ng per mouse. systemic leukocyte concentration was analyzed using a hematocytometer and expressed as number of leukocytes per l of whole blood. microvascular parameters (venular diameter, venular vessel segment length, and leukocyte rolling velocity) were measured using an image - processing system. venular centerline red blood cell velocity was measured during the experiment via a dual photodiode and a digital online cross - correlation program (circusoft instrumentation, hockessin, usa). an empirical factor of 0.625 was used to convert centerline velocities to mean blood flow velocities. wall shear rates (w) were estimated as 4.9 (8vb / d), where vb is mean blood flow velocity and d the diameter of the vessel [20, 21 ]. to differentially count intravascular and extravascular leukocytes, cremaster muscle - whole mounts briefly, while the cremaster muscle was still mounted on the stage for intravital microscopy, the tissue was fixed with 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4). the cremaster muscle was removed and mounted flat on a superfrost glass slide (menzel, braunschweig, germany), air dried for 510 min, and fixed in 4% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4) for 24 h at 4c. after fixation, the tissue was washed three times in 0.1 m phosphate buffer with 5% ethanol, stained with giemsa (sigma) at room temperature for 5 min, and differentiated in 0.01% acetic acid for contrast. the differentiated slides were washed in water, 75% ethanol, 95% ethanol, 100% ethanol, and fresh xylene, followed by mounting in mounting media (agar scientific). the giemsa - stained cremaster muscles were observed using a leica dmrb upright microscope and a 25/0,75 na oil immersion objective (both leica, germany). intravascular and interstitial leukocytes were counted and differentiated into neutrophils, eosinophils, and mononuclear cells. to investigate the endothelial expression of icam1 on unstimulated cremaster muscle venules or during trauma - induced inflammation, we performed immunohistochemical analysis of whole - mount cremaster muscles as described [24, 25 ]. briefly, after insertion of a catheter into the carotid artery, primary antibodies against icam1 (yn-1, monoclonal rat anti - mouse ; 30 g / mouse, atcc, wesel, germany) were systemically injected and incubated for 10 minutes. because of the intravascular antibody application, which was performed before surgical preparation of the cremaster muscle, binding of antibodies is mostly restricted to surface expressed antigens within the vasculature. for unstimulated venules, excess antibody was washed out from the circulation with normal saline solution before the cremaster muscle was obtained. for trauma - stimulated venules, washing out of excess antibody occurred after exteriorization of the cremaster muscle and 20 minutes of superfusion with superfusion buffer. cremaster muscle whole mounts were surgically prepared as reported previously and transferred onto adhesive slides (superfrost, menzel, braunschweig, germany). after air - drying over 510 min, cremaster muscles were fixed in acetone (18c) overnight. permeabilization of the tissue was conducted in 0.05 mol / l tris buffer containing 0.003% saponine. after washing, cremaster muscle tissue was incubated with biotin - conjugated secondary goat anti - rat antibody (ebioscience, usa) over 45 min in a humid chamber. blocking of endogenous peroxidase activity was performed with h2o2 in methanol over 1 h, followed by incubation of the tissue with peroxidase - conjugated streptavidin. staining of tissue samples was performed using a commercially available kit (dab, vector lab, burlingame, usa). for counter - staining mayer 's hemalaun thereafter, the samples were incubated with an increasing concentration of alcohol followed by xylene (carl roth, karlsruhe, germany). sealing of tissue samples was performed with mounting medium (dpx mounting medium, agar scientific, stansted essex, uk). analysis of stained slides was conducted on a leica dmrb upright microscope and a 25/0.75 na oil immersion objective (both leica, wetzlar, germany). photographs of the samples were taken using a color ccd camera (kappa, germany). after isolation, they were loaded on top of a discontinuous percoll gradient (52%/64%/72%) and centrifuged at 1000 g for 30 minutes. pmns were harvested from the 64%/72% interface, washed in pbs, and cultivated for 24 hours in rpmi 1640 medium supplemented with 20% wehi-3b - conditioned medium. pmn viability was greater than 95% as assessed by the trypan blue exclusion test, and purity was greater than 98% as analyzed by microscopy using hemacolor staining (merck, darmstadt, germany). the expression of mac1 and lfa1 on bone marrow - derived neutrophils was assessed by flow cytometry. after red blood cell lysis, 10 leukocytes / ml were stimulated for 15 min with 10 mg glucose at 37c. next, cells were incubated in the dark with fitc - conjugated anti - mac1 mab m1/70 (1 g/10 cells, rat igg2b ; ebioscience, san diego, usa), fitc - conjugated anti - lfa1 mab m17/4 (1 g/10 cells, rat igg2a ; ebioscience, san diego, usa), or respective fitc - conjugated isotype control antibodies (1 g/10 cells, rat igg2b or rat igg2a ; ebioscience, san diego, usa) to detect anti - mac1 and anti - lfa1 signals, respectively. mac1 and lfa1 expression was assessed on 10.000 cells per mouse within the neutrophil cluster defined by forward - side scatter analysis using lsrii with diva software package (becton dickinson, san jose, usa). expression of mac1 and lfa1 upon stimulation with different glucose concentrations was compared to unstimulated cells and their respective isotype controls. flow chamber experiments were conducted as described [23, 27 ]. in brief, rectangular microglass capillaries (vitrocom, mountain lakes, usa) were coated with rmp - selectin (2 g / ml), rmcxcl1 (5 g / ml), and icam1 (1 g / ml) and connected via pe tubing to a 2 ml syringe containing freshly isolated bone marrow neutrophils from lysegfp mice. in lysegfp mice the enhanced gfp (egfp) is knocked into the murine lysozyme m (lys) locus leading to the expression of egfp in myelomonocytic cells. the cell suspension (0.25 10 gfp positive cells) was perfused through the flow chamber and adhesion of gfp - positive cells observed by fluorescence microscopy (bx51 wi with a saline immersion objective 20/0.95 na, olympus hamburg) for 10 minutes under constant flow conditions using a high precision perfusion pump (harvard instruments, march - hugstetten, germany ; wall shear stress 0.1 pa). images were recorded via a ccd camera system (cf8hs ; kappa) on a panasonic s - vhs recorder. in some experiments, cell suspensions were incubated with d - glucose (10 mg/10 cells) 15 minutes before the perfusion through the flow chamber. sigma stat 3.5 (systat software, erkrath, germany) was used for statistical analysis. leukocyte counts, vessel diameters, leukocyte adhesion, leukocyte - rolling flux fractions, wall shear rates, and in vitro leukocyte adhesion between groups and treatments were compared with one - way anova followed by a multiple pairwise comparison test (dunn 's test) or by wilcoxon rank - sum test, as appropriate. the first objective of our experiments was triggering a significant increase of the murine blood glucose concentration by intravenous glucose application. as described earlier, stress (induced by anesthesia and surgical preparation) resulted in high basal blood glucose levels in all investigated mice. as expected one minute after glucose injection blood glucose concentration increased significantly in a dose - dependent manner (see supplemental table 1 available online at doi:10.1155/2012/739176). in contrast, recent studies [7, 8 ] did not detect elevation of systemic blood glucose levels presumably due to different experimental setup (intraperitoneal injection of glucose and longer - time interval until microscopic observation). because we observed a rapid and significant increase of systemic blood glucose concentration after a dose of 0,5 g / kg glucose, which is in the range clinically used to treat severe hypoglycemia, we continued with that dose in all further experiments. surgical preparation of the cremaster muscle induces leukocyte adhesion mainly via the chemokine cxcl1-cxcr2 pathway and 2 integrins lfa1 and mac1 in the short - term model of trauma - induced inflammation [23, 27 ]. using this model we analyzed the number of adherent leukocytes in wt mice in postcapillary venules of the cremaster muscle before and after intravenous injection of different doses of glucose. the results obtained from these experiments were compared to injection of normal saline and l - glucose. (vascular diameter, blood flow velocity, wall shear rate, and white blood cell count) before and after glucose or saline injection (table 1). thereby, we ruled out that alterations of leukocyte recruitment might be caused by hemodynamic changes in response to fluid injection. administration of a low dose of 0,25 g / kg glucose did not lead to any significant changes of leukocyte adhesion. however, increasing doses of glucose (0,5 g / kg and 1 g / kg, resp.) lead to a significant, dose - dependent increase of leukocyte adhesion in wt mice within 15 minutes after glucose administration (figure 1(a)) compared to controls injected with normal saline or l - glucose. these results suggest that glucose triggers leukocyte adhesion which is not attributable to hemodynamic changes. to investigate the impact of glucose on neutrophil transmigration, we also performed giemsa staining of whole - mount cremaster muscles in this model and classified leukocytes into neutrophils, eosinophils, and mononuclear cells as described. accordingly, the number of perivascular neutrophils was higher after administration of glucose compared to saline controls suggesting that glucose - triggered leukocyte adhesion translates into increased transmigration of leukocytes, too (figures 1(b)1(d)). taken together, leukocyte recruitment during trauma induced inflammation can be rapidly enhanced by intravenous glucose injection. despite different experimental design, this observation is consistent with intravital microscopic studies on mesenterial vessels of schffler., booth., and azcutia. importantly, neither changes of hemodynamic conditions nor osmolarity [6, 8 ] are causative for the observed glucose induced effects. moreover, there is evidence that glucose triggered leukocyte recruitment is reversible through insulin treatment. as a potent proinflammatory agent, we administered tnf in a dose of 500 ng 3 h prior to exteriorization of the cremaster muscle and observed leukocyte adhesion in cremaster muscle venules of wt mice, either treated with glucose or normal saline. in this model, transition from leukocyte rolling to firm adhesion after tnf pretreatment is mediated in an overlapping fashion involving cxcr2 and e selectin. microvascular parameters for the groups are presented in table 2 and show similar vessel diameters, centerline velocities, wall shear rates, and wbc counts. administration of 0,5 g / kg glucose did not significantly increase leukocyte adhesion in the tnf model when compared to injection of normal saline (figure 2(a)). to investigate whether glucose alters neutrophil transmigration, we performed giemsa staining of whole mounts of tnf-treated cremaster muscles and classified leukocytes into neutrophils, eosinophils, and mononuclear cells as described. in mice injected with glucose, the number of extravasated neutrophils was hardly increased compared to normal saline - treated mice, indicating that proinflammatory stimulation with tnf is not further enhanced by glucose (figures 2(b)2(d)). thus, glucose exerts proinflammatory effects with regard to leukocyte recruitment in the short - term trauma model, but not in the long - term tnf model. we hypothesize that in contrast to the short term model of trauma - induced inflammation tnf induces a very strong nfb - triggered proinflammatory stimulation over 3h that is not further augmentable by glucose administration. this theory is supported by the fact that the level of leukocyte adhesion and transmigration is about the same after tnf stimulation compared to trauma - induced inflammation plus glucose. using icam1-ko and lfa1-ko mice we addressed the question whether icam1 and lfa1 play a role for the observed glucose - induced effects during leukocyte recruitment during trauma - induced inflammation (hemodynamic and microvascular parameters are presented in table 3). in line with previous reports [31, 32 ] we found comparable baseline leukocyte adhesion in cremaster muscle venules of wt, lfa1-ko, and icam1-ko mice. in contrast to wt mice, injection of 0.5 g / kg glucose in icam1 and in lfa1 mice did not lead to significant changes of adherent leukocytes compared to baseline values (figure 1(a)). accordingly, transmigration of neutrophils as observed in giemsa - stained cremaster muscle whole mounts of icam1-ko and in lfa1-ko mice was similar after administration of glucose compared to normal saline (figures 1(b), 1(f), and 1(g)). these findings suggest that icam1 and lfa1 are crucially involved in mediating glucose - induced leukocyte recruitment, which is in line with previous observations [8, 33 ]. in order to gain further insight into underlying mechanisms, we next investigated the role of the cxcl1-cxcr2 pathway for glucose - triggered leukocyte recruitment in our experimental setting. we and others demonstrated that injection of cxcr2-binding chemokine cxcl1 induces a significant increase of leukocyte adhesion which is mostly attributed to lfa1 activation and its subsequent icam1 binding [23, 31 ]. we first asked whether systemically injection of 600 ng cxcl1 in addition to 0.5 g / kg glucose would be able to further enhance leukocyte adhesion and transmigration during trauma - induced inflammation (hemodynamic and microvascular parameters are presented in table 3). however, the number of adherent and transmigrated cells was not further increased in glucose - injected mice after costimulation with cxcl1 compared to glucose injection only (figures 1(a), 1(b), and 1(h)), indicating that cxcl1 might be involved in glucose - mediated leukocyte recruitment. next, we used pertussis toxin (ptx) to unspecifically block the chemokine receptor cxcr2 by disruption of g - protein - coupled receptor signaling. notably, ptx pretreatment completely abolished leukocyte adhesion or transmigration in response to glucose in our experimental model (figures 1(a), 1(b), and 1(e)) suggesting that the cxcl1-cxcr2 pathway is involved in mediating glucose - induced leukocyte recruitment. to further explore underlying mechanisms for the observed glucose - induced effects we analyzed the expression of the 2 integrins lfa1 and mac1 on neutrophils in response to glucose using flow cytometry. as depicted in figure 3, expression of lfa1 and mac1 on bone marrow - derived mouse neutrophils was not altered after incubation with 10 mg / ml glucose for 15 minutes when compared to control neutrophils. this finding indicates that upregulation of 2 integrins does not account for the observed glucose - induced augmentation of leukocyte recruitment. therefore, the changes of leukocyte recruitment are unlikely to be attributable to leukocyte - driven mechanism, as signaling of glucose - incubated leukocytes is identical to leukocytes incubated with saline. glucose - evoked changes of integrin avidity or activity are difficult to exclude in our flow cytometric approach, as respective antibodies are not available in mice. next, we aimed to further dissect leukocyte- from endothelium - driven mechanisms mediating glucose - induced adhesion of leukocytes and performed flow chamber experiments. therefore, microflow chambers were coated with p selectin, icam1, and kc and constantly perfused with isolated glucose - stimulated or saline control bone marrow neutrophils of lysegfp mice. under control conditions we observed a significant number of adherent gfp - positive cells (mainly neutrophils) in fully coated flow chamber compared to uncoated flow chambers (figure 4). noteworthy, glucose stimulation (10 mg / ml for 15 minutes) did not affect leukocyte adhesion in this dynamic flow chamber approach, indicating that leukocyte - born mechanisms are unlikely to be connected to glucose - induced changes of leukocyte recruitment. we next ask about the impact of glucose on the regulation of endothelial adhesion molecules. since interaction of lfa1 with icam1 is involved in mediation of glucose - induced effects of leukocyte recruitment, we assessed endothelial icam1 expression in postcapillary venules of trauma - stimulated cremaster muscles using immunohistochemistry. endothelial icam1 expression in cremaster muscle venules of glucose treated mice was significantly increased compared to control mice (figure 5, also illustrated by micrographs). these findings confirm previous observations of azcutia. and others [8, 33 ], strongly suggesting that glucose - dependent enhancement of leukocyte recruitment is at least in part entailed to icam1 upregulation. however, future studies are necessary to investigate whether glucose regulates other endothelial leukocyte adhesion molecules acting in concert with icam1. in addition, humoral factors cytokines and chemokines could be involved in the glucose - induced proinflammatory responses, as described by azcutia. and ling. based on our presented results we hypothesize that glucose induces cxcl1-cxcr2-dependent activation of lfa1 which in turn binds to upregulated icam1 in order to mediate leukocyte recruitment. however, up to date it remains unclear whether and how glucose - dependent signaling, that is, glucose receptors or channels are involved in upstream signalling pathways. to our knowledge, this is the first study that investigated the immediate effects of intravenously administered glucose on the leukocyte recruitment cascade in vivo, so that our results may stimulate future studies further investigating underlying mechanisms of the observed glucose - dependent proinflammatory signaling. leukocyte adhesion and transmigration are strongly augmented by intravenous glucose injection during trauma - induced inflammation but not during 3h - tnf stimulation. the glucose - induced leukocyte recruitment is mediated by cxcl1-triggered interaction of lfa1 and icam1. thus, our results also indicate that the proinflammatory properties of glucose are stimulus - dependent and might open new perspectives for the development of strategies targeting hyperglycemia - related inflammatory conditions. | it is well acknowledged that proinflammatory stimulation during acute hyperglycemia is able to aggravate inflammatory diseases. however, the mechanisms of proinflammatory effects of glucose are controversially discussed. we investigated leukocyte recruitment after intravenous injection of glucose in different inflammatory models using intravital microscopy. flow chamber experiments, expression analysis, functional depletion, and knockout of key adhesion molecules gave mechanistic insight in involved pathways. we demonstrated that a single injection of glucose rapidly increased blood glucose levels in a dose - dependent manner. notably, during tumor necrosis factor (tnf) -induced inflammation leukocyte recruitment was not further enhanced by glucose administration, whereas glucose injection profoundly augmented leukocyte adhesion and transmigration into inflamed tissue in the trauma model, indicating that proinflammatory properties of glucose are stimulus dependent. experiments with functional or genetic inhibition of the chemokine receptor cxcr2, intercellular adhesion molecule 1 (icam1), and lymphocyte function antigen 1 (lfa1) suggest that keratino - derived - chemokine cxcl1-triggered interactions of icam1 and lfa1 are crucially involved in the trauma model of inflammation. the lacking effect of glucose on 2 integrin expression and on leukocyte adhesion in dynamic flow chamber experiments argues against leukocyte - driven underlying mechanisms and favours an endothelial pathway since endothelial icam1 expression was significantly upregulated in response to glucose. |
for several decades there has been a demographic shift towards an ageing population taking place across the european continent, albeit at varying speeds. the number of children being born has universally decreased across europe and the birth rate is falling below replacement levels in some member states such as germany and italy. in the european commission 2010 demography report a slight improvement to fertility rates was reported but at 1.6 it is still far below the replacement rate of 2.1 and will, at best, slow the rate of population decline in the eu. in italy, one of europe s oldest nations with one of the lowest fertility rates, the population is projected to decline from the current 57 million to 41 million by 2050 (european commission 2011). this coupled with the positive fact that people are now living longer and often healthier lives has resulted in a uniquely large proportion of the population reaching old age. increased female involvement in the formal labour force coupled with changing family structures have put a strain on the ability of member states to provide care for elderly who need assistance. traditionally, one female member of the family would provide care. the continued idea that a family member and in particular a female one will provide care is in direct conflict with the lisbon and 2020 strategy targets of increased labour force participation. additionally, since families are having fewer children and there is greater need for everyone to engage in the formal workforce the pool of potential carers has diminished or put another way this means that children are more likely to be needed as carers of their elderly parents. the major challenges now facing long - term care provision across europe are related to the supply of carers, both formal and informal, and the high cost associated with long - term care service provision. in 2007 the eu average for the number of people aged 65 or over in receipt of institutional care was just over 3 %. altogether, about 8 % of people aged 65 and over in the eu are receiving some formal long - term care at home - but this ranges from 25 % in denmark and over 20 % in the netherlands to 7 % in ireland, only 1 % in lithuania and even less in poland (huber. carer migration is defined as the movement of care workers or immigrants involved in the provision of care assistance to older people. migrants are already filling the labour gaps and providing essential care services in many european member states. in the united kingdom and in italy the proportion of migrants among the paid care work force is as high as 1 in 4 and 1 in 2 respectively (oecd 2011). within the formal sector migrant workers make up a large proportion of available and employed carers. similarly in an attempt to overcome the shortages and strain of long - term care many families are employing migrant workers within private homes to provide care. this is unregulated but often preferred as it is less expensive than formal care for the recipient and their family and it means that the care recipient can remain at home which has consistently been found to be the preferred place of care for the recipient. there are three drivers of population change : fertility rates, life expectancy and migration (european commission 2010). within the eu27 there are about 5 million births a year, which outnumbers deaths by only several hundred thousand according to the european commission 2010 demography report. net migration for the eu is over a million a year which makes migration the single greatest driver of population growth in the eu (european commission 2010 : 2). demographic changes affect international migration in several ways : rapid population growth combined with economic difficulties at home is likely to push people to move in search of work, and a declining and ageing population pressures countries to accept migrants. population ageing is a global issue effecting america, japan, korea and australia but it is most pronounced within the european context. figure 1 illustrates the growth predictions of the global working age population from 2010 to 2050. it clearly shows a north south divide with younger, growing populations in the southern hemisphere, much smaller growth predicted in the northern hemisphere and a reduction of 23 % in europe s working age population by 2050.fig. 1working age population forecast for growth 20102050 working age population forecast for growth 20102050 long - term care informal care is usually provided by family or friends, while formal care has evolved to include state, private and non - for - profit agencies (timonen and doyle 2007). in general the formal care providers are paid and the informal unpaid but a range of arrangements exists including care allowances and long - term care insurance. the number of migrants working in the formal care sector has grown but to understand the extent of migrant care work the large numbers working in the grey market as unregulated carers in private homes also need to be taken into consideration (oecd 2011). the need for long - term care and the responsibility for its provision have changed greatly over the last four decades. the demographic shift towards an ageing population has been accompanied by changes in the labour market and also in the family unit, so the role of women in society has dramatically changed. traditionally care giving has been overwhelmingly unpaid women s work, but it is increasingly recognised that care for the elderly is a larger societal concern. demography and dependency ratios will play an important part in determining the sustainability of welfare states pensions. across europe over the last 40 years more women have joined the labour force, birth rates have declined, (although a very slight increase was reported in 2010), and divorce has become more common. social developments have changed the ability of women, in particular, to provide such extensive informal care (dhner and eickhoff 2008). male breadwinner model has been replaced by an adult worker model which not only encourages but often requires the participation of both men and women in the formal labour market (lutz and palenga - mollenbeck 2010 : 425). this is reinforced in both the lisbon and 2020 strategies which aim to boost labour force activity. this shift has forced a re - organisation and re - distribution of care responsibilities within societies (lutz and palenga - mollenbeck 2010). one outcome of the changing family model and role of women in society in the context of demographic ageing is that more elderly people are living alone. within small families it is now more likely that an adult child will have to undertake some carer responsibilities but also that many older people are providing care to spouses. a recent study about family carers3 showed that there is still a strong desire to provide care to older relatives. however, eldercare is often a full - time job with little social or financial recognition and it has also been shown that people who care are more likely to need care themselves in later life (dhner and eickhoff 2008). there has been a commodification of care ; demand has been outsourced to the formal and paid migrant sectors and there has been a globalisation of care, as women migrate across the globe to cater for this growing market. as female labor force participation has increased demand for domestic workers has risen. inadequate state provision and a move towards providing cash benefits for care services are intensifying this demand. much of the new demand for care in private households is being met by non - eu nationals who are estimated to account for over 10 % of workers in this sector (schwenken and heimeshoff 2011) however, since much of this work is undocumented, its contribution to the european economy is likely to be much greater than reported. it was estimated that in 2008 about 100,000 female migrants were providing care to elderly people in germany (dhner and eickhoff 2008). in 2010 it was reported that about 700,000 migrant care workers were employed in italy ; almost all of them by households in the unregulated sector (interlinks 2010). policy makers and the general public are only starting to become aware of the large number of older people in need of (affordable) care and the migrant carers who provide it (dhner and eickhoff 2008). by looking at the past and present provision of care in some of the old european countries (sweden, the netherlands, france, germany, the united kingdom and italy) it is possible to explore how different countries and their welfare models are responding to the demands of the demographic shift. the provision of long term care is complex, divided between state, market and family providers ; nowhere can or does the state alone act as the sole provider of care (banks 1998). the extent to which different sectors are relied upon is largely dependent on the ideology of the country s welfare state (timonen and doyle 2007). women s working choices are, of course, strongly affected by the dominant care - provision ideology of their society (lutz and palenga - mollenbeck 2010). in western europe a spectrum of care provision exists ; at one end lies the informal carer - led model, while at the other end the service - led model. the informal carer - led model is defined by limited governmental responsibility. in this model the state acts as a regulator rather than a provider of care ; it is the users responsibility to find care, the government may then subsidise some of the financial cost., the service - led model aims to provide extensive service provision and to reduce the responsibility of the family. in this model direct financial support to users is more limited ; care is provided through the use of extensive services, with the aim to make formal female employment and care compatible. problems have arisen with both models ; in the former the growing number of dependent older people living alone has increased the financial strain on the state and with more women in formal employment there has been a declining supply of informal carers. in the service - led model the high cost involved in providing extensive services and the demands for quality and flexibility of such services have put increasing strain on the capacity of the state. in the 1990s countries such as sweden and the netherlands had relatively high levels of service provision and uptake, with high numbers of over 65s receiving the services provided. meanwhile germany, france and especially italy had fewer services and attempted to aid family support through a limited provision of cash programmes. sweden had adopted the services - led model to the greatest extent with particular importance attached to home care services. similarly the netherlands provided a large number of services but also had a large investment in residential care. both italy and germany encouraged more informal provision of care, although germany did provide more residential care services. there has been a growing trend in the use and importance of cash benefits to support both dependent individuals and their family carers. although little used in sweden, where emphasis was on providing services, cash programmes had developed particularly in the uk, germany and the netherlands. regardless of the position of countries on the care provision spectrum 20 years ago, there has been a clear convergence among the new policies of these member states. as financial constraints have increased with the growing numbers of dependent elderly, governments have begun to implement policies to support care at home ; encouraging those who need care to look for in - house help. such policies encourage the autonomy of the elderly, the caring capacities of their families and emphasise, at least in principle, greater freedom of choice. new policies have sought to combat the inefficiencies and limitations of previous cash programmes and service provision by rejecting the idea of two independent and mutually exclusive forms of care ; in fact formal and informal care are now considered this begs the question of where the migrant care workers fit in particularly with regard to the eligibility for carers benefits. public provision is seen as one element of care which aims to support informal care provision and increase the user s freedom of choice (pavolini and ranci 2008). countries which began as predominantly informal care - led providers have increased public funding of services and support for informal carers. meanwhile predominantly service - led countries have restructured their care services and concentrated the provision of care on those with higher dependencies while also increasing support for informal carers. therefore both ends of the spectrum have taken steps to provide a more inclusive, effective and mixed provision of care (pavolini and ranci 2008). it is important to note that this can not be seen as a general overview of the care situation across the european union and developments in other countries will reflect their own history, resources and values. alber and kohler (2004) underline the reliance upon family care in the new member states but argue it is not merely due to a lack of public services but an indicator of the strength of family values in those countries. italy has made little effort to reform its system of care ; there is still a heavy reliance on family responsibility either to provide care themselves or to outsource it to a private provider. the italian system is so reliant on migrant carers that it could be argued that rather than being family dependent it is a migrant dependent model (pavolini and ranci 2008 : 257) of course italy is not the only country which is already heavily reliant on migrant care workers ; in 1991 16 % of employed home care workers in europe were foreign and this figure increased to 86 %, in 2005, the majority being employed as carers for the elderly (european commission 2009). clearly budgets are one factor limiting the development of services and supply but so too is the availability of human resources in the member states. increased demand for adequate long - term care services has put increasing pressure on governments to fund such services. in response governments are implementing policies which in theory aim to cater for the growing number of service users and to curb their growing financial strain on the state. emphasis is often put on the fact that the new policies aim to support informal carers by providing benefits and cash programmes but it is not clear to what extent these cash - for - care programmes are naturally reaching family carers. the introduction of cash - for - care policies seek to establish a new balance between formal services and informal care, but also have implications for private funding of migrant carers in the unregulated market these new initiatives also recognise the major role of family care and consider how to support it rather than exploit it (pavolini and ranci 2008). it is often presumed that care work is an unskilled job but on the contrary it requires skills such as patience, empathy, emotional intelligence trust and high frustration management are all required and hard skills to learn (lutz and palenga - mollenbeck 2010). informal, family carers have been undervalued and unacknowledged in the care policy documents of many countries until relatively recently. da roit found, in her study caring beyond borders - migrant care work in europe, that the higher the rate of care allowance and the looser the government regulations on payment the greater the number of migrants providing informal care.4 in germany care insurance is highly regulated and paid to the care recipient. the payment aims to increase the recipient s freedom of choice and sense of control. however, it can result in a lack of payment actually reaching informal care providers. timonen and mcmenamin (2002) concluded that there were somewhere in the region of 100,000 people in ireland providing informal care on a full - time or almost full - time basis and recent estimates are even higher. the vast majority, the disproportionate number of women providing informal care is likely to have detrimental long - term financial consequences by prolonging the discrimination of women in the labour force and reinforce the trap of women in low paid and undervalued employment (glendinning. even though the provision of cash - for - care payments can be perceived as a positive recognition of care work it can also trap carers, women, into undervalued physically and mentally demanding work. this overview provides a look at the policies being implemented but not at how well they work in practice. looking at the direction in which long - term care policies are moving, does not explore how, in reality, countries are meeting long - term care needs. some policies that are put in place, while being sound in theory, may fail due to inflexibility or low carer and user take up. many of the policies being put in place to cater for the growing older population are reliant upon a steady supply of available workers or family carers yet the question of how to maintain this supply is often not discussed. there is a need for governments to introduce policies which help workers maintain a work / life balance so as to encourage more people, specifically women, to enter the workforce and also to implement policies which curb the growing cost of formal care provision. family care is not only the expressed preference for many elderly people it is also seen as a cost containing measure for the government (timonen and mcmenamin 2002). as fig. 2 shows, expectations and preferences for different types of care provision are fairly evenly matched across the 27 eu member states with a strong preference for home care. however, there are big differences between countries ; the expectation to be looked after at home by a relative is very high in eastern europe ; in poland 70 % of respondents chose this option, while in denmark only 20 % stated this as their preference (eurobarometer 2007). political discourses reinforce the cultural desire that care should be provided by the family at home (lutz and palenga - mollenbeck 2010).fig. 2expected and preferred way of getting assistance if one becomes dependent and needs regular help and long - term care % eu27 : (special eurobarometer 2007 : 95) expected and preferred way of getting assistance if one becomes dependent and needs regular help and long - term care % eu27 : (special eurobarometer 2007 : 95) in reality the preference for home care often demands a much larger supply of carers than is available in that country and this leads to a heavy reliance on and use of the grey labour market. grey labour market or unregulated work can be defined as involving people who do not have the legal right to employment in that country and in the case of the eu these are usually people from third countries (dhner and eickhoff 2008). it has been found that dependent persons would prefer to be cared for in their own home (lutz and palenga - mollenbeck 2010). various studies report this, such as the european dialog project, in which 14 different european countries participated (hhn. when asked about help from informal, family carers (which could also include migrant care workers) 4060 % stated that they would prefer that option, with the highest figure in austria (77 %) and the lowest in finland (39 %) (hhn. there is a heavy reliance on foreign labour in the care sector in western european countries (piperno 2010). long - term care for the elderly is often provided by migrants coming from eastern european countries (dhner and eickhoff 2008). western europe s care services are sustained by workers from eastern europe (dhner and eickhoff 2008 ; lutz and palenga - mollenbeck 2010). it can been observed that governments are content to turn a blind eye to the problem of unregulated and undeclared migrant workers providing long - term care as it is a short - term solution to the problem of increasing demand for care and limited finances (lutz and palenga - mollenbeck 2010). host governments and societies are benefiting from the cheap migrant labour that is providing essential services without having to provide many social services to the carers in turn. tonken has argued that to prevent the exploitation and invisibility of migrant care workers the issue needs to go beyond the jurisdiction of individual member states.5 by ignoring the situation of migrant care workers the government achieves two things;cheap and flexible care provision;a continued societal misunderstanding about the important and positive role migrants play within societies. cheap and flexible care provision ; a continued societal misunderstanding about the important and positive role migrants play within societies. a large number of people migrate into europe either as carers (with and without qualifications) or they later become carers in both the formal and unregulated sectors. in a british study ; migrant care workers in ageing societies, it was found that in 2008 that 19 % of care workers and 35 % of nurses working in formal older care were migrants (cangiano. 2009). by not properly addressing the role of migrant care workers governments are reinforcing the invisibility of this group within society. carer migration is responding to the growing demand for workers aided by new migrant networks created through globalisation. migrant flows are far from static and quickly respond to socio - economic needs ; the rapid growth and transformation of countries means that their status as a sender or recipient of migrants can change quickly. poland has been transformed since 2004 and is now experiencing both in and out flows of migrants simultaneously (hoff. there is evidence of an extensive benefit available to host countries and to the immigrant workers themselves. in the host countries, the immigration of care workers is positively able to respond to the growing demand for care services. yet the use of grey market and irregular employees endangers not only the quality of working life but also the quality and standards of these services (piperno 2010). it has been widely documented that remittances are an important source of income for migrant s families and communities in the sender countries (hoff. 2010). however, it would also appear that many sender countries are losing valuable and often skilled human capital at a time when they are needed in their home countries. development in some third countries is continually defeated by the fact that after being educated and invested in by the taxpayers in their home country migrants leave to find higher wages and a better standard of living (howse 2007). people are less willing to offer a care service (to a non - relative) in their own country if they can be paid more to do it in different location and this can lead to the issue of care drain (piperno 2010). there are three drivers of population change : fertility rates, life expectancy and migration (european commission 2010). within the eu27 there are about 5 million births a year, which outnumbers deaths by only several hundred thousand according to the european commission 2010 demography report. net migration for the eu is over a million a year which makes migration the single greatest driver of population growth in the eu (european commission 2010 : 2). demographic changes affect international migration in several ways : rapid population growth combined with economic difficulties at home is likely to push people to move in search of work, and a declining and ageing population pressures countries to accept migrants. population ageing is a global issue effecting america, japan, korea and australia but it is most pronounced within the european context. figure 1 illustrates the growth predictions of the global working age population from 2010 to 2050. it clearly shows a north south divide with younger, growing populations in the southern hemisphere, much smaller growth predicted in the northern hemisphere and a reduction of 23 % in europe s working age population by 2050.fig. 1working age population forecast for growth 20102050 working age population forecast for growth 20102050 informal care is usually provided by family or friends, while formal care has evolved to include state, private and non - for - profit agencies (timonen and doyle 2007). in general the formal care providers are paid and the informal unpaid but a range of arrangements exists including care allowances and long - term care insurance. the number of migrants working in the formal care sector has grown but to understand the extent of migrant care work the large numbers working in the grey market as unregulated carers in private homes also need to be taken into consideration (oecd 2011). the need for long - term care and the responsibility for its provision have changed greatly over the last four decades. the demographic shift towards an ageing population has been accompanied by changes in the labour market and also in the family unit, so the role of women in society has dramatically changed. traditionally care giving has been overwhelmingly unpaid women s work, but it is increasingly recognised that care for the elderly is a larger societal concern. demography and dependency ratios will play an important part in determining the sustainability of welfare states pensions. across europe over the last 40 years more women have joined the labour force, birth rates have declined, (although a very slight increase was reported in 2010), and divorce has become more common. social developments have changed the ability of women, in particular, to provide such extensive informal care (dhner and eickhoff 2008). the male breadwinner model has been replaced by an adult worker model which not only encourages but often requires the participation of both men and women in the formal labour market (lutz and palenga - mollenbeck 2010 : 425). this is reinforced in both the lisbon and 2020 strategies which aim to boost labour force activity. this shift has forced a re - organisation and re - distribution of care responsibilities within societies (lutz and palenga - mollenbeck 2010). one outcome of the changing family model and role of women in society in the context of demographic ageing is that more elderly people are living alone. within small families it is now more likely that an adult child will have to undertake some carer responsibilities but also that many older people are providing care to spouses. a recent study about family carers3 showed that there is still a strong desire to provide care to older relatives. however, eldercare is often a full - time job with little social or financial recognition and it has also been shown that people who care are more likely to need care themselves in later life (dhner and eickhoff 2008). there has been a commodification of care ; demand has been outsourced to the formal and paid migrant sectors and there has been a globalisation of care, as women migrate across the globe to cater for this growing market. as female labor force participation has increased demand for domestic workers has risen. inadequate state provision and a move towards providing cash benefits for care services are intensifying this demand. much of the new demand for care in private households is being met by non - eu nationals who are estimated to account for over 10 % of workers in this sector (schwenken and heimeshoff 2011) however, since much of this work is undocumented, its contribution to the european economy is likely to be much greater than reported. it was estimated that in 2008 about 100,000 female migrants were providing care to elderly people in germany (dhner and eickhoff 2008). in 2010 it was reported that about 700,000 migrant care workers were employed in italy ; almost all of them by households in the unregulated sector (interlinks 2010). policy makers and the general public are only starting to become aware of the large number of older people in need of (affordable) care and the migrant carers who provide it (dhner and eickhoff 2008). by looking at the past and present provision of care in some of the old european countries (sweden, the netherlands, france, germany, the united kingdom and italy) it is possible to explore how different countries and their welfare models are responding to the demands of the demographic shift. the provision of long term care is complex, divided between state, market and family providers ; nowhere can or does the state alone act as the sole provider of care (banks 1998). the extent to which different sectors are relied upon is largely dependent on the ideology of the country s welfare state (timonen and doyle 2007). women s working choices are, of course, strongly affected by the dominant care - provision ideology of their society (lutz and palenga - mollenbeck 2010). in western europe a spectrum of care provision exists ; at one end lies the informal carer - led model, while at the other end the service - led model. the informal carer - led model is defined by limited governmental responsibility. in this model the state acts as a regulator rather than a provider of care ; it is the users responsibility to find care, the government may then subsidise some of the financial cost., the service - led model aims to provide extensive service provision and to reduce the responsibility of the family. in this model direct financial support to users is more limited ; care is provided through the use of extensive services, with the aim to make formal female employment and care compatible. problems have arisen with both models ; in the former the growing number of dependent older people living alone has increased the financial strain on the state and with more women in formal employment there has been a declining supply of informal carers. in the service - led model the high cost involved in providing extensive services and the demands for quality and flexibility of such services have put increasing strain on the capacity of the state. in the 1990s countries such as sweden and the netherlands had relatively high levels of service provision and uptake, with high numbers of over 65s receiving the services provided. meanwhile germany, france and especially italy had fewer services and attempted to aid family support through a limited provision of cash programmes. sweden had adopted the services - led model to the greatest extent with particular importance attached to home care services. similarly the netherlands provided a large number of services but also had a large investment in residential care. both italy and germany encouraged more informal provision of care, although germany did provide more residential care services. there has been a growing trend in the use and importance of cash benefits to support both dependent individuals and their family carers. although little used in sweden, where emphasis was on providing services, cash programmes had developed particularly in the uk, germany and the netherlands. regardless of the position of countries on the care provision spectrum 20 years ago, there has been a clear convergence among the new policies of these member states. as financial constraints have increased with the growing numbers of dependent elderly, governments have begun to implement policies to support care at home ; encouraging those who need care to look for in - house help. such policies encourage the autonomy of the elderly, the caring capacities of their families and emphasise, at least in principle, greater freedom of choice. new policies have sought to combat the inefficiencies and limitations of previous cash programmes and service provision by rejecting the idea of two independent and mutually exclusive forms of care ; in fact formal and informal care are now considered this begs the question of where the migrant care workers fit in particularly with regard to the eligibility for carers benefits. public provision is seen as one element of care which aims to support informal care provision and increase the user s freedom of choice (pavolini and ranci 2008). countries which began as predominantly informal care - led providers have increased public funding of services and support for informal carers. meanwhile predominantly service - led countries have restructured their care services and concentrated the provision of care on those with higher dependencies while also increasing support for informal carers. therefore both ends of the spectrum have taken steps to provide a more inclusive, effective and mixed provision of care (pavolini and ranci 2008). it is important to note that this can not be seen as a general overview of the care situation across the european union and developments in other countries will reflect their own history, resources and values. alber and kohler (2004) underline the reliance upon family care in the new member states but argue it is not merely due to a lack of public services but an indicator of the strength of family values in those countries. italy has made little effort to reform its system of care ; there is still a heavy reliance on family responsibility either to provide care themselves or to outsource it to a private provider. the italian system is so reliant on migrant carers that it could be argued that rather than being family dependent it is a migrant dependent model (pavolini and ranci 2008 : 257) of course italy is not the only country which is already heavily reliant on migrant care workers ; in 1991 16 % of employed home care workers in europe were foreign and this figure increased to 86 %, in 2005, the majority being employed as carers for the elderly (european commission 2009). clearly budgets are one factor limiting the development of services and supply but so too is the availability of human resources in the member states. increased demand for adequate long - term care services has put increasing pressure on governments to fund such services. in response governments are implementing policies which in theory aim to cater for the growing number of service users and to curb their growing financial strain on the state. emphasis is often put on the fact that the new policies aim to support informal carers by providing benefits and cash programmes but it is not clear to what extent these cash - for - care programmes are naturally reaching family carers. the introduction of cash - for - care policies seek to establish a new balance between formal services and informal care, but also have implications for private funding of migrant carers in the unregulated market these new initiatives also recognise the major role of family care and consider how to support it rather than exploit it (pavolini and ranci 2008). it is often presumed that care work is an unskilled job but on the contrary it requires skills such as patience, empathy, emotional intelligence trust and high frustration management are all required and hard skills to learn (lutz and palenga - mollenbeck 2010). informal, family carers have been undervalued and unacknowledged in the care policy documents of many countries until relatively recently. da roit found, in her study caring beyond borders - migrant care work in europe, that the higher the rate of care allowance and the looser the government regulations on payment the greater the number of migrants providing informal care.4 in germany care insurance is highly regulated and paid to the care recipient. the payment aims to increase the recipient s freedom of choice and sense of control. however, it can result in a lack of payment actually reaching informal care providers. timonen and mcmenamin (2002) concluded that there were somewhere in the region of 100,000 people in ireland providing informal care on a full - time or almost full - time basis and recent estimates are even higher. the vast majority, the disproportionate number of women providing informal care is likely to have detrimental long - term financial consequences by prolonging the discrimination of women in the labour force and reinforce the trap of women in low paid and undervalued employment (glendinning. even though the provision of cash - for - care payments can be perceived as a positive recognition of care work it can also trap carers, women, into undervalued physically and mentally demanding work. this overview provides a look at the policies being implemented but not at how well they work in practice. looking at the direction in which long - term care policies are moving, does not explore how, in reality, countries are meeting long - term care needs. some policies that are put in place, while being sound in theory, may fail due to inflexibility or low carer and user take up. many of the policies being put in place to cater for the growing older population are reliant upon a steady supply of available workers or family carers yet the question of how to maintain this supply is often not discussed. there is a need for governments to introduce policies which help workers maintain a work / life balance so as to encourage more people, specifically women, to enter the workforce and also to implement policies which curb the growing cost of formal care provision. family care is not only the expressed preference for many elderly people it is also seen as a cost containing measure for the government (timonen and mcmenamin 2002). as fig. 2 shows, expectations and preferences for different types of care provision are fairly evenly matched across the 27 eu member states with a strong preference for home care. however, there are big differences between countries ; the expectation to be looked after at home by a relative is very high in eastern europe ; in poland 70 % of respondents chose this option, while in denmark only 20 % stated this as their preference (eurobarometer 2007). political discourses reinforce the cultural desire that care should be provided by the family at home (lutz and palenga - mollenbeck 2010).fig. 2expected and preferred way of getting assistance if one becomes dependent and needs regular help and long - term care % eu27 : (special eurobarometer 2007 : 95) expected and preferred way of getting assistance if one becomes dependent and needs regular help and long - term care % eu27 : (special eurobarometer 2007 : 95) in reality the preference for home care often demands a much larger supply of carers than is available in that country and this leads to a heavy reliance on and use of the grey labour market. grey labour market or unregulated work can be defined as involving people who do not have the legal right to employment in that country and in the case of the eu these are usually people from third countries (dhner and eickhoff 2008). it has been found that dependent persons would prefer to be cared for in their own home (lutz and palenga - mollenbeck 2010). various studies report this, such as the european dialog project, in which 14 different european countries participated (hhn. when asked about help from informal, family carers (which could also include migrant care workers) 4060 % stated that they would prefer that option, with the highest figure in austria (77 %) and the lowest in finland (39 %) (hhn. there is a heavy reliance on foreign labour in the care sector in western european countries (piperno 2010). long - term care for the elderly is often provided by migrants coming from eastern european countries (dhner and eickhoff 2008). western europe s care services are sustained by workers from eastern europe (dhner and eickhoff 2008 ; lutz and palenga - mollenbeck 2010). it can been observed that governments are content to turn a blind eye to the problem of unregulated and undeclared migrant workers providing long - term care as it is a short - term solution to the problem of increasing demand for care and limited finances (lutz and palenga - mollenbeck 2010). host governments and societies are benefiting from the cheap migrant labour that is providing essential services without having to provide many social services to the carers in turn. tonken has argued that to prevent the exploitation and invisibility of migrant care workers the issue needs to go beyond the jurisdiction of individual member states.5 by ignoring the situation of migrant care workers the government achieves two things;cheap and flexible care provision;a continued societal misunderstanding about the important and positive role migrants play within societies. cheap and flexible care provision ; a continued societal misunderstanding about the important and positive role migrants play within societies. a large number of people migrate into europe either as carers (with and without qualifications) or they later become carers in both the formal and unregulated sectors. in a british study ; migrant care workers in ageing societies, it was found that in 2008 that 19 % of care workers and 35 % of nurses working in formal older care were migrants (cangiano. 2009). by not properly addressing the role of migrant care workers governments are reinforcing the invisibility of this group within society. carer migration is responding to the growing demand for workers aided by new migrant networks created through globalisation. migrant flows are far from static and quickly respond to socio - economic needs ; the rapid growth and transformation of countries means that their status as a sender or recipient of migrants can change quickly. poland has been transformed since 2004 and is now experiencing both in and out flows of migrants simultaneously (hoff. there is evidence of an extensive benefit available to host countries and to the immigrant workers themselves. in the host countries, the immigration of care workers is positively able to respond to the growing demand for care services. yet the use of grey market and irregular employees endangers not only the quality of working life but also the quality and standards of these services (piperno 2010). it has been widely documented that remittances are an important source of income for migrant s families and communities in the sender countries (hoff. however, it would also appear that many sender countries are losing valuable and often skilled human capital at a time when they are needed in their home countries. development in some third countries is continually defeated by the fact that after being educated and invested in by the taxpayers in their home country migrants leave to find higher wages and a better standard of living (howse 2007). people are less willing to offer a care service (to a non - relative) in their own country if they can be paid more to do it in different location and this can lead to the issue of care drain (piperno 2010). care is not a patriarchal concern for women, a type of secondary moral question or the work of the least well off in society. it is time we begin to change our political and social institutions to reflect this truth. (tronto, 1993:180) care is not a patriarchal concern for women, a type of secondary moral question or the work of the least well off in society. it is time we begin to change our political and social institutions to reflect this truth. (tronto, 1993:180) europe s demographic shift and the increasing demand for long - term care are becoming more evident and calling for urgent policy attention. however, the policy debate must be informed by a clear understanding of the subject. this paper has illustrated the need for clearer concepts of long - term care and for the categories of care providers. the growing demand for migrant care workers is a result of economic and social needs at both the formal and informal level. it is only possible to make relevant policy recommendations once the issues of formal / informal, contracted / unregulated and paid benefits in kind are clearly understood. the growing numbers of migrant care workers can not be understood outside of the social care system, policy regulations and culture of care within which they work. the role of migrant care workers within this web of care provision is a result of number of supply and demand factors. care and care workers have gradually become a more prominent policy issue ; care is no longer solely a private, family or female concern. on the demand side, the need for carers has increased as the numbers of old and dependent people has risen. in addition, the traditional supply of informal carers has been reduced through declining family sizes, more geographical mobility, as women have moved into the formal work force and are either unable or unwilling to provide long - term care to older relatives. yet, at - home care migrant care workers have to been pivotal to both the formal and unregulated supply of care in ireland ; they have enabled a number of potential carers to enter the formal work force. similar to the complex nature of care provision, care is not a single policy issue but rather involves immigration, employment and health policies. at both the eu and national level immigration policies have been tightened in response to the recession and only skilled migration is being encouraged. minimum income restrictions and work permit quotas have resulted in fewer qualified migrant nurses being employed below their skill level in the unregulated sector and the employment of migrant care workers has been pushed further into the grey market. and this is against a background of falling numbers in the working age population of the eu in the near future. on a final note it has been found that the migration of care workers will be important to europe s economic recovery and demographic revival. however, this relationship can only be beneficial to all involved and in the long run when the policies in place promote an equitable and efficient relationship. | this paper is an examination of the recent restructuring and subsequent convergence of european long - term care models. this paper also aims to highlight the increased role of migrant care workers and the need for great social and governmental recognition for all care providers. the provision of long term care is complex, divided between state, market and family providers ; the state alone could not and does not act as the sole provider of care (banks 1998). the extent to which different sectors are relied upon is largely dependent on the ideology of the country 's welfare state (timonen and doyle 2007). |
cloacogenic carcinomas (ccs) are relatively common neoplasms of the anal canal and are thought to arise from cloacogenic remnants at this site. histological and ultrastructural studies have indicated that these neoplasms may also arise from transitional cloacogenic epithelium lining the anal ducts or from the basal layer of anal squamous epithelium. outside the anal canal, it has been postulated that these tumors arise from cloacogenic embryologic remnants, squamous metaplastic epithelium, or totipotential basal cells. we report a patient with cc of the recto - sigmoid junction presenting as intraabdominal abscess. for this reason, a 23-year - old man visited the emergency unit of our hospital with rectal bleeding, severe suprapubic abdominal pain, fever, poor general condition and fluid discharge from a median subumbilical incision. physical examination showed generalized peritonitis in the abdomen and his general condition was moderately dehydrated. the systolic and diastolic blood pressures were 80 and 50 mm hg respectively, his pulse rate was 110/min, and his respiratory rate was 22/min. laboratory results were as follows : blood urea nitrogen 65 mg / dl, creatinine 2.1 mg / dl, c - reactive protein 198 mg / l, potassium (k) 2.2 meq / dl, and albumin 2.3 g / dl. blood cell counts revealed leukocytosis (215,000/l) ; his hemoglobin was 10.5 g / dl and the platelet count was 408,000/l. we performed abdominal computed tomography (ct) with intravenous contrast, which showed diffuse wall thickening 10 - 11 cm above the rectosigmoid junction, and an 8 8.5 cm soft tissue lesion that included cystic areas in the pelvis (fig. 1, fig. multiple lymph nodes were seen in the abdomen ; the biggest measured 3 2 cm. air vesicles and images concordant with a fistula tract in the anterior abdominal wall were detected. an 8 5 cm abscess was seen related to the fistula tract on the anterior wall of the abdomen. no intestinal content was observed in the abdomen. a 10 cm mass attached to nearby tissues that contained necrotic and caseated areas covered the distal sigmoid and proximal rectum. as resection was not possible, hartman - type proximal end colostomy was performed and the operation was ended leaving a distal stubby. as adenoid structures in patches were observed by histopathological examination, the differential diagnosis was made between poorly differentiated adenocarcinoma and cc. immunohistochemically, staining with high - molecular - weight cytokeratin (hmwck) (fig. ca 19 - 9 levels were normal (1.1 ng / ml and 2 iu / ml, respectively). three weeks after discharge from the hospital, a 5-fluorouracil + folinic acid + irinotecan regimen was started. in total, three cycles of radiotherapy and a concurrent three - drug chemotherapy regimen (folfiri ; folinic acid, fluorouracil, irinotecan) were administered to the patient over 6 weeks. in ct scans 3 months after therapy, an increase in mass size and hydroureteronephrosis were observed. at the end of the fifth month, in this case, failure in detecting the mass in the pelvis during laparatomy can depend on two reasons : either the mass was not formed at the time of the laparatomy or the mass could not be seen. probably it was the colonic mass that caused symptoms of appendicitis in his first surgery. however, it is normal that colon carcinoma is not suspected initially in an 18-year - old patient presenting with abdominal pain without any family history, since without the presence of familial colon cancer or ulcerative colitis history, incidence of colon cancer is very low at < 40 years of age. in this case, neither in family history nor in colonoscopic examination was any sign of polyposis coli present. furthermore, because of aggressive progression but nearly normal cea levels, no sign of liver metastasis, and no clinical answer to radiotherapy and chemotherapy, we thought that it was either undifferentiated carcinoma arising from the colonic mucosa or anoderm originated cc which rarely occurs in the rectosigmoid area. in the literature, most papers report positive reactions with hmwck and p63 immunohistochemical antibodies in cc. there are only 5 cases of cc located outside the anal canal in the literature. presented the first case of cc originating from the sigmoid colon and located outside the anal canal. newell. reported a cc case originating from the splenic flexure of the colon with liver metastases. presented a 24-year - old female patient with cc originating from the descending colon. as a result, although cc generally occurs under the dentate line, rarely it can root from the other sides of the colon. tumor size < 2 cm, patient age < 40 years, being poor in cells and having high fibroblastic activity are bad prognostic factors. | cloacogenic carcinoma (cc, basaloid carcinoma) generally occurs around the dentate line, rarely it can arise from the other sides of the colon. there are only 5 cases of cc located outside the anal canal in the literature. the first occurrence of a cc presents as intraabdominal abscess. we describe a 23-year - old male patient who was admitted with fever and severe abdominal pain. computed tomography imaging showed diffuse wall thickening about 1011 cm above the rectosigmoid junction, intraabdominal abscess and a soft tissue lession covering the pelvis with a size of 8 8.5 cm including cystic necrotic areas. we performed hartman procedure since the mass was nonresectable. histopathological examination showed cc. in total, three times radiotherapy and a concurrent three - drug regimen of irinotecan, fluorouracil and folinic acid chemotherapy were administered for 6 weeks. as a result, the patient was lost because of multiple organ dysfunction syndrome that developed 3 months after radio - chemotherapy. |
gastric antral vascular ectasia (gave) is a rare disorder associated with chronic upper gastrointestinal bleeding that mainly affects elderly women. it has a unique endoscopic appearance characterized by multiple longitudinal stripes of red vessels (watermelon stomach) or a diffuse spread of red vessels (diffuse antral vascular ectasia), which is largely limited to the gastric antrum. the typical histological findings are superficial hyperplastic gastric antral mucosa, capillary ectasia with thrombosis and fibromuscular hyperplasia in the lamina propria, and abnormal submucosal vasculature. gave is sometimes found in patients with chronic kidney disease (ckd), regardless of the maintenance hemodialysis therapy administered. recently, argon plasma coagulation (apc) has been frequently used to treat gave. however we herein present 3 cases of gave associated with ckd that were not previously treated with dialysis. a 67-year - old woman with ckd stage v [serum creatinine (cr) = 3.14 mg / dl ; egfr = 12.3 ml / min/1.73 m ] caused by biopsy - proven chronic mesangial proliferative glomerulonephritis suffered from loss of appetite and edema with uremia. laboratory findings showed severe anemia [hemoglobin (hb) = 5.8 g / dl ] despite treatment with recombinant human erythropoietin (rhuepo) and intravenous iron administration. upper gastrointestinal endoscopy showed watermelon stomach at the antrum without active bleeding. despite repeated blood transfusions, six months later, the patient was treated with apc, but the anemia persisted. one month after the first apc administration, a second round of apc treatment was administered, and hemodialysis therapy was initiated (fig. her anemia improved, and no additional transfusions have since been performed. a 61-year - old woman with alcoholic liver cirrhosis (child - pugh class b) and ckd stage v (cr = 3.14 mg / dl ; egfr = 12.6 ml / min/1.73 m) caused by hepatorenal syndrome was admitted to the hospital. she suffered from loss of appetite with uremia and had severe anemia (hb = 4.8 g / dl) despite administration of rhuepo and intravenous iron. she also had severe melena, and upper gastrointestinal endoscopy revealed diffuse antral vascular ectasia without esophageal varices or active bleeding. she was treated with apc twice, but frequent blood transfusions were required. five months after the second apc treatment, the patient was started on hemodialysis because of volume expansion (fig. she no longer requires blood transfusions. a 66-year - old woman with ckd stage v (cr = 6.4 mg / dl ; egfr = 5.7ml / min/1.73 m) presented with severe anemia (hb = 4.8 g / dl) and suffered from lassitude, loss of appetite, and dyspnea with uremia. the primary cause of ckd was an autosomal dominant polycystic disease with numerous and massive cysts in both the kidneys and liver. after 8 months of apc sessions, she was treated with another round of apc and started on hemodialysis (fig. 1). after the induction of hemodialysis, her loss of appetite and lassitude disappeared. a 67-year - old woman with ckd stage v [serum creatinine (cr) = 3.14 mg / dl ; egfr = 12.3 ml / min/1.73 m ] caused by biopsy - proven chronic mesangial proliferative glomerulonephritis suffered from loss of appetite and edema with uremia. laboratory findings showed severe anemia [hemoglobin (hb) = 5.8 g / dl ] despite treatment with recombinant human erythropoietin (rhuepo) and intravenous iron administration. upper gastrointestinal endoscopy showed watermelon stomach at the antrum without active bleeding. despite repeated blood transfusions, six months later, the patient was treated with apc, but the anemia persisted. one month after the first apc administration, a second round of apc treatment was administered, and hemodialysis therapy was initiated (fig. a 61-year - old woman with alcoholic liver cirrhosis (child - pugh class b) and ckd stage v (cr = 3.14 mg / dl ; egfr = 12.6 ml / min/1.73 m) caused by hepatorenal syndrome was admitted to the hospital. she suffered from loss of appetite with uremia and had severe anemia (hb = 4.8 g / dl) despite administration of rhuepo and intravenous iron. she also had severe melena, and upper gastrointestinal endoscopy revealed diffuse antral vascular ectasia without esophageal varices or active bleeding. she was treated with apc twice, but frequent blood transfusions were required. five months after the second apc treatment, the patient was started on hemodialysis because of volume expansion (fig. a 66-year - old woman with ckd stage v (cr = 6.4 mg / dl ; egfr = 5.7ml / min/1.73 m) presented with severe anemia (hb = 4.8 g / dl) and suffered from lassitude, loss of appetite, and dyspnea with uremia. the primary cause of ckd was an autosomal dominant polycystic disease with numerous and massive cysts in both the kidneys and liver. after 8 months of apc sessions, she was treated with another round of apc and started on hemodialysis (fig. 1). after the induction of hemodialysis, her loss of appetite and lassitude disappeared. moreover, her anemia improved, and gave has not recurred. written informed consent was obtained from all 3 patients. gave is often associated with systemic illnesses such as cirrhosis of the liver, autoimmune connective tissue disorders, bone marrow transplantation, and ckd. although the pathogenic link remains unclear, vascular ectasia is a significantly more common cause of upper gastrointestinal bleeding in patients with ckd than in those with normal renal function (13 vs. 1.3%, respectively ; p < 0.01). it has been reported that the antral area half - time in these patients is significantly increased when compared with that of healthy controls. this finding supports the idea that mechanical stress plays an important role in the pathogenesis of gave. in patients with ckd, increased mechanical stress of the antrum in uremia - induced weakening of gastric emptiness is thought to contribute to the development of gave. according to another theory, vasoactive mediators have an important pathogenic role in gave. enhanced gastrin levels, which are known to increase gastric vasodilation, have been observed in patients with gave. however, the pathogenic role of gastrin is not supported by all studies because hypergastrinemia is not invariably present in patients with gave. prostaglandin e2 is another hormone with vasoactive functions ; in a single study, it was found to be significantly higher in cirrhosis patients with gave than in those without. in another study, histological examination showed that neuroendocrine cells containing 5-hydroxytryptamine and vasoactive intestinal peptide were found close to the ecstatic gastric mucosal vessels [6, 7, 8 ]. disturbed renal excretion and/or catabolism of these vasoactive mediators in patients with ckd might also be associated with gave. uremia - induced platelet dysfunction might also be incriminated as a risk factor for antral bleeding. volume overload has not been reported to be associated with gave, which is sometimes confused with portal hypertensive gastropathy. however, gave did not improve with treatment for portal hypertension and congestive heart failure because intra - abdominal venous pressure was not necessarily parallel with circulating volume. moreover, it was reported that gave in a patient with end - stage renal disease improved following a change to continuous ambulatory peritoneal dialysis from hemodialysis. therefore, volume overload may not be associated with gave. in the 3 patients with ckd reported in this study, the development of severe anemia was obviously associated with gave before the initiation of hemodialysis, and the patients required frequent blood transfusions despite apc therapy (fig. 1). none of the 3 patients experienced a relapse of gave after the initiation of hemodialysis. interestingly, all of the patients experienced severe loss of appetite, and gastrointestinal symptoms disappeared with the initiation of hemodialysis. it was reported that hemodialysis might improve impaired gastric motility and reduce gastrointestinal symptoms in patients with chronic renal failure. therefore, the improvement in upper gastrointestinal motility is assumed to have resulted in the improvements in appetite and gave. in addition, the resolution of gave due to the improvement in gastric motility may have resulted in further appetite increase. a single endoscopic examination often fails to identify gave, which may be a more common complication among patients with end - stage renal disease than is generally considered. it is known that a number of patients, even those undergoing dialysis, have severe anemia due to gave. in these patients, gave might be associated with multiple factors. however, mechanical stress in the antrum due to underdialysis might be one of the causes. in such cases, improvement in dialysis efficiency might contribute to the remission of gave. according to our theory, a recurrence of gave after the initiation of hemodialysis should be a rare event. however, we need longer follow - up periods for these patients, because the follow - up periods for our 3 patients (28 months) would certainly be considered short. we conclude that initiation of dialysis may induce the remission of intractable gave in patients with ckd. although the cause is still unknown, improvement in uremia - induced mechanical stress in the antrum might contribute to the remission of gave. we suggest that in patients with renal insufficiency who suffer from gave relapse, it is important to introduce dialysis treatment. | gastric antral vascular ectasia (gave) is currently recognized as an important cause of gastrointestinal bleeding. chronic kidney disease (ckd) is associated with a high incidence of gave. we report 3 patients with ckd who presented with severe anemia and were diagnosed with gave ; they were resistant to endoscopic argon plasma coagulation. however, remission of anemia and improvement in gave lesions were observed after the initiation of hemodialysis. the pathogenesis of gave remains largely unknown, but mechanical stress of the antrum could play an important role. this stress may be reduced by hemodialysis through improvement of uremia - associated gastrointestinal symptoms. therefore, the initiation of hemodialysis might be effective for intractable gave in ckd patients. |
wilms tumor is the most common primary malignant renal tumor in children, accounting for 6% of all pediatric malignant tumors 1, however, it is very rare in adults, with an incidence of about 0.2 per million per year in the usa and europe 2. only approximately 300 well - documented cases of adult invasion of the renal vein is present in about 12% of cases. up to 4% of patients with wt have tumor thrombus involving the vena cava, and the incidence of cardiac involvement is about 0.8% to 1% 4, 5. in adults, tumor extension into the vena cava and the right side of the heart only one case of an adult with caval involvement has been reported in the english - language literature 6. in this report, a case of favorable histology wt with invasion of the inferior vena cava and extension to the right atrium is discussed with a review of the literature. a 54-year - old man presented to a local hospital with right flank pain that was believed to be caused by cholecystitis. and abdominal ultrasound revealed a hypo - echoic area measuring 2.5 cm 2.3 cm in the right renal collecting system. the patient denied back pain, gross hematuria, urinary frequency, urinary urgency, and urodynia. computerized tomography (ct) scan and magnetic resonance imaging (mri) revealed a mass in the right kidney with extension into the right renal vein and inferior vena cava, and further extension into the right atrium (fig. retrograde urography confirmed that the right renal pelvis was compressed and distorted, but was unobstructed. digital subtraction angiography revealed tumor thrombus in the inferior vena cava extending from the level of the right renal vein to the right atrium (figs 1b). the patient was transferred to our hospital for further evaluation and treatment. based on the clinical findings and investigations, the patient underwent a right radical nephrectomy in a thoraco - abdominal procedure. at the same time, the tumor thrombus was removed from the inferior vena cava and the right atrium. the resected right kidney measured 14cm8cm6 cm. near the renal pelvis was a tumor measuring 7cm5cm4.5 cm with extension into the renal vein. on cut section, the mass had a solid and multicystic appearance with focal invasion of the renal parenchyma. the cysts measured from 0.5 cm to 1.5 cm in diameter, and were filled with clear colorless fluid. macroscopically, the tumor thrombus measured 25cm1.5cm1.5 cm, and was gray with a myxoid cut surface. histologically, the tumor exhibited a triphasic pattern of blastema, epithelium, and stroma with myxoid background in the solid area and in the septa of the cystic area (figs 2). the lining cells of the cysts ranged from flattened to columnar, occasionally of hobnail type, and showed positive staining for cytokeratin (fig 3a) and ema. interestingly, the majority of the cysts had distinct walls composed of spindle or oval cells that expressed muscle - derived markers, such as smooth muscle actin (1a4) and muscle actin (hhf35) (fig 3b). thus, the final diagnosis was favorable histology wt (partially differentiated type), with renal vein invasion and extension to the inferior vena cava and right atrium. the patient received nine courses of adjuvant chemotherapy postoperatively, including two courses with dactinomycin, adriamycin, and vincristine, two courses with paclitaxel and carboplatin, four courses with paclitaxel, ifosfamide, and mesna, and one course with vinorelbine and dactinomycin. multiple metastases to the liver, lung, and chest wall were occurred within 12 months after surgery, and the patient was dead from the disease 14 months later. wt is the most common primary malignant renal neoplasm of childhood, but it is rarely found in adults. the preoperative diagnosis of adult wt is extremely difficult because there are no specific radiographic findings that can distinguish it from the more common adult malignant renal neoplasms 7. both characteristically have triphasic histology, with components of blastemal, epithelial, and stromal structures, as were noted in our case. pathological diagnosis of adult wt is also more difficult than in pediatric one, because there are many other adult renal tumors that should be considered in the differential diagnosis. insufficient sampling of the tumor for histologic evaluation, and the absence of a systemic work - up can lead to misidentification of the tumor. kilton 8 have applied six rigid diagnostic criteria for adult wt, which remain in use : 1) primary renal neoplasm, 2) primary blastematous spindle- or round - cell component, 3) formation of abortive or embryonal, tubular epithelial, or glomeruloid structure, 4) no areas of tumor diagnostic of renal cell carcinoma, 5) pictorial confirmation of histology and 6) age > 15 years. according to these strict diagnostic criteria, only about 300 cases of adult wt have been reported in the english literature to date 4. in the current case, the classic triphasic pattern was clear, with no areas suggestive of renal cell carcinoma. 4% children wt have caval involvement, and 0.8 - 1% of these patients also have atrial extension of the tumor 4, 5, however, the incidence of caval involvement in adults is unknown, and only one adult case with right atrial extension has been reported in english - language literature 6. echocardiography and color flow doppler ultrasound are helpful in detecting inferior vena caval and right atrial tumor if the wt has spread through the inferior vena cava into the right atrium 9. the present case was diagnosed as inferior vena cava and right atrium involvement by the ct and mri examination, although tumor type could not define before surgery. cases with intravascular involvement may represent a daunting challenge for the surgeon. in mart 's 10 experience, the surgical strategy for these cases depends on the length of the thrombus and whether or not the thrombus has infiltrated the wall of the vena cava. if the thrombus can be easily removed, complete resection is the treatment of choice, however, in cases of atrial thrombus, and more particularly if the thrombus involves the intima, these authors suggest that the thrombus not be touched, and the tumor should be treated by preoperative and postoperative chemotherapy. this observation is only partially explained by the fact that more adults seem to have advanced stage at the time of diagnosis. even when comparing tumors with same stage, the prognosis still appears to be worse in adults than in children. 11 have suggested that venous invasion correlates with the children 's prognosis. in cases staged as n0m0v2a (in which thrombus is free in the vena cava, without infiltration of the wall), nephrectomy and unblocking of the abdominal vena cava may achieve remarkably long survival, however tumors of substage v2b (in which the thrombus infiltrates the wall of the vena cava) should be treated as tumors with visceral metastases. due to the rarity of wt in adults, it was agreed that all stages of adult wt warrant aggressive multimodal therapy [surgery, chemotherapy, and radiotherapy ] because of its poor prognosis. the national wilms ' tumor study group (nwtsg) in the united state recommended that all adult patient with favorable histology should be treated with stage - appropriate combined therapy, as is done for wt in children 12. the presented patient died from multiple metastases within 14 months although treated with adjuvant chemotherapy, implying that adult wt has poor prognosis and aggressive multimodal therapy should be scheduled. | wilms tumor (wt) occurs infrequently in adults. even rarer is adult wt with extension by direct intravascular spread into the right side of the heart. the present report describes a partially differentiated wt with intracaval and intracardiac extension in a 54-year - old man. the morphologic and immunohistochemical findings confirmed the diagnosis. |
many studies provide evidence pointing to a clear role of vitamin d in the pathogenesis of autoimmune diseases, especially autoimmune thyroid diseases (aitds), as vitamin d deficiency is linked with high levels of anti - thyroid antibodies, abnormal thyroid function, increased thyroid volume, and increased tsh levels [14 ]. nevertheless, at the present time vitamin d levels above 30 ng / ml are considered sufficient and confer protection from bone disease, whereas lower levels induce elevation of parathyroid hormone and are associated with other hazardous systemic effects. however, it is unclear whether low vitamin d levels are closely associated with the development of autoimmune thyroid disease. some case - control studies have suggested that lower serum vitamin d levels or a higher prevalence of vitamin d insufficiency existed in patients with aitds compared with that in healthy controls [1, 2, 6, 7 ]. however, another study reported no significant association between serum vitamin d levels and thyroid autoimmunity. thus, we aim to estimate the associations between vitamin d deficiency and/or insufficiency and autoimmune thyroid diseases, especially to clarify the relationship between vitamin d and serum thyroid autoantibody, in a relative rather than quantitative manner by comparing levels of vitamin d in a chinese population. from may to june 2013 this study was performed in rmqi of xinjiang province. adults aged 16 years and above who were chinese citizens and lived in current residence for 3 years or longer were selected and invited into our study. those with severe communication problems and acute illness and unwilling to participate were excluded from the study. we excluded participants who had missing lab results (n = 140) and questionnaire data (n = 153) and were younger than 16 years old (n = 4) and other ethnic groups (n = 242). finally, 1714 subjects were enrolled, including han and uyghur. the study protocol was approved by the ethics committee of people 's hospital of xinjiang uyghur autonomous region. informed consent was obtained from all subjects before data collection. in our study site, all the data collection was performed by the same staff group from department of endocrinology in people 's hospital of xinjiang uyghur autonomous region. they were trained according to a standard protocol that made them familiar with the specific tools and methods used. venous blood samples were drawn after an overnight fast of at least 8 h and centrifuged on the spot after collection. serum 25(oh)d levels and thyroid parameters, serum tsh, and the levels of tgab and tpoab were measured with a roche electrochemiluminometric analyzer (e601), with an interassay variance of 35 iu / ml and/or tgab of > 116 iu / ml were considered autoantibody positivity (roche). continuous variables are presented as means standard deviation for continuous normally distributed variables and median (interquartile range) for the nonnormally distributed variables. student 's t - test and mann - whitney u test were used for comparison of mean values between groups. linear regression analysis was used to examine the relationship between log - transformed tgab / tpoab titer and age, ethnicity, 25(oh)d, and other biochemical variables. all calculations were performed using spss 19.0 for windows (chicago, il, usa). a probability (p) value of < 0.05 was considered statistically significant for all tests. a total of 1,714 subjects (969 han and 745 uyghur) including 1,197 females (652 han and 545 uyghur) and 517 males (317 han and 200 uyghur) were enrolled in this study. the mean level of total serum 25(oh)d was 16.55 8.53 ng / ml. vitamin d deficiency and insufficiency were noted in 1250 (72.9%) and 344 (20.1%) subjects, respectively. females had higher serum tsh levels and tpoab and tgab titres but lower serum 25(oh)d levels than males (p < 0.05) (table 1). ethnic subgroup studies showed that uyghurs had higher tpoab and tgab titres but lower serum tsh and 25(oh)d levels compared to hans (p 0.001). han subjects were significantly older than uyghurs (48.34 15.46 versus 43.78 12.81 years, p < 0.01). vitamin d insufficiency was noted in 28.3% of han and 9.3% of uyghur residents and vitamin d deficiency was seen in 61.6% of hans versus 87.6% of uyghurs. as shown in figure 2, uyghurs had a lower mean 25(oh)d level compared to hans (19.40 8.37 versus 12.84 7.23, p < 0.001). in the ethnic han subgroup, females had a lower mean 25(oh)d level compared to males (19.18 8.88 versus 19.86 7.18 ng / ml, p = 0.002) ; similarly uyghur females had lower 25(oh)d levels than uyghur males (11.67 7.28 versus 16.04 6.01 ng / ml, p < 0.001). in the present study, 240 (14.0%) subjects were positive for serum tpoab and a total of 106 (6.2%) subjects were positive for serum tgab, of which 209 (12.2%) were positive for both serum tpoab and serum tgab. there were a significant high proportion of females compared to males in tpo(+), tg(+), and tpo(+)tg(+) groups (73.3% versus 26.7%, p = 0.203 ; 85.8% versus 14.2%, p < 0.001 ; 86.6% versus 13.4%, p < 0.001 ; resp.), while the proportion of uyghurs was higher as compared to hans (p < 0.05) only in the tg(+) group but not in the tpo(+) and tpo(+)tg(+) groups. tgab(+) group had a lower mean 25(oh)d level compared to the tgab() group (p < 0.01). the prevalence of vitamin d deficiency in tgab positive subjects was higher than that observed in tgab negative subjects, while no statistical difference was found (78.3% versus 72.6%, p = 0.199). the serum tsh levels in the tpo(+)tg(+) group were significantly higher than those in the tpo(+) group and tg(+) group (p < 0.01). however, there was no significant difference between tpo(+) group and tg(+) group (table 2). to account for the probable interaction between vitamin d status and anti - thyroid antibody level, it was found that higher tgab titres were associated with lower 25(oh)d levels independent of age, ethnicity, and tsh levels in females (beta = 0.121, p = 0.014, table 3) but not in males. linear regression analysis showed a weak relationship between tpoab titres and serum 25(oh)d levels independent of age, ethnicity, and tsh levels (males, beta = 0.005, p = 0.002 ; females, beta = 0.006, p = 0.000, table 4). age, tsh, and thyroid - globulin antibody titres, but not 25(oh)d or ethnicity, were associated with tpoab titres in men and women. given the findings of lower serum 25(oh)d levels in female subjects in the tgab(+) group, we evaluated the adjusted odds ratios (ors) for women using multivariate logistic regression model. the multivariate analysis using logistic regression revealed that 25(oh)d levels were an independent determinant of the presence of tgab (odds ratio : 1.156, 95% confidence interval : 1.0041.331, p = 0.043 ; see table 5) after adjusting for age, gender, and ethnicity. in this analysis, in the current study, we found that a low circulating 25(oh)d level was significantly associated with a higher risk of prevalent aitds in females. to the best of our knowledge, this is the first study to investigate the association between circulating 25(oh)d and anti - thyroid antibodies in community based men and women from xinjiang province of china. according to our study performed, the mean serum vitamin d level in the xinjiang population is 16.55 8.53 ng / ml, and the prevalence of vitamin d deficiency is 72.9%. this prevalence is remarkably high in the uyghur population, especially in uyghur females compared to males. wearing covered clothing the fact that vitamin d deficiency is predominantly found in uyghur females signifies a certain association between these two conditions ; it seems that the lower 25(oh)d level in uyghurs is due to headscarves and clothing that covers the neck, arms, and legs. in contrast, han populations are more mindful of their health and often exercise outdoors before and after work and with ordinary clothing, thereby increasing their sunshine exposure. lower vitamin d levels have been found in several autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, type 1 diabetes mellitus, multiple sclerosis, inflammatory bowel diseases, autoimmune thyroid diseases (i.e., hashimoto 's thyroiditis and graves ' disease), and autoimmune gastritis. the mechanisms underlying the link between vitd and autoimmunity are not completely understood but probably are associated with its anti - inflammatory and immunomodulatory functions, as well as the presence of vdrs on most immune cells [10, 11 ]. aitds are multifactorial diseases in which autoimmunity plays a fundamental role with infiltration of the gland by t- and b - cells and production of specific autoantibodies, reactive to thyroid antigens [antithyroglobulin, anti - thyroid peroxidase (tpoab), and anti - tsh receptor (trab) ].. reported, for the first time, a link between vitamin d deficiency and the presence of anti - thyroid antibodies. in recent years, evidence has emerged pointing to an involvement of vitamin d in the development of autoimmune thyroid disease. the immune modulator properties of vitamin d are ascribed to its effect on cells of the innate and adaptive systems, including macrophages, dendritic cells, and t and b lymphocytes, all of which harbor vdr. recently, several genetic studies have demonstrated an association between thyroid autoimmunity susceptibility and gene polymorphisms of numerous proteins and enzymes that are associated with vitd functions, including vdr, dbp, cyp27b1, and cyp2r1 [11, 12, 14 ]. activated vitamin d modulates autoimmune reactions by regulating t lymphocytes to inhibit both the production and activity of cytokines. vitamin d directly regulates t lymphocyte functions by inhibiting the proliferation of th1 cells and increasing the number of th2 cells. vitamin d also suppresses the production of il-2, il-5, ifn - c, and tnf - a and increases the production of il-4 and transforming growth factor in th2 cells. there are no further experimental studies to reveal whether there is a casual relationship and what are the mechanisms between low serum vitamin d and anti - thyroid antibody presence. hence, from the results of our study, we supposed that vitamin d may enhance the responsiveness in thyroid autoimmune reaction which induced tgab secretion of thyrocytes. in the present study, the prevalence of vitamin d deficiency (78.3%) and insufficiency (20%) is higher in tgab positive subjects. in addition, we explored the probable interaction between vitamin d status and thyroid - globulin antibodies in female xinjiang chinese population. similar to the study in other populations, it has been observed that women have higher serum antithyroglobulin antibody (tgab) levels but lower serum 25(oh)d levels than men. it is reported that vitamin d deficiency is more closely related to anti - thyroid antibody titer rather than thyroid function itself in humans. our study not only supports the existing argument on the association between vitamin d and aitds but also further solidifies the association by revealing that 25(oh)d has a statistical correlation with tgab presence. our current results suggest a possible crosstalk between vitamin d and x chromosome in the development of aitds. the association of lower vitamin d levels with the higher prevalence of tgab positivity was found to be significant only in women and not in men, which is likely to more accurately reflect the mean reference value of serum 25(oh)d and the relationship with tgab. aitds have a female : male ratio of approximately 9 : 1. there is also some studies that provide strong evidence for female predominance and x chromosome involvement in aitds, such as the finding that the x chromosome contains a considerable number of sex and immune - related genes that are essential in determining sex hormone levels and more importantly in immune tolerance, such as ar, il-2 receptor gamma chain, cd40 ligand, and foxp3 [2022 ]. an alternative explanation has been recently proposed, that is, an enhanced skewed xci in peripheral blood cells of female patients with autoimmune diseases [2326 ]. results from our study showed a significant high proportion of women in tg(+) group, which is consistent with previous studies, making better understanding of aitds occurrence risks from the perspective of sex determination genetics in females compared to males. the x chromosome has been a recurrently suspicious contributor to disease genetics, and many reports provide strong evidence that the prevalence of vitamin d deficiency or insufficiency was more severe in female individuals. further clinical and experimental investigations of the effect of vitamin d supplementation on thyroid antibodies, particularly in female individuals, are needed to elucidate an effect of vitamin d on either the thyrocytes or the thyroid follicular epithelium and genetic differences that might provide insight into this relationship. most of the studies supported the inverse relation between low levels of 25(oh)d and autoimmune disease [1012 ]. interestingly, in our study multivariate logistic regression analysis revealed that 25(oh)d was an independent risk factor of tgab. we suppose that this result of the study indicated that both low and high levels of vitamin d may result in an increased prevalence of autoimmune thyroid diseases. some studies have investigated the association between mortality and higher levels of 25(oh)d and found both high and low levels of 25(oh)d were associated with increased risk of overall mortality [27, 28 ]. suggested a u - shaped relationship between serum 25(oh)d concentration and the frequency of certain pathologies. unfortunately, the appropriate range for vitamin d that will be safe and sufficient to modulate immunological homeostasis is yet to be determined. it is generally agreed that high serum 25(oh)d levels are associated with low circulating tsh. recently, population - based studies have reported that high vitamin d status in younger individuals and middle - aged and elderly males is associated with low circulating thyroid - stimulating hormone (tsh) [3, 30 ]. however, our study did not find a link between vitamin d status and thyrotropin after controlling for age, gender, ethnicity, and thyroid autoantibody. the lack of statistical significance may be due to the relatively small number of subjects, especially after subgroup analysis in different ethnics or genders. our study is limited by the fact that we did not record several factors affecting sun exposure, such as outdoor activity, difference in the use of sunscreens, and vacation activities. in our study, serum 25(oh)d was measured at the end of spring season in the xinjiang region of china ; the average amount of sunshine for may and june was up to 13 - 14 hours. however, our findings strongly suggest that we likely underestimate vitamin d deficiency in the population. another limitation is the lack of information on differences in socioeconomic status between hans and uyghurs in this cohort. besides, because of the cross - sectional nature of the present study, the causative effect of vitamin d on serum anti - thyroid - globulin antibody titres could not be readily determined. 25(oh)d status was much worse among uyghur (especially in females) populations, the prevalence of vitamin d deficiency and insufficiency was higher in tgab positive individuals, and low serum 25(oh)d was associated with tgab presence only in female subjects. taken together, these data suggest a pathogenic relationship between vitamin d status and aitds. future studies are warranted to clarify the potential contribution of the low serum vitamin d to the development of autoimmune thyroid diseases. | objectives. some evidence has pointed out that vitamin d plays a significant role in reducing the incidence of autoimmune diseases, especially autoimmune thyroid diseases. the authors aimed to examine the relationship between circulating 25-hydroxyvitamin d and thyroid autoantibody in a population - based health survey of xinjiang chinese population. subjects and methods. a total of 1714 chinese adults were analyzed. 25(oh)d, anti - thyroid antibodies, and thyroid function were measured. results. the prevalence of vitamin d insufficiency was 28.3% in hans and 9.3% in uyghurs, and the prevalence of vitamin d deficiency was 61.6% in hans and 87.6% in uyghurs. overall prevalence of tgab positivity was 6.2% (0.9% males ; 5.3% females). in female subjects, mean serum 25(oh)d levels were significantly lower in hans and uyghurs compared with males, and the difference was statistically significant. importantly, after adjusting for age and ethnicity, a negative correlation (r = 0.121, p = 0.014) was recognized between 25(oh)d and tgab levels only in female subjects. conclusion. vitamin d insufficiency and deficiency are prevalent among chinese adults. low serum 25(oh)d is related to the presence of tgab in females. the causal effect of low vitamin d level on thyroid autoimmunity should be studied further more. |
asthma and copd have been regarded as two distinct disease entities that often overlap.13 asthma and copd overlap has a prevalence of 15%20% in patients with obstructive airway disease (asthma or copd)46 and is associated with significant health status impairment,4 increased exacerbations,5 and increased hospitalizations.6 in 2014, the global initiative for asthma (gina) and the global initiative for chronic obstructive lung disease (gold) issued a joint document describing that asthma - copd overlap syndrome (acos) is characterized by persistent airflow limitation with several features usually associated with both asthma and copd.7 the clinical phenotypes and underlying mechanisms of acos have attracted interest during recent years. however, acos remains somewhat controversial, and there is no consensus on the best definition of acos.8,9 the clinical phenotypes, including biomarkers, of acos remain elusive. inflammatory biomarkers, such as fractional exhaled nitric oxide (feno), blood eosinophils, and allergen - specific immunoglobulin e (ige), are sometimes used to distinguish between asthma and copd.7 typically, asthma is characterised by inflammation predominantly involving eosinophils, whereas copd is characterized by inflammation by neutrophils.1,2 feno and blood eosinophil count have been considered as biomarkers of local and systemic eosinophilic inflammation, which increase in patients with asthma.10,11 total serum ige and antigen - specific ige levels are also elevated in those with allergic asthma.12 however, the significance of these inflammatory biomarkers in diagnosis of acos remains unclear. we conducted a cross - sectional study to 1) analyze prospectively collected data from a japanese copd registry and 2) compare copd patients with and without acos, focusing on inflammatory biomarkers, to investigate clinical phenotypes of acos. we conducted a cross - sectional study to analyze the prospectively collected data of consecutive scheduled visits or newly registered patients from the ishinomaki copd network (icon) registry13 between may 2015 and january 2016. briefly, icon is a regional medical liaison system aimed at comprehensive care of patients with copd and has a multi - center interdisciplinary collaboration with health care providers in ishinomaki, japan. patients registered in icon are regularly treated by general practitioners in ishinomaki and surrounding cities, and receive scheduled examinations and education at the japanese red cross ishinomaki hospital (a 452-bed tertiary community hospital in ishinomaki) every 612 months. this study is part of an ongoing copd cohort study registered with the university hospital information network clinical trials registry (identifier : umin000017376). all patients provided written informed consent, and the study was approved by the ethics committee of japanese red cross ishinomaki hospital (approval number : 12 - 14 - 1). patients with stable copd, who were aged 4090 years and former smokers with a smoking history of at least 10 pack - years, were included in this study. all patients were diagnosed with copd according to gold criteria.14 persistent airflow limitation, defined as postbronchodilator forced expiratory volume in 1 second (fev1)/forced vital capacity (fvc) ratio 12% and 200 ml from baseline, or increase in fev1 > 12% and 200 ml (or in peak expiratory flow > 20%) from baseline after 4 weeks of anti - inflammatory treatment, in the absence of respiratory infections. we evaluated these diagnostic features based on the medical record review prior to biomarker measurement. sociodemographic characteristics, smoking status, presence of rhinitis, and maintenance treatments were recorded for each patient. dyspnea was evaluated by using the modified medical research council dyspnea scale.16 copd - related health status was assessed by using the copd assessment test.17 frequency of severe exacerbations requiring hospitalization in the previous year was evaluated based on direct patient interviews, diaries kept by patients or caregivers, and medical record review. pulmonary function tests were conducted by a well - trained technician following the guidelines under a stable condition.18 severity of airflow limitation was classified in accordance with gold staging.14 feno level was measured by using the niox mino device (aerocrine, morrisville, nc, usa) according to the standard operating procedures recommended by the manufacturer. feno level was classified as follows : normal, 50 ppb.19 blood samples were obtained to determine blood eosinophil count and percentage and total serum ige level. presence of antigen - specific ige was determined by using the immunocap phadiatop test (thermo fisher scientific, waltham, ma, usa), an in vitro assay for antigen - specific ige antibodies to common inhalant allergens. antigen - specific ige was considered to be present when the results were positive for at least one of the following : house dust mite, cat, dog, alternaria tenuis, aspergillus fumigatus, or ragweed. the cutoff value of high blood eosinophil count was set at 500 cells / mm,3,20 while the cutoff value of total serum ige level was set at 173 iu / ml according to the reference range of the clinical laboratory at japanese red cross ishinomaki hospital. data are shown as median (interquartile range) or mean standard deviation unless otherwise specified. differences between groups were assessed by using the student s t - test or mann whitney u - test for continuous variables, while associations between categorical variables were evaluated by using fisher s exact test. the value of total serum ige level was log transformed because the variables were not normally distributed ; the results are expressed as geometric mean values. distribution of gold staging between patients with and without acos was analyzed by using the kruskal receiver operating characteristic (roc) curves were plotted in order to estimate the diagnostic cutoff values. all statistical analyses were performed by using ezr (saitama medical center, jichi medical university, saitama, japan), a graphical user interface for r (the r foundation for statistical computing, vienna, austria).21 p - values 12% and 200 ml from baseline, or increase in fev1 > 12% and 200 ml (or in peak expiratory flow > 20%) from baseline after 4 weeks of anti - inflammatory treatment, in the absence of respiratory infections. we evaluated these diagnostic features based on the medical record review prior to biomarker measurement. sociodemographic characteristics, smoking status, presence of rhinitis, and maintenance treatments were recorded for each patient. dyspnea was evaluated by using the modified medical research council dyspnea scale.16 copd - related health status was assessed by using the copd assessment test.17 frequency of severe exacerbations requiring hospitalization in the previous year was evaluated based on direct patient interviews, diaries kept by patients or caregivers, and medical record review. pulmonary function tests were conducted by a well - trained technician following the guidelines under a stable condition.18 severity of airflow limitation was classified in accordance with gold staging.14 feno level was measured by using the niox mino device (aerocrine, morrisville, nc, usa) according to the standard operating procedures recommended by the manufacturer. feno level was classified as follows : normal, 50 ppb.19 blood samples were obtained to determine blood eosinophil count and percentage and total serum ige level. presence of antigen - specific ige was determined by using the immunocap phadiatop test (thermo fisher scientific, waltham, ma, usa), an in vitro assay for antigen - specific ige antibodies to common inhalant allergens. antigen - specific ige was considered to be present when the results were positive for at least one of the following : house dust mite, cat, dog, alternaria tenuis, aspergillus fumigatus, or ragweed. the cutoff value of high blood eosinophil count was set at 500 cells / mm,3,20 while the cutoff value of total serum ige level was set at 173 iu / ml according to the reference range of the clinical laboratory at japanese red cross ishinomaki hospital. data are shown as median (interquartile range) or mean standard deviation unless otherwise specified. differences between groups were assessed by using the student s t - test or mann whitney u - test for continuous variables, while associations between categorical variables were evaluated by using fisher s exact test. the value of total serum ige level was log transformed because the variables were not normally distributed ; the results are expressed as geometric mean values. distribution of gold staging between patients with and without acos was analyzed by using the kruskal receiver operating characteristic (roc) curves were plotted in order to estimate the diagnostic cutoff values. all statistical analyses were performed by using ezr (saitama medical center, jichi medical university, saitama, japan), a graphical user interface for r (the r foundation for statistical computing, vienna, austria).21 p - values 12% and 400 ml from baseline was observed in four patients (10.8%). patients with acos tended to be younger (p=0.003) and have a shorter smoking history (p=0.019). sex, bmi, spirometric results, dyspnea, copd - related health status, and incidence of severe exacerbations requiring hospitalization in the previous year were not significantly different between the groups. patients with acos tended to use more respiratory medications, especially inhaled corticosteroids (ics) and theophylline, compared with patients without acos (86.5% vs 36.8%, 27.0% vs 9.5%, respectively ; both p 2%) was observed in those with acos (p=0.014) (table 3). no significant association with ics use and blood eosinophil count or percentage was observed in patients with (p=0.42 and p=0.78, respectively) and without acos (p=0.11 and p=0.61, respectively). total serum ige level was significantly higher in patients with acos than in those without acos (2989 iu / ml vs 451 iu / ml, p=0.004) (figure 1d). presence of antigen - specific ige also was observed more frequently in those with acos (56.8% vs 28.6%, p=0.001). roc curve analysis demonstrated that 23 ppb was the best diagnostic cutoff value of feno level for acos (area under the curve [auc ] 0.74 ; 95% confidence interval [ci ] 0.630.84). sensitivity and specificity of 23 ppb for the diagnosis of acos were 73.0% and 68.2%, respectively. in addition, roc curve analysis showed that 156.2/mm was the best diagnostic cutoff value of eosinophil count (auc 0.70 ; 95% ci 0.610.78) and that 434 iu / ml was the best diagnostic cutoff value of total serum ige level (auc 0.64 ; 95% ci 0.540.75). sensitivity and specificity of 156.2/mm and 434 iu / ml for the diagnosis of acos were 83.8% and 49.5%, and 45.9% and 85.9%, respectively. combination of feno > 23 ppb and ige > 434 iu / ml showed 94.1% specificity, 37.8% sensitivity, 51.9% positive predictive value (ppv), and 90.0% negative predictive value (npv). combination of feno > 23 ppb and eosinophil count > 156.2/mm showed 85.5% specificity, 59.5% sensitivity, 40.7% ppv, and 92.6% npv. combination of ige > 434 iu / ml and eosinophil count > 156.2/mm showed 92.3% specificity, 40.5% sensitivity, 46.9% ppv, and 90.2% npv. triple combination (feno > 23 ppb, ige > 434 iu / ml, and eosinophil count > 156.2/mm) showed 96.8% specificity, 37.8% sensitivity, 66.7% ppv, and 90.3% npv. a total of 334 consecutive patients with copd were recruited, and 257 eligible patients (236 males, 21 females ; median age, 75 years) were identified. characteristics of the study participants are shown in table 1. among the 257 patients with copd, 48 (18.7%) had a history of variable respiratory symptoms, 57 (22.2%) had variable expiratory airflow limitation, and 37 (14.4%) had both features ; these 37 patients were diagnosed as having acos. among the 37 patients with acos, no patient had respiratory symptoms in childhood, but two (5.4%) had symptoms before the age of 40 years. marked reversibility with postbronchodilator increase in fev1 > 12% and 400 ml from baseline was observed in four patients (10.8%). patients with acos tended to be younger (p=0.003) and have a shorter smoking history (p=0.019). sex, bmi, spirometric results, dyspnea, copd - related health status, and incidence of severe exacerbations requiring hospitalization in the previous year were not significantly different between the groups. patients with acos tended to use more respiratory medications, especially inhaled corticosteroids (ics) and theophylline, compared with patients without acos (86.5% vs 36.8%, 27.0% vs 9.5%, respectively ; both p 2%) was observed in those with acos (p=0.014) (table 3). no significant association with ics use and blood eosinophil count or percentage was observed in patients with (p=0.42 and p=0.78, respectively) and without acos (p=0.11 and p=0.61, respectively). total serum ige level was significantly higher in patients with acos than in those without acos (2989 iu / ml vs 451 iu / ml, p=0.004) (figure 1d). presence of antigen - specific ige also was observed more frequently in those with acos (56.8% vs 28.6%, p=0.001). roc curve analysis demonstrated that 23 ppb was the best diagnostic cutoff value of feno level for acos (area under the curve [auc ] 0.74 ; 95% confidence interval [ci ] 0.630.84). sensitivity and specificity of 23 ppb for the diagnosis of acos were 73.0% and 68.2%, respectively. in addition, roc curve analysis showed that 156.2/mm was the best diagnostic cutoff value of eosinophil count (auc 0.70 ; 95% ci 0.610.78) and that 434 iu / ml was the best diagnostic cutoff value of total serum ige level (auc 0.64 ; 95% ci 0.540.75). sensitivity and specificity of 156.2/mm and 434 iu / ml for the diagnosis of acos were 83.8% and 49.5%, and 45.9% and 85.9%, respectively. combination of feno > 23 ppb and ige > 434 iu / ml showed 94.1% specificity, 37.8% sensitivity, 51.9% positive predictive value (ppv), and 90.0% negative predictive value (npv). combination of feno > 23 ppb and eosinophil count > 156.2/mm showed 85.5% specificity, 59.5% sensitivity, 40.7% ppv, and 92.6% npv. combination of ige > 434 iu / ml and eosinophil count > 156.2/mm showed 92.3% specificity, 40.5% sensitivity, 46.9% ppv, and 90.2% npv. triple combination (feno > 23 ppb, ige > 434 iu / ml, and eosinophil count > 156.2/mm) showed 96.8% specificity, 37.8% sensitivity, 66.7% ppv, and 90.3% npv. we identified patients with acos presenting features of asthma, including both variable respiratory symptoms and variable expiratory airflow limitation, in a copd outpatient cohort, and demonstrated that airway inflammatory biomarkers, including feno, blood eosinophil count, and ige, are increased in those with acos. the results of the present study demonstrated that inflammatory biomarkers can be used to support the diagnosis of acos. previous studies have attempted to identify the acos phenotypes by using various criteria.8 thus, there is an increased awareness of the importance of recognizing acos by using biomarkers.2225 recent studies reported that increased feno levels were identified in a subset of patients with copd.23,24 our findings are congruent with the observation by donohue, who reported increased feno levels in patients previously clinically diagnosed with both copd and asthma.23 in addition, the findings of our study are compatible with those of kitaguchi, who observed increased blood and sputum eosinophil counts in copd patients with asthmatic symptoms.25 the results of this study indicated that inflammatory biomarkers provide additional diagnostic information for acos. although the sensitivity and specificity of these biomarkers were relatively low, combinations of these biomarkers showed high specificity for acos diagnosis. total serum ige level was not affected by ics therapy ; however, treatment with ics was reported to decrease feno level26,27 and blood eosinophil count.28 half of the patients in this study used ics. furthermore, airway eosinophilia is not exclusive to asthma and is present in patients with copd.29 therefore, we propose that overconfidence in such biomarkers may lead to diagnostic errors. our finding that 14% of patients with copd fulfilled the acos phenotype is in line with the results of well - characterized copd cohorts, such as the copdgene study5 and chain cohort.30 in this study, patients with acos tended to be younger, have a shorter smoking history, and use more respiratory medications, which is compatible with previous studies.46,30,31 however, discrepancies between this and previous studies may have been caused by the older age of the patients in this study, since the participants were enrolled from a regional copd registry in japan, one of the most rapidly aging societies in the world.13 to date, a standardized treatment for acos has not been established because patients with asthma and copd overlap have been excluded from randomized controlled trials. treatment with ics is provisionally recommended by the gina / gold joint document.7 high feno level32 and blood eosinophilia33,34 have been identified as surrogate markers of the response to steroids in patients with copd. the findings of our study support the view that ics treatment may be beneficial in patients with acos and encourage the development of a standardized treatment for acos. the primary strength of our study is that we applied precise diagnostic criteria to identify patients with acos. previous reports included patients with a medical history of physician - diagnosed asthma as the main inclusion criterion. in this study, we confirmed the diagnosis in all patients, with or without features of asthma, according to both asthmatic symptoms and documented reversibility, because we found that some patients had received an inappropriate diagnosis of asthma by a nonrespiratory specialist. in addition, we excluded patients who had characteristics possibly affecting biomarker measurement, including current smokers and oral corticosteroid users. a potential weakness of our study is that the results were not confirmed by a validation cohort. thus, the results can not be generalized directly to a different setting, such as an asthma cohort. further studies are required to evaluate the diagnostic value of inflammatory biomarkers in untreated patients. the results of this study provide evidence that inflammatory biomarkers, including feno, blood eosinophil count, and ige, can be used to support the diagnosis of acos. the results of our study may guide toward better recognition of acos and encourage development of specific interventions for acos. | backgroundthe clinical phenotypes and underlying mechanisms of asthma - copd overlap syndrome (acos) remain elusive. this study aimed to investigate a comparison of copd patients with and without acos, focusing on inflammatory biomarkers, in an outpatient copd cohort.methodswe conducted a cross - sectional study analyzing prospectively collected data from the ishinomaki copd network registry. all participants were diagnosed with copd, confirmed by using spirometry, and were aged 4090 years and former smokers. patients with features of asthma including both variable respiratory symptoms and variable expiratory airflow limitation were identified and defined as having acos. then, the inflammatory biomarkers such as fractional exhaled nitric oxide level, blood eosinophil count and percentage, total immunoglobulin e (ige) level, and presence of antigen - specific ige were evaluated.resultsa total of 257 patients with copd were identified, including 37 (14.4%) with acos. patients with acos tended to be younger, have a shorter smoking history, and use more respiratory medications, especially inhaled corticosteroids and theophylline. mean fractional exhaled nitric oxide level was significantly higher in those with acos than in those without acos (38.5 parts per billion [ppb ] vs 20.3 ppb, p<0.001). blood eosinophil count and percentage were significantly increased in those with acos (295/mm3 vs 212/mm3, p=0.032 ; 4.7% vs 3.2%, p=0.003, respectively). total ige level was also significantly higher, and presence of antigen - specific ige was observed more frequently in patients with acos. receiver operating characteristic curve analysis indicated that the sensitivity and specificity of these biomarkers were relatively low, but combinations of these biomarkers showed high specificity for acos diagnosis.conclusionthese results provide evidence that these inflammatory biomarkers can be used to support the diagnosis of acos. |
porcine parvovirus (ppv), first isolated from sows in germany, has been found to occur worldwide [24 ]. this syndrome is characterized by stillbirth, mummified fetuses, early embryonic and fetal death, delayed return to estrus, and infertility (abbreviated as smedi) [5, 6 ]. ppv is also shown to be an agent able to increase the effects of porcine circovirus type 2 infection in the clinical course of postweaning multisystemic wasting syndrome, which is a significant disease in global swine production. five different groups of porcine parvoviruses (ppv) have been identified : classic ppv (ppv1), ppv2, ppv3 (known as porcine parv4, hokovirus, or partetravirus), and ppv4 and porcine bocaviruses, which all have substantial genetic divergence [912 ]. recently, a new parvovirus provisionally proposed to be named as ppv5 was discovered in the united states. classic ppv has one serotype subdivided into four clinical genotypes (biotypes) according to their pathogenicity. the nadl-8 strain can cause viremia and crosses the placenta to infect fetuses, leading to fetus death. in contrast, nonpathogenic nadl-2 strain is currently widely used as an attenuated vaccine and causes only limited viremia without crossing the placental barrier in experimental infections. the other two groups are the kresse and iaf - a83 strains, which are associated with dermatitis and enteric diseases, respectively. this group of viruses also infects rodents and carnivores and belongs to the parvoviridae subfamily within the parvoviridae family. ppv has a negative, single - stranded dna of about 5 kb with distinct hairpin termini. the genome contains two major open reading frames (orfs), each located in the same frame of the complementary strand., vp2, and vp3 are encoded in orf2. vp1 and vp2 are translated from differently spliced rnas, whereas vp3 is formed by proteolytic cleavage of vp2 [17, 18 ]. vp2 consists of an eight - stranded antiparallel -barrel motif with 4 large loops between -strands. these loops are shown to possess many b - cell epitopes and can tolerate insertions [19, 20 ] that make ppv vp2 a potential antigen carrier and play a key role in ppv diagnosis and immune prophylaxis. the structure of ppv capsid composed of baculovirus system generated recombinant vp2 is available in 3.5 resolution (pdb accession number 1k3v). immunogenic major capsid protein vp2 of ppv has been synthesized in several expression systems including bacteria [21, 23 ]. ppv vp2 protein expressed using the baculovirus expression vector system was shown to assemble into virus - like particles (vlps) similar in size and morphology to the original virions. vlps generated in baculovirus system exhibit positive immunoreactivity for ppv and are used in most commercial elisa tests. most recently, immunogenic ppv vp2 protein was synthesized in yeast pichia pastoris the formation of recombinant antigenic human parvovirus capsid protein vlps in s. cerevisiae has been recently demonstrated [28, 29 ]. regarding costs, yield, and ease of handling, vlp production in yeast represents an alternative to the recombinant baculovirus expression system, which is so far the dominating source of vp2-derived vlps of parvoviruses [27, 28 ]. in the current study, we have generated the ppv vp2 protein as vlps in s. cerevisiae expression system, demonstated their structural and antigenic similarity with viral capsids and developed a new indirect igg elisa based on the use of ppv vp2-derived vlps. moreover, we have developed a panel of ppv vp2 protein - specific monoclonal antibodies and demonstrated their reactivity with ppv - infected cells. one hundred and eighty - three swine serum samples from farms in lithuania (n = 160), romania (n = 14), and ukraine (n = 13) were collected in years 20082010 and used in this study. viral nucleic acids (nas) were extracted from porcine kidney cell culture pk-15 (atcc ccl-33) infected with porcine parvovirus strain nadl-2. nas were extracted using commercial qiaamp ultrasens virus kit (qiagen gmbh, hilden, germany) following the manufacturer 's manual and stored at 70c until use. the ppv vp2 gene was amplified using high fidelity enzyme mix (fermentas / thermo fisher scientific, vilnius, lithuania) directly from extracted nas using the following pair of primers (idt, munich, germany) : ppv - vp2-f 5-tctactagtacaatgagtgaaaatgtggaacaa-3 ppv - vp2-r 5-gagactagtctagtataattttcttggtataagt-3 ppv - vp2-f 5-tctactagtacaatgagtgaaaatgtggaacaa-3 ppv - vp2-r 5-gagactagtctagtataattttcttggtataagt-3 the primers used for amplification incorporated bcui site (underlined) for subcloning into the yeast vector pfx7. the thermal cycle conditions were the following : initial denaturation for 3 min at 95c, followed by 30 cycles of 94c for 1 min, 55c for 1 min, and 72c for 2 min, and then the final elongation at 72c for 10 min. the pcr amplification product was digested with bcui and inserted into xbai - linearized and dephosphorylated yeast expression plasmid pfx7 under control of yeast gal1 - 10 promoter and confirmed by pcr and subsequent dna sequence analysis. the nucleotide sequence of the amplified ppv vp2 was compared with those in genbank using the basic local alignment search tool (blast). all dna manipulations were performed according to standard procedures using enzymes and kits from fermentas / thermo fisher scientific. recombinant constructs were screened in escherichia coli dh5 f. recombinant construct containing ppv vp2 sequence was screened in e. coli dh5f cells. saccharomyces cerevisiae haploid strain ah22 mata (leu2 his4 pep4) was used for the expression of ppv vp2 protein. selection of yeast transformants resistant to formaldehyde was carried out on the yepd (1% yeast extract, 2% peptone, 2% dextrose, difco, sparks, md, usa) agar supplemented with 5 mm formaldehyde. s. cerevisiae transformants were grown in yepd medium supplemented with 5 mm formaldehyde or in yepg induction medium (1% yeast extract, 2% peptone, 2% galactose, difco). cultivation of transformed yeast cells, expression and purification of ppv vp2 was performed as previously described [32, 33 ]. after purification, the total protein concentration was determined by the bradford assay (roth, karlsruhe, germany) with bovine serum albumin (bsa) used as a standard. the samples were boiled in a reducing sample buffer and separated in gel electrophoresis in sds - tris - glycine buffer. proteins were visualized by staining with coomassie brilliant blue (sigma - aldrich co., st. blotting, proteins were electrotransferred to immobilon p membrane (millipore, bedford, ma, usa) as described by sambrook and russell. the membranes were blocked with 5% milk in phosphate buffered saline (pbs) for 2 h. the blocking solution was removed and the blots were incubated with the mabs against ppv vp2 protein (undiluted hybridoma supernatants). secondary antibodies conjugated to horseradish peroxidase (hrp) (bio - rad, hercules, ca, usa) were used for detection of specific antibody binding. the blots were stained with 3,3,5,5-tetramethylbenzidine (tmb) ready - to - use chromogenic substrate (clinical science products inc., mansfield, ma, usa). after purification by cscl ultracentrifugation, suspension of the recombinant ppv vp2 protein was placed on 400-mesh carbon coated copper grids (agar scientific, stansted, uk). the protein samples were stained with 2% aqueous uranyl acetate solution (reachim, moscow, russia) and examined with a morgagni-268 electron microscope (fei, eindhoven, the netherlands). porcine serum samples were assayed for the presence of anti - ppv antibodies using a commercial ingezim ppv compact kit (ingenasa, madrid, spain). this is an enzymatic assay based on the blocking elisa technique which uses mab specific for porcine parvovirus vp2 protein, and baculovirus expression systems generated recombinant capsid of vp2. two blocking percentage (bp) values were used for result interpretation : samples with bp higher than 30% were considered as positive and samples with bp lower than 25% were considered as negative. polystyrene microtiter plates (nerbe plus, winsen / luhe, germany) were coated with 50 ng per well of recombinant ppv vp2 protein, diluted in 100 l of 0.05 m carbonate - bicarbonate coating buffer (ph 9.6) and incubated overnight at 4c. plates were washed three times with pbst (phosphate buffered saline with 0.05% (v / v) tween 20, (bio - rad, richmond, ca, usa)) and then blocked by the addition of 150 l of blocking buffer per well (1x roti - block, carl roth gmbh & co.) and incubation at room temperature for 1 hour. after blocking, the plates were washed three times with pbst and 100 l aliquots of serum specimens, diluted 1 : 400 in pbst with 1% bsa, were added to the wells. antigen concentration and serum dilution level for this assay were determined by titration to reach optimal conditions for sensitivity and specificity (data not shown). after 2 h of incubation at 37c, the plates were rinsed three times with pbst. hrp - conjugated rabbit anti - pig igg (sigma - aldrich biosciences, seattle, usa) diluted 1 : 30 000 in pbst, containing 1% bsa, were added to the wells in 100 l aliquots and incubated for 1 h at 37c. binding of specific antibodies was visualized by the addition of 100 l / well of tmb substrate (clinical science products inc., mansfield, ma, usa). after 10 min of incubation at the room temperature, the reaction was stopped by adding 100 l / well of 10% sulphuric acid and the optical density (od) was measured at 450 nm (reference filter 620 nm). eight - week - old female balb / c mice (obtained from a breeding colony at the center for innovative medicine, vilnius, lithuania) were immunized at days 0, 28, and 56 by a subcutaneous injection of 50 g of recombinant ppv vp2 protein. for an initial immunization, three days after the final injection, mouse spleen cells were fused with sp2/0-ag 14 mouse myeloma cells using polyethylene glycol 1500 (peg / dmso solution, hybri - max, sigma - aldrich). hybrid cells were selected in growth medium supplemented with hypoxanthine, aminopterin, and thymidine (50hat media supplement, sigma - aldrich). samples of supernatant from wells with viable clones were screened by an indirect elisa (as described above) using goat anti - mouse igg (bio - rad) diluted 1 : 5000 to detect specific antibodies to ppv vp2. hybridomas secreting specific antibodies to ppv vp2 protein were subcloned twice by a limiting dilution assay. hybridoma cells were maintained in complete dulbecco 's modified eagle 's medium (dmem, biochrom, berlin, germany) containing 15% fetal calf serum (biochrom) and antibiotics. antibodies in hybridoma culture supernatants were isotyped using the mouse monoclonal antibody isotyping kit (pierce, thermo scientific) in accordance with the manufacturer 's protocol. the reactivity of the mabs with ppv - infected cells was analyzed by immunofluorescence assay (ifa) using porcine parvovirus fa substrate slides (vmrd, inc., pullman, usa) containing fixed swine testicle cells infected and noninfected with porcine parvovirus strain ky-11. the slides were treated according to the manufacturer 's protocol, incubated with undiluted hybridoma supernatants and developed with fluorescein isothiocyanate- (fitc-) labelled secondary antibody (bd biosciences, franklin lakes, nj, usa). the immunostained slides were observed by fluorescent microscope olympus ix-70 (olympus, tokyo, japan). the gene encoding ppv vp2 was derived from the nucleic acids extract obtained from ppv - infected cell culture. the 1700 bp sequence amplified by pcr was sequenced and confirmed to be identical to vp2 gene from porcine parvovirus strain nadl-2 (genbank entry number nc001718). the gene was cloned into s. cerevisiae expression vector pfx7 under the galactose - inducible promoter. a sds - page analysis of the lysate of induced yeast biomass revealed a major protein band of approximately 64 kda (figure 1, lane 2). no additional protein bands were observed in crude lysates of s. cerevisiae harboring empty yeast vector pfx7 (figure 1, lane 1). after centrifugation of lysates through 30% sucrose cushion, the pellets harboring recombinant proteins were subjected to cscl - gradient centrifugation. cscl gradients revealed recombinant ppv vp2 protein (figure 1, lane 3) in fractions with buoyant density of 1.2861.308 g / ml. in several preparative procedures, the yield of purified recombinant ppv vp2 protein was found to be 8 - 9 mg per liter of induced yeast culture. after cscl gradient purification, the recombinant vp2 protein was dialyzed against pbs and stored at 20c in pbs containing 50% glycerol. typical icosahedral structures of parvoviruses with a diameter of approximately 2530 nm were observed indicating that ppv vp2 protein is self - assembled to vlps (figure 2(a)). vlps of ppv produced in s. cerevisiae expression system were similar to those previously generated in insect cells or native ppv particles. treatment with 25 mm edta or 10 mm egta did not cause the dissociation of recombinant vlps indicating that the assembled structures do not require divalent ions (data not shown). the recombinant ppv vp2-derived vlps were found to be stable when lyophilized and stored at 20c longer than a year and vlps remained intact when resolubilized in pbs as no pentamers or disrupted particles were observed by electron microscopy (figure 2(b)). moreover, the elisa results using freshly prepared and resolubilized ppv vp2 antigen were fully concordant (data not shown). the stability of vlps is crucial to ensure their successful transportation and possible application in the point - of - care tests. therefore, it is successfully employed for the serodiagnostics as well as epidemiological studies of ppv infection. moreover, vp2 protein is the major agent for developing vaccines. to meet the need for stable recombinant vlps of ppv, several expression systems were tested as an alternative to baculovirus expression system that is a major source of the antigen for the market. e. coli, lactobacillus casei, and recently yeast pichia pastoris were reported to have been successfully used for producing ppv vp2 protein, but vlp formation in these expression systems has not been confirmed. to our knowledge, our study provides the first evidence of stable recombinant ppv vp2 vlps not produced in baculovirus expression system. s. cerevisiae expression system has been shown to be efficient in producing antigenic vlps of diagnostic relevance of human parvoviruses [28, 29 ] and porcine circovirus. therefore, it represents a potential system to meet the need for vlp - forming antigens to detect and differentiate a number of newly discovered porcine. ppv vp2 protein - derived vlps generated in s. cerevisiae were used to develop an indirect elisa for the detection of ppv - specific igg antibodies in swine serum specimens. in order to test the antigenic properties of yeast - derived vlps, 187 serum samples were tested using ingezim ppv compact test as a gold standard and further retested with the newly developed indirect igg ppv elisa. both assays were performed in parallel for every serum sample to determine the sensitivity and specificity of the new indirect igg ppv elisa. the cut - off value for the new assay was calculated as the mean od value of the 39 negative sera (identified with the commercial kit) plus 2 standard deviations (x-+2sd) resulting in 95% confidence. therefore, sera with od values above 0.330 were considered positive (n = 129) and those with od value below this cut - off were assessed as negative (n = 58) in the newly developed indirect igg ppv elisa. thirty - eight out of the 39 sera tested as negative with a commercial kit were assessed as negative by the indirect igg ppv elisa. nine out of 137 positive and all 11 doubtful serum samples by ingezim assay showed the od value below the cut - off in the indirect igg ppv elisa and were considered as negative (table 1). thus, the calculated specificity and sensitivity for the new indirect igg ppv elisa were 97.4% (38/39) and 93.4% (128/137), respectively. all 9 false - negative samples of the new assay were weak positive in ingezim kit showing blocking percentage in the 3345% range. all samples above bp equal to 30% were considered positive in this commercial kit. the only false - positive sample in the indirect igg ppv elisa showed od = 0.354 that is just above the cut - off od of 0.330. to obtain more precise estimation of the sensitivity and specificity of the new assay, additional evaluation with more serum samples and alternative assays must be done in the future. alternatively, the precision of the test can be improved using other formats of elisa. in summary, results of the current study are promising to the use of ppv vp2 antigen synthesized in yeast s. cerevisiae in diagnostic kits. purified recombinant ppv vp2 protein was used to immunize mice and generate ppv vp2-specific mabs. after screening and cloning of positive hybridoma clones, six mabs produced by hybridoma clones were of igg1 subtype and the remaining three were found to be of igg2a subtype. all mabs reacted specifically with recombinant ppv vp2 protein in elisa and did not react with other yeast - expressed proteins used as a negative control (table 2). to characterize the nature of the epitopes recognized by the mabs, their reactivity in western blotting was analyzed. the mabs 4f11, 16g11, 25c5, 6d1, and 10a7 recognized sds - denatured ppv vp2 protein in western blotting assay (figure 3, lanes 2). this result indicates that these mabs recognize sds - denatured epitopes of the ppv vp2 protein. the other four mabs (clones 1f8, 16a1, 22g2, and 23a7) did not recognize sds - denatured ppv vp2 protein in western blotting assay (data not shown), suggesting that these mabs recognize conformation - dependent epitopes. the specificities of the mabs were further analyzed by ifa to verify the ability of the mabs to recognize native virion. for this purpose, commercial porcine parvovirus control slides containing virus - infected and noninfected fixed cells were used. none of the mabs reacted with noninfected cells, which confirms the specificity of the assay (figure 4, negative control). both groups of mabs recognizing linear or conformational epitopes reacted with infected cells ; however, only the latter ones produced images with sharp nucleus - shaped patterns. in contrast, the mabs recognizing linear epitopes produced signal outside the nuclei but in lesser intensity (figure 4). this difference could be explained by the possibility that ppv vlps finish their assembly in the nucleus forming conformational epitopes. taking into consideration trimer translocation model for other parvoviruses, conformational epitopes might be available only in intact capsid but not in trimmers or pentamers formed by vp2 protein. this possibility emphasizes the importance of properly assembled vlps to elicit strong immune responses when using recombinant antigens as potential vaccines. further epitope mapping needs to be done to answer if linear epitopes remain accessible on the intact vlp surface or are hidden within the structure. however, our generated mabs represent an attractive tool for studying intracellular ppv infection and capsid formation process. the ppv vp2-derived vlps generated in s. cerevisiae have not been yet tested for a possible use as a vaccine in pigs ; however, considering results on the antigenic structure and the immunogenicity in mice described in this study, this is an attractive alternative to the currently used recombinant ppv vaccines. in previous studies, ppv vp2-derived vlps have been shown to be effective epitope carriers to elicit a strong immune response in mice [41, 42 ]. furthermore, yeast expression system does not require additional contaminant elimination procedure as described for baculovirus expression system for such recombinant subunit vaccine preparation. therefore, ppv vp2-derived vlps generated in yeast s. cerevisiae are a promising platform for new ppv vaccine development. in this study, we have demonstrated that yeast s. cerevisiae is a suitable host for the production of recombinant ppv vp2 protein as stable immunogenic vlps. the recombinant yeast - derived ppv vp2 protein can be employed in an indirect elisa for detection of ppv - specific igg antibodies in swine sera with high specificity and sensitivity. the mabs raised against yeast - derived ppv vp2 vlps recognize virus - infected cells and differentiate conformational and linear epitopes of ppv vp2 protein. | porcine parvovirus (ppv) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. the gene coding for the major capsid protein vp2 of ppv was amplified using viral nucleic acid extract from swine serum and inserted into yeast saccharomyces cerevisiae expression plasmid. recombinant ppv vp2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. electron microscopy analysis of purified ppv vp2 protein revealed the self - assembly of virus - like particles (vlps). nine monoclonal antibodies (mabs) against the recombinant ppv vp2 protein were generated. the specificity of the newly generated mabs was proven by immunofluorescence analysis of ppv - infected cells. indirect igg elisa based on the recombinant vlps for detection of ppv - specific antibodies in swine sera was developed and evaluated. the sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. in conclusion, yeast s. cerevisiae represents a promising expression system for generating recombinant ppv vp2 protein vlps of diagnostic relevance. |
the prevalence of as ranges from 2% to 9% of aged population over 65 years old, and it is increasingly diagnosed in the contemporary era of aging society.1) given no established medical treatment to improve prognosis of as patients, decision to proceed with corrective surgery is crucial. progression of as is usually longitudinally followed using transthoracic echocardiography, and the estimation of effective orifice area of aortic valve (eoaav) is considered the most important parameter to monitor as patients.2) eoaav is calculated by the transvalvular pressure gradient (tpg) and transvalvar flow, and tpg is associated with systemic vascular resistance (svr).3) in the presence of systemic hypertension or peripheral arterial disease, svr increases and this svr alteration might possibly change the parameters that are frequently used to determine as severity. this hypothesis is corroborated by notion that high left ventricular (lv) afterload can result in paradoxical low - flow, low - gradient severe as, highlighting the notion that lv afterload should be considered in terms of assessing severe as.4) furthermore, in contrast to traditional belief that blood pressure was thought to be decreased in case of severe as, recent studies reported that hypertension is common even in severe as patients and one of the important risk factors of significant as.1) therefore, consideration of hypertension is a commonly encountered clinical situation in estimating as severity.5) the aim of this study was to investigate the impact of lv afterload on the assessment of as severity. patients diagnosed as moderate or severe as (eoaav calculated from continuity equation of less than 1.5 cm) were consecutively enrolled from march 2008 to february 2009. exclusion criteria were as follows ; patients with inadequate image quality due to poor echo window, any valvular regurgitation more than mild degree, any valvular stenosis other than aortic valve, severe systemic hypertension [systolic blood pressure (bp) of > 180 mmhg, and/or diastolic bp of > 110 mmhg ], severe lv dysfunction defined as lv ejection fraction (ef) of less than 30%, and diagnosis of acute coronary syndrome within a month. patients with peripheral artery disease with claudication transthoracic echocardiography was performed using commercially available echocardiography machine (vivid 7, ge medical systems, milwaukee, wi, usa). if the patient was confirmed to be eligible, baseline echocardiography was performed after 20 minutes of rest. non - invasive bp and heart rate (hr) were measured just before echocardiography examination. peak early (e) and late (a) diastolic velocities of the mitral inflow were measured using a pulsed - wave doppler at the tip of mitral valve leaflets, and peak early (e ') and late (a ') diastolic mitral annular velocities were acquired at the septal side in the apical 4 chamber view. recording of aortic valve maximal systolic velocity (av vmax) was acquired from multiple views including suprasternal, right parasternal, apical and subcostal transducer positions with a continuous - wave doppler echocardiographic technique, among which only the highest peak velocity was chosen for subsequent analyses. pulsed - wave doppler at lv outflow tract (lvot) was also obtained at the apical 5-chamber view. lv end - systolic wall stress was calculated as follows ; (lv end - systolic wall stress is in g / cm, pes, which stands for lv end - systolic pressure plus maximal pressure gradient of aortic valve, is in mmhg, des and he s are in cm, and 0.34 is the factor for converting pes from mmhg to g / cm.) specially designed pneumatic trousers without the bladder for compression of the lower abdomen were put on the patients as previously described.6) after baseline echocardiography data acquisition, a specially designed compressor inflated the pneumatic trousers up to a pressure of 100 mmhg on both lower extremities and this pressure was maintained throughout the examination. echocardiography was performed including doppler parameters 3 minutes after pneumatic compression of the lower extremities (pcom). doppler measurements of av vmax, transvalvular peak and mean pressure gradient (av peak pg and av mean pg), and time - velocity integral of lv outflow tract (tvilvot) and aortic valve (tviav) were also made. eoaav was calculated by continuity equation as previously described.7) doppler velocity index (tvilvot / tviav) was calculated, as well. lvot area (csalvot) was calculated from the diameter obtained at the level of the aortic annulus during systole with the assumption of a circular shape of lvot. lv stroke volume was calculated by multiplying csalvot by tvilvot as previously described.8) cardiac output (co) was calculated by multiplying sv and hr. systemic vascular resistance (svr) was estimated as : svr = 80 mean bp / co and systemic arterial compliance (sac) was calculated as : sac = sv / (sbp - dbp) numerical data are expressed as means sd or median (interquartile range) where appropriate. paired t - test was adopted for comparison of echocardiographic parameters between before and after application of pcom. all statistical analyses were performed using pasw 18.0 (spss inc., chicago, il, usa), and p - values of 180 mmhg, and/or diastolic bp of > 110 mmhg ], severe lv dysfunction defined as lv ejection fraction (ef) of less than 30%, and diagnosis of acute coronary syndrome within a month. patients with peripheral artery disease with claudication were also excluded. transthoracic echocardiography was performed using commercially available echocardiography machine (vivid 7, ge medical systems, milwaukee, wi, usa). if the patient was confirmed to be eligible, baseline echocardiography was performed after 20 minutes of rest. non - invasive bp and heart rate (hr) were measured just before echocardiography examination. peak early (e) and late (a) diastolic velocities of the mitral inflow were measured using a pulsed - wave doppler at the tip of mitral valve leaflets, and peak early (e ') and late (a ') diastolic mitral annular velocities were acquired at the septal side in the apical 4 chamber view. recording of aortic valve maximal systolic velocity (av vmax) was acquired from multiple views including suprasternal, right parasternal, apical and subcostal transducer positions with a continuous - wave doppler echocardiographic technique, among which only the highest peak velocity was chosen for subsequent analyses. pulsed - wave doppler at lv outflow tract (lvot) was also obtained at the apical 5-chamber view. lv end - systolic wall stress was calculated as follows ; (lv end - systolic wall stress is in g / cm, pes, which stands for lv end - systolic pressure plus maximal pressure gradient of aortic valve, is in mmhg, des and he s are in cm, and 0.34 is the factor for converting pes from mmhg to g / cm.) specially designed pneumatic trousers without the bladder for compression of the lower abdomen were put on the patients as previously described.6) after baseline echocardiography data acquisition, a specially designed compressor inflated the pneumatic trousers up to a pressure of 100 mmhg on both lower extremities and this pressure was maintained throughout the examination. echocardiography was performed including doppler parameters 3 minutes after pneumatic compression of the lower extremities (pcom). doppler measurements of av vmax, transvalvular peak and mean pressure gradient (av peak pg and av mean pg), and time - velocity integral of lv outflow tract (tvilvot) and aortic valve (tviav) were also made. eoaav was calculated by continuity equation as previously described.7) doppler velocity index (tvilvot / tviav) was calculated, as well. lvot area (csalvot) was calculated from the diameter obtained at the level of the aortic annulus during systole with the assumption of a circular shape of lvot. lv stroke volume was calculated by multiplying csalvot by tvilvot as previously described.8) cardiac output (co) was calculated by multiplying sv and hr. systemic vascular resistance (svr) was estimated as : svr = 80 mean bp / co and systemic arterial compliance (sac) was calculated as : sac = sv / (sbp - dbp) numerical data are expressed as means sd or median (interquartile range) where appropriate. paired t - test was adopted for comparison of echocardiographic parameters between before and after application of pcom. all statistical analyses were performed using pasw 18.0 (spss inc., chicago, il, usa), and p - values of < 0.05 were considered statistically significant. mean bp was significantly increased from 92 13 to 98 14 mmhg (p svr significantly increased from 1351.0 370.2 to 1450.3 476.0 dyns / cm (p = 0.004) and accordingly, sac was decreased from 1.57 0.53 to 1.49 0.55 ml / mmhg (p = 0.07). however, hr remained unchanged (67.23 13.4 bpm at baseline vs. 67.1 12.8 bpm under pcom, p = 0.69). pcom application exerted no significant effect on lv end - systolic dimension and lv ef (table 2). likewise, lv sv and co under pcom were comparable to those measured at baseline. in contrast, lv end - diastolic dimension was slightly increased under pcom (47.4 5.9 mm vs. 48.9 4.9 mm, p < 0.02). e wave velocity was also significantly increased after pneumatic compression (0.74 0.21 m / s vs. 0.82 0.26 m / s, p < 0.001), and e / e ' tended to be slightly increased with a borderline statistical significance (16.9 7.6 vs. 17.8 8.6, p = 0.06). neither s ' nor calculated end - systolic wall stress under pcom showed significant changes. doppler velocity index (tvilvot / tviav) was not changed under pcom (p = 0.48) (table 3). however, av vmax and av peak pg displayed a small, but significant decline under pcom however absolute difference between the two different afterload status was only 0.10 0.24 m / s and 1.73 6.08 mmhg, respectively (table 3). mean bp was significantly increased from 92 13 to 98 14 mmhg (p < 0.001) (fig. svr significantly increased from 1351.0 370.2 to 1450.3 476.0 dyns / cm (p = 0.004) and accordingly, sac was decreased from 1.57 0.53 to 1.49 0.55 ml / mmhg (p = 0.07). however, hr remained unchanged (67.23 13.4 bpm at baseline vs. 67.1 12.8 bpm under pcom, p = 0.69). pcom application exerted no significant effect on lv end - systolic dimension and lv ef (table 2). likewise, lv sv and co under pcom were comparable to those measured at baseline. in contrast, lv end - diastolic dimension was slightly increased under pcom (47.4 5.9 mm vs. 48.9 4.9 mm, p < 0.02). e wave velocity was also significantly increased after pneumatic compression (0.74 0.21 m / s vs. 0.82 0.26 m / s, p < 0.001), and e / e ' tended to be slightly increased with a borderline statistical significance (16.9 7.6 vs. 17.8 8.6, p = 0.06). neither s ' nor calculated end - systolic wall stress under pcom showed significant changes. doppler velocity index (tvilvot / tviav) was not changed under pcom (p = 0.48) (table 3). however, av vmax and av peak pg displayed a small, but significant decline under pcom however absolute difference between the two different afterload status was only 0.10 0.24 m / s and 1.73 6.08 mmhg, respectively (table 3). in the present study, we modulated lv afterload using specially designed pneumatic compression trousers without any significant effect on hr to evaluate the impact of lv afterload modification on the assessment of as severity. pcom successfully increased lv afterload, as evidenced by increased svr as well as decreased sac without any change of hr. although tpg of aortic valve slightly decreased with lv afterload rise, this was not translated into significant changes in eoaav and doppler velocity index obtained with routine echocardiography. increased afterload may have a potential to affect the accuracy of assessment of as severity. increased afterload increases svr, which subsequently elevates the ascending aortic pressure. in case of significant as, the effect of increased systemic central bp on intraventricular pressure might be minimal, and thus, tpg between lv and ascending aorta might be decreased because of relatively increased central aortic pressure in comparison with intra - lv pressure. however, if the transmission of increased afterload into lv cavity per se were considerable, lv systolic wall stress would rise and lead to a decrease in lv systolic performance. in addition, increased lv afterload along with a decrease in sac can attenuate lv co,9) and finally result in a decrease in av mean pg, av vmax, and av peak pg. it may be possible that aortic root expansion also can increase csalvot during systole, and subsequently lead to commissural separation and finally stretch the free edge of the aortic valve leaflets. although the effect of aortic root expansion was previously reported in normal aortic valve,10)11) it appears to be unclear whether thickened and less mobile aortic valve and annulus can change their shape during lv afterload changes as normal aortic valve does. in this study, we found that lv ef, lv sv and co did not change significantly with a maneuver leading to a rise in lv afterload. in order to achieve effective lv afterload increment without any influence of hr although we could did not directly measure intracardiac pressure, pcom was considered very effective in modifying lv afterload, as demonstrated by a significant increase in svr. as shown in our representative cases in fig. 2, tpg of av, as represented by 2 parameters of av vmax and av peak pg, was decreased under pcom, suggesting the fact that an increase of aortic pressure was even greater than an increase of intra - lv pressure during systolic phase in the setting of lv afterload increment. on the other hand, eoaav and doppler velocity index did not change, indirectly highlighting that increased afterload did not modulate aortic root size or commissural opening of stenotic aortic valve in this special as population. in this study, we used pcom for experimental change in afterload. pcom successfully increased lv afterload without a significant change in hr ; however, pcom possibly increases the venous return from lower extremities, which could explain, to some extent, increased e velocity, and slightly increased lv end - diastolic dimension. increased preload can increase the stroke volume and possibly increase the av vmax of aortic valve. our data showed no significant change of stroke volume, av peak pg and av vmax before and after pcom ; thus the effects of pcom on lv preload and stroke volume, and furthermore as severity assessment appears negligible. a previous study by little.12) showed decreased ava by acute bp elevation induced by phenylephrine infusion and hand grip exercise. they suggested that the impact of bp change is associated with transvalvular flow rate through aortic valve without relation to svr or sac. in this study, we adopted pcom instead of hand grip exercise or phenylephrine infusion because these methods augment heart rate and induce a significant change in ejection time, as previously described. because our study was free from changes in hr and lv ejection time under pcom, an impact of lv afterload change on ava assessment can be exclusively assessed, which is, we believe, a significant advantage of the current study. pcom is a useful method to increase lv afterload without change of preload or hr when compared to other methods, such as hand grip exercise or phenylephrine infusion.12) however, pcom possibly increases the venous return from lower extremities, which could explain, to some extent, increased e velocity, and slightly increased lv end - diastolic dimension. however, a previous study clearly demonstrated that the primary mechanism whereby pcom induced changes in hemodynamics is through an acute increase in lv afterload.13) in addition, a change in lv co was minimal, highlighting the impression that hemodynamic effect by venous return for cardiac performance was, if any, negligible. simulation of arterial bp elevation by pcom also has a limitation for svr increase alone, and can not increase aortic stiffness which is more important in essential hypertension in elderly patients. we also can not directly measure lv systolic wall stress and central bp, because we did not perform invasive monitoring of intra - lv pressure and central bp, however, svr and sac that were employed in the current study were previously validated against invasively obtained hemodynamic data as indirect indexes for lv afterload.14)15) assessment of as severity by routine transthoracic echocardiography was not significantly influenced by a change in lv afterload. av vmax could be slightly decreased when lv afterload rises, but these changes did not seem to exert a significant influence on clinical decision making in managing as patients. eoaav and doppler velocity index is more stable method for evaluation of as severity than av vmax, and therefore these 2 indexes should be used in the determination of as severity, rather than av vmax or tpg of av. pcom is a useful method to increase lv afterload without change of preload or hr when compared to other methods, such as hand grip exercise or phenylephrine infusion.12) however, pcom possibly increases the venous return from lower extremities, which could explain, to some extent, increased e velocity, and slightly increased lv end - diastolic dimension. however, a previous study clearly demonstrated that the primary mechanism whereby pcom induced changes in hemodynamics is through an acute increase in lv afterload.13) in addition, a change in lv co was minimal, highlighting the impression that hemodynamic effect by venous return for cardiac performance was, if any, negligible. simulation of arterial bp elevation by pcom also has a limitation for svr increase alone, and can not increase aortic stiffness which is more important in essential hypertension in elderly patients. we also can not directly measure lv systolic wall stress and central bp, because we did not perform invasive monitoring of intra - lv pressure and central bp, however, svr and sac that were employed in the current study were previously validated against invasively obtained hemodynamic data as indirect indexes for lv afterload.14)15) assessment of as severity by routine transthoracic echocardiography was not significantly influenced by a change in lv afterload. av vmax could be slightly decreased when lv afterload rises, but these changes did not seem to exert a significant influence on clinical decision making in managing as patients. eoaav and doppler velocity index is more stable method for evaluation of as severity than av vmax, and therefore these 2 indexes should be used in the determination of as severity, rather than av vmax or tpg of av. | backgroundaortic stenosis (as) is increasingly diagnosed in current aging society. echocardiography is the most important tool in the assessment of as and its severity. however, load - dependency of doppler measurement could affect the accuracy of as severity assessment. we tried to evaluate the impact of afterload on the assessment of as severity by modification of afterload using pneumatic compression (pcom).methodsforty patients diagnosed as moderate or severe as [effective orifice area of aortic valve (eoaav) by continuity equation of < 1.5 cm2 ] were consecutively enrolled. patients with severely uncontrolled hypertension, severe left ventricular (lv) dysfunction, and other significant valve disease were excluded. comprehensive echocardiography was performed at baseline to assess as severity. then, pneumatic compression of the lower extremities by 100 mmhg was applied to increase lv afterload. after 3 minutes, echocardiography was repeated to assess as severity.resultsmean blood pressure was significantly increased under pcom (p < 0.001), while heart rate remained unchanged. peak aortic valve velocity (vmax) was slightly, but significantly decreased under pcom (p = 0.03). however, doppler velocity index and eoaav by continuity equation were not affected by pcom.conclusionas severity assessment by echocardiography was not dependent on the change of lv afterload imposed by pcom. av vmax was slightly decreased with lv afterload increment, but these changes were too small to alter treatment plan of as patients. eoaav and doppler velocity index are more stable parameters for as severity assessment. |
migraine is typically characterized by a pulsating headache with or without aura, nausea, and vomiting. the drugs commonly used in acute migraine attacks are sumatriptan (which is one of the triptans chemically grouped under serotonin agonists) and ergot alkaloids, while propranolol and calcium channel blockers are used prophylactically. however, recently topiramate (tpm), an anti - epileptic drug, has gained popularity as a first line drug for use in prophylaxis of migraine. the use of tpm for epilepsy and migraine has been approved by the us food and drug administration (fda) and it also has a number of off - label indications, such as bulimia nervosa, alcohol dependence, smoking, and possibly a depressive phase in bipolar disorders. the most significant adverse effects associated with tpm include psychomotor slowing, difficulty in concentration, somnolence, and fatigue. additional non - specific central nervous system (cns) adverse effects are dizziness, confusion, and memory problems. the dose and duration - dependent use include renal stones, weight loss, and paresthesia. recently, evidence has been accumulating on a rare but serious adverse event on tpm - induced acute myopia with acute angle - closure glaucoma (aacg) both as spontaneous case reports and case series [47 ]. warning was, therefore, issued by the fda in 2004, regarding its potential to induce aacg as a precaution for practicing physicians. the world health organization (who) has estimated that india has a 1% prevalence of blindness. of the estimated 8.9 million blind in india, 12.8% are due to glaucoma. while there is population - based data available on primary glaucoma from south asia, especially india, data on secondary drug - induced glaucoma are lacking, except for spontaneous reports. the authors report an observation of a case, where a female patient developed aacg after 8 days, following tpm 25 mg / day, once a day (od), for 3 days as an adverse reaction to tpm. she was started on tpm 25 mg / day ; however, she stopped the treatment after 3 days without consultation as her headache was not relieved. after 5 days of stopping the treatment with tpm, she presented at the emergency clinic of a hospital with complaints of blurred vision and severe pain in both the eyes, which were of a few hours in duration. she also complained of colored halos and headache associated with nausea with no family history of eye - related disorders. on ophthalmic examination, the visual acuity was found to be 3/60 in both the eyes, and did not show improvement in visual acuity in the pinhole test. both anterior chambers were found to be shallow, appeared occluded in the periphery, and pupils were reactive. applanation tonometry revealed high intraocular pressure (iop) of 34 and 32 mmhg, in the right and left eyes, respectively (fig. 1).fig. 1a photograph of normal (a) and glaucomatous (b) eyes a photograph of normal (a) and glaucomatous (b) eyes a diagnosis of aacg, precipitated following oral administration of tpm, was made. while, laser peripheral iridotomy would have been an ideal procedure for aacg, due to presence of choroidal effusion along with anterior migration of anterior structures this treatment option was not considered in this case. she was hence started on acetazolamide tablets 250 mg four times a day (qid), pilocarpine 2% eye drops qid, travoprost 0.004% od, and dorzolamide 2% eye drops three times a day (tid). since she had already stopped tpm, she was advised not to take it again and was reviewed the next day. the repeat ophthalmic examination on the second day showed improved vision (6/60) in both the eyes, reduction in conjuctival chemosis, and improved depth of anterior chamber, while it continued to be shallow peripherally. iop measurements were repeated using applanation tonometry and were found to be 20 and 18 mmhg, in the right and left eyes, respectively. on the third day, her vision improved to 6/12 in the right eye and 6/6 partial (p) in the left with iop 10 and 14 mmhg, respectively. subsequent examination on the fifth day showed improved visual acuity 6/6 p in both the eyes and iop was 14 and 12 mmhg and the anterior chamber appeared well formed. she was advised to taper the anti - glaucoma medication and was examined a month later when her visual acuity was 6/6, with iop 14 mmhg in both the eyes and was then advised via evaluation by a glaucoma specialist. all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of 1975, as revised in 2008. written informed consent was obtained from the patient for which identifying information is included in this case report. there have been 115 case reports of ocular side effects, 86 cases of secondary angle - closure glaucoma, and 7 cases of permanent visual loss reported with tpm, suggesting an association between acute onset of aacg after tpm therapy [11, 12 ]. the present case report adds evidence to show symptoms of acute onset of blurred vision associated with ocular pain to be due to adverse event associated with tpm prescribed for migraine. the ciliary body edema, uveal effusion, and relaxation of zonules along with anterior rotation of the ciliary body resulting in forward shift of the iris - lens apparatus are reported to contribute to tpm - induced aacg. in addition, these latter effects are said to produce shallow anterior chamber, leading to an increase in iop and thus precipitating the acute glaucomatous attack secondary to the increase in iop. tpm primarily introduced for the treatment of epilepsy is said to act by modulation of the voltage gated sodium channels, strengthening gamma - aminobutyric acid (gaba)ergic activity and reduction in the effects of activation of glutamatergic receptor of alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (ampa)-kainate subtype. interestingly, tpm is also known to share weak carbonic anhydrase (ca)-inhibitor activity like acetazolamide, hence paradoxically, is an effective anti - glaucoma drug. however, as a ca inhibitor it is linked with the occasional development of renal calculi and also being a sulfa - based drug by itself is reported to induce aacg through an idiosyncratic reaction [13, 14 ]. the potential to cause glaucoma may, therefore, be attributed to a mechanism independent of the well - known pharmacodynamic effects involved in anti - convulsant activity of tpm. several studies do report on the genetic basis of primary open - angle glaucoma (poag) and juvenile onset open - angle glaucoma (joag) [15, 16 ]. the genetic predisposition may also be considered as a cause for aacg in some individuals, although several reports propose on usage of tpm to induce secondary aacg [4, 5, 17, 18 ]. it is to be noted that ideally patients presenting with increased iop will be subjected to ophthalmic examination like gonioscopic examination with ultrasound biomicroscopy which are recommended routinely [5, 18 ]. however, these were not carried out in this particular patient, which may be due to the fact that the presenting history clearly revealed a diagnosis of tpm - induced aacg. thus, the presenting nature of the symptoms has possibly warranted initiation of anti - glaucoma therapy immediately. the other tests for glaucoma work - up like glaucoma diagnosis - variable cornea compensation (gdx - vcc) and optical coherence tomography (oct), which are both expensive and not mandatory in the acute phase of aacg, were not carried out. it is to be noted that ideally the present case could have been subjected to complete ophthalmic examination to include gonioscopic examination along with ultrasound biomicroscopy and iris configuration as recommended routinely [5, 18 ]. the diagnosis in this case was primarily based on detailed history taking as these tests are not carried out routinely due to economic constraints in the indian context. while tpm was discontinued, this patient was prescribed miotics like pilocarpine as eye drops, which is reported to be of doubtful benefit in the treatment of tpm - induced glaucoma along with sulfa drugs like acetazolamide and dorzolamide. it is hence important to note that administration of these medications appears inappropriate as these are likely to exacerbate the condition and could have been avoided. in addition, the treatment regimen did not include anti - inflammatory medication such as prednisolone as a topical steroid in this case [7, 18 ]. it is recommended that treatment with tpm for migraine be started with a low dose and gradually increased to the required dosage depending on improvement of the condition and patient tolerability profile. but in this particular patient, reaction appeared to be independent of the dose and duration as the patient had received the minimum therapeutic dose of 25 mg / day and tpm reaction developed within a short duration of treatment [5, 19 ]. while this is difficult to explain, despite discontinuation of tpm, the cumulative effect may be considered responsible for development of aacg. the mechanism by which tpm causes ciliary body swelling and anterior rotation of the ciliary body is still not clear. based on the above information, it may be suggested that it is important and appropriate to avoid treatment of aacg with miotics and medications chemically related to sulfa drugs. initiation of treatment with tpm over 3 days induced aacg on day 8 and instituting treatment reversed the signs and symptoms of aacg and patient was normal after discontinuation of tpm. based on this fact, the eventual causality as tpm - induced aacg can be confirmed. however, in this particular patient although history of medication intake revealed treatment with anti - cholinergics for other reasons, patient did not exhibit eye symptoms. therefore, the mechanisms implicated in development of aacg in this case need further evaluation. lastly, the authors wish to point out that the patient in this case is 25 years old, which is young for poag as the onset is normally seen after 40 years of age. the limitations of this study were that this is a single case report and not a case series, which are preferred. in addition, causality using a standard scale was not used to confirm association between the offending drug and adverse reaction. tpm is frequently used in the treatment of migraine and is reported to increase iop and as a result has a potential to precipitate aacg in a genetically predisposed person. a single dose may precipitate aacg as an idiosyncratic reaction which is a rare but a very serious adverse reaction and may progress to blindness. it is, therefore, recommended that the physician before prescribing tpm should warn and advise the patient to report any cases of visual disturbances immediately. it is also advisable to monitor the iop at baseline before initiating treatment with tpm and regularly at weekly intervals thereafter for a month from the time of commencement of medications to prevent glaucoma as one of its serious adverse reactions. although this is a widely reported adverse effect of tpm, there are a few questions which remain unanswered. one aspect that could be examined is how exactly sulfa - derived medications such as tpm can cause swelling of ciliary body and eventual anterior rotation of ciliary body. it would be worth investigating to see if patients who developed aacg with tpm showed a similar reaction to other sulfa - containing compounds. in addition, considering age of this patient it is unlikely to be poag. with recent advances in the field of drug discovery and development, there appears to be a possibility to minimize such undesirable, unexpected, and serious adverse events using safer treatment options in patients with migraine. finally, with exponential advancement in pharmacogenetics, every patient taking tpm could be considered as a potential candidate for developing aacg, and be screened adequately and appropriately using suitable biomarkers to prevent serious adverse events. chanda kulkarni, urmimala ray chaudhuri, and annalakshmi jagathesan declare no conflict of interest. all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of 1975, as revised in 2008. written informed consent was obtained from the patient for which identifying information is included in this case report. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | introductionthis case report adds supportive evidence to the development of acute angle - closure glaucoma (aacg), a rare but serious adverse effect following the use of topiramate (tpm) for a severe headache.case reporta 25-year - old female reported with severe headache, suspected to be migraine, and was started on tpm 25 mg / day on the first day. however, she presented at the emergency clinic of a hospital with sudden blurring of vision and colored halos 5 days after stopping the drug, i.e., day 8. she was subjected to ophthalmic examination and was diagnosed with aacg. the intraocular pressure (iop) was found to be elevated and she was hence started on acetazolamide 500 mg instantly, maintained on tablet acetazolamide 250 mg four times a day (qid), pilocarpine 2% eye drops qid, travoprost 0.004% once a day (od), and dorzolamide 2% eye drops three times a day (tid). after a week s treatment, there was rapid improvement with return of iop to normal.conclusiontpm-induced aacg is a rare serious adverse event leading to blindness but is preventable, when diagnosed early and by instituting appropriate treatment. |
the data from the articles by goldstein. suggest that both cardiologists and their patients tend to think that once a device such as an icd is in place, one ought not stop it. as a general rule, philosophers have suggested that the conditions under which one could justify withholding a treatment are those under which one could justify withdrawing a treatment. for example, if the patient were irreversibly and imminently dying of a painful cancer and had recurrent ventricular tachycardia, one would be perfectly justified in not placing an icd. rationally, if the patient had an icd implanted 2 years ago and now develops a painful cancer and death is imminent, deactivating the icd seems just as justifiable as withholding it. tell us, however, is that what seems equivalent according to the logic of ethics continues to feel psychologically different to both patients and practitioners. does the fact that the icd is required intermittently rather than continuously mark a moral difference ? the discontinuation of an intermittent treatment such as hemodialysis, should it become burdensome, has been judged morally acceptable by persons holding a wide variety of ethical viewpoints. reports, the very fact that it functions only intermittently might make it psychologically easier to deactivate an icd than to deactivate the pacemaker of a patient with complete heart block. some might consider the duration of therapy morally important. but is this true ? if the icd in the case of the patient with cancer that i discussed above had been in place for 20 years instead of 2, with no changes except for batteries, would the duration alone make us think that it would be immoral to deactivate it if the patient were imminently dying, in great pain, and the device might only prolong that state ? consider a patient who has been ventilator - dependent for 30 years after contracting polio, is not depressed, and comes to the conclusion, might we not be more willing to accept his request to discontinue the ventilator as a well thought out and morally acceptable choice than if the same request were made by the patient after only 1 week of ventilator support ? all therapies are restorative in intent,10 but different therapies restore patients in 2 broadly different ways. some therapies are regulative. that is to say they coax the body back toward its own homeostatic equilibrium. they take over a function that the body can no longer provide for itself. might this distinction mark a moral difference between killing patients and allowing them to die ? if anything, it would seem that the discontinuation of constitutive therapies would raise more questions than the discontinuation of regulative therapies. mimicking physiology, doing what the body no longer can do for itself, seems closer to being a part of the patient than does a therapy that nudges the body into healing itself. despite the psychological differences that this distinction might raise, however, we still regularly accept the morality of discontinuing constitutive therapies such as ventilatory support. the fact that a treatment is constitutive does not seem to mark a moral difference between killing and allowing to die. further, the fact that icds are regulative, not constitutive, suggests that this distinction can not explain any special moral worries about deactivating an icd. does the fact that a technology has become internal to the body mark the boundary between killing and allowing to die ? this question seems to come closer to explaining the concerns raised by the patients and practitioners about deactivating icds, but does it stand up to ethical analysis ? does the fact that many new medical technologies are inside the body mean that they have thereby become part of the person so that deactivating an icd or a pacemaker becomes morally equivalent to discontinuing the function of a natural heart by injecting kcl ? elsewhere,11 i have defined killing as an act in which an agent performs an action that creates a new, nontherapeutic, lethal pathophysiological state in a human being with the intention of thereby causing that human being s death. by contrast, i have defined allowing to die as an act in which an agent either performs an action to remove a treatment for a preexisting fatal disease or refrains from action that would treat a preexisting fatal disease, either intending that this person should die by way of that act or not so intending. the acts of allowing to die that have traditionally been thought morally permissible are those in which the physician s intentions are to aim not at making the patient dead, but simply at stopping the treatment. these definitions make no reference to internal or external. by these definitions, deactivating an external pacemaker is morally equivalent to deactivating an internal pacemaker, and deactivating an external defibrillator is morally equivalent to discontinuing an icd. but is this correct ? does not having a device inside a patient make it a part of the patient, part of her physiology, so that stopping its function is killing ? certainly, this intuition seems correct if one is talking about a heart transplant. stopping the function of a transplanted heart with an injection of kcl seems morally no different from stopping a native heart with an injection of kcl. and is not a heart transplant every bit as much a technological intervention as is an icd ? upon further reflection, the fact that a treatment is inside the body does not, of itself, seem to do the moral work some might think it does. consider and compare the following technological interventions : an lhrh agonist implant for prostate cancer and a skin transplant after a severe burn. if one were to discontinue these therapies, however, the mere fact that one treatment is placed under the patient s skin, whereas the other is placed over the patient s skin does not constitute the difference between killing and allowing to die. if the patient with prostate cancer were experiencing hot flashes from the lhrh action of the drug and having pain at the site of the implant, his request to have it removed should be honored. but what if the burn patient were to ask that the skin transplant be removed, saying that she had grown tired of the need to take anti - rejection medication ? without an intact integument, the patient would experience sepsis and die. most plastic surgeons would refuse to do this on the grounds that they would be mutilating, if not killing, the patient, even if she were otherwise dying from some other comorbid disease. the mere fact that a technological intervention has been placed under the skin does not seem to mark the moral difference between killing and allowing to die. so, then, perhaps those who have approached the question as a standard application of the principles typically used in bioethics are right after all. jansen has argued that the questions raised by advances in medical technology such as icds are challenging our notions about the boundaries of the self.12 particularly, as biotechnological treatments become more bio than techno, we need to think clearly about the characteristics that render a treatment a part of the patient s self, so that its discontinuation is morally indistinguishable from killing. jansen has offered 3 rules of thumb for helping to judge whether a particular technological intervention ought to be considered a part of the patient. she suggests that there are more serious grounds for considering the technological intervention a part of the patient if it is located inside the patient, has been in the patient for a long time, and plays something akin to what i have called a constitutive therapeutic role. i have explained above, however, why i believe none of these 3 factors can bear the weight of the distinction between killing and allowing to die. and, whereas jansen s insight is correct that some treatments must truly be considered within the ontological boundaries of the patient s while i further agree with her that these will necessarily be rules of thumb and that a bright line will be hard to draw, i think we can press for greater clarity. i would like to suggest 2 alternative guiding principles that, while provisional hypotheses, might advance the discussion. these interventions are distinct from the organism and extrinsic to its function, whether administered inside or outside the body. one of the patients quoted by goldstein. illustrates the psychology of distinguishing between icd and self nicely when she describes how she talks to her icd, treating it as something other than her self although it is located within her.7 regulatory therapies, no matter how sophisticated, and whether located inside the body or not, can be thought about just as one would think about withholding or withdrawing more standard forms of therapy at the end of life. they may be forgone if they are futile or if the burdens of treatment become disproportionate to the benefits. second, some, but not all, constitutive therapies are distinct from the self. i am not certain how best to characterize this distinction, but my preliminary hypothesis is that we can distinguish between constitutive therapeutic interventions that have replaced the pathologically disordered function and those that are substitutes for the pathologically disordered function. the distinction between these 2 kinds of constitutive therapies will not always be clear, but i hope to make it clear enough that it can be clinically and morally useful. in my view, in the proper circumstances, it is morally permissible to withhold or withdraw substitutive therapies, but the more an intervention can be understood as a replacement therapy, the less it seems morally appropriate to withdraw it. what i mean by a replacement therapy is a technological intervention that participates in the organic unity of the patient as an organism. this is what it really means to say that a technological intervention has become a part of the patient. a replacement therapy is one that has become part of the patient s restored physiology. the most important feature of a replacement therapy is that it provides the function that has been pathologically lost, more or less in the same manner in which the patient was once able to provide this function when healthy. thus, for instance, a renal transplant is a replacement therapy, whereas peritoneal dialysis (although it also takes place inside the body) is a substitutive therapy. additional signs suggestive of an intervention being a replacement therapy might include : (1) its responsiveness to changes in the organism or its environment, (2) properties such as growth and self - repair, (3) independence from external energy sources or supplies, (4) independence from external control by an expert, (5) immunologic compatibility, (6) physical integration into the patient s body. the paradigmatic replacement therapy is thus a well - functioning organ transplant from an identical twin. the more a technological intervention meets the conditions for being a replacement therapy, the harder it is to contend that it is extrinsic to the patient s identity. this distinction between replacement and substitutive types of constitutive therapies might help us to say how far someone who is opposed to euthanasia could reasonably go in classifying certain cases as morally appropriate withdrawal of life - sustaining treatment rather than morally unacceptable cases of killing. whereas there is no absolute standard for judging whether something is a replacement or a substitute, the more clearly a technology can be classified as a replacement therapy, the greater the case for judging that its discontinuation would constitute an immoral act of killing. replacement therapies become part of the restored physiology of the patient, part of the integrated unity of the patient as an intact individual organism. to discontinue such therapies is better understood as introducing a new lethal pathophysiological state rather than discontinuing a treatment that is merely substituting for a preexisting lethal pathophysiological lack of that function. to illustrate, consider the fact that there are cases in which discontinuing insulin injections in a diabetic patient would be morally appropriate. if the patient were imminently dying of a malignancy, even the burdens of being injected with insulin or having finger - stick checks for blood glucose might be considered disproportionate to the benefits. it provides the normal physiological function in an abnormal way and the treatment is not part of some new organic unity that has altered the identity of the patient. if the same patient had previously undergone an islet cell transplant as a treatment for his diabetes, it would seem highly morally problematic to discontinue insulin therapy under these same end - of - life conditions by injecting streptozosin to kill the transplanted islet cells. islet cell therapy is a replacement therapy and, as such, the injection of streptozocin would be mischaracterized if one were to describe it as the mere discontinuation of a life - sustaining therapy. the cells would have become part of the patient an integrated aspect of her restored physiology and to destroy these cells would be ethically indistinguishable from destroying the native islet cells of a healthy person. those who are opposed to euthanasia but supportive of the withholding and withdrawing disproportionately burdensome life - sustaining treatments need not demur at the idea of deactivating icds. the analysis i have presented shows that deactivating an icd can be ethically distinguished from killing and considered a part of good palliative care. however, the argument necessary to reach this conclusion will doubtless prove challenging for persons unaccustomed to philosophical thinking. the data from goldstein. describe in detail how much effort will be needed in making this argument clear for cardiologists and patients. it is critically important, however, that we begin thinking seriously and carefully about what makes an intervention a part of the patient, rather than a treatment that is extrinsic to the patient s self, even if it is located inside the patient s body. the rapid pace of technological progress assures us that these sorts of questions will continue to surface in clinical practice. ethics, as the most practical branch of philosophy, must be prepared to keep pace with these challenges. | as implantable cardioverter defibrillators (icds) have become more common, ethical issues have arisen regarding the deactivation of these devices. goldstein., have shown that both patients and cardiologists consider icd deactivation to be different from the discontinuation of other life - sustaining treatments. it can not be argued ethically that icds raise new questions about the distinction between withholding and withdrawing treatment, and neither the fact that they are used intermittently, nor the duration of therapy, nor the mere fact that they are located inside the body can be considered unique to these devices and morally decisive. however, frequent allusions to the fact that they are located inside the body might provide a clue about what bothers patients and physicians. as technology progresses, some interventions seem to become a part of the patient as a unified whole person, completely replacing body parts and lost physiological functions rather than merely substituting for impaired structure and function. if a life - sustaining intervention can be considered a replacementa part of the patient as a unified whole person then it seems that deactivation is better classified as a case of killing rather than a case of forgoing a life - sustaining treatment. icds are not a replacement therapy in this sense. the deactivation of an icd is best classified, under the proper conditions, as the forgoing of an extraordinary means of care. as technology becomes more sophisticated, however, and new interventions come to be best classified as replacements (a heart transplant would be a good example), discontinuing these interventions should be much more morally troubling for those clinicians who oppose euthanasia and assisted suicide. |
total joint arthroplasty, in particular tha, has had a revolutionary role in improving the quality of life. the use of bone cement for the implant stability versus biologic fixation with bone ingrowth in cementless tha has been a controversial issue for years. while immediate cement fixation in very old people or in those with poor bone stock might provide a quicker return to daily activity, cementless implants have gained more popularity over the years. the relative superiority of cementless acetabular component over cemented ones is nowadays a well - accepted fact. the femoral stem, however, has very good long - term reports both in cemented and cementless forms. efforts to decrease the rate of loosening have included the use of newer materials, improvement in the design of the implant ; and modification of operative techniques. after the midterm follow - up results of cementless tha, the long - term results with impressive survival rates, are being reported more and more. the purpose of this study is to evaluate the efficacy and prosthesis survival in an iranian society, with its unique cultural lifestyle and social differences from western societies. the cases of cementless total hip arthroplasty performed in nemazee hospital by a single surgeon from may 1997 to june 2007 were included in a retrospective outcome study. from the total of 63 hips in 52 consecutive patients, 3 patients had died at the time of the last follow up due to problems unrelated to the operation and 7 patients could not be reached. the information from medical records of all the cases, including radiographs, was collected and the patients were called in for an interview, physical examination, and radiographic assessment. the patients filled the general - health assessment form, short form 36 (sf-36) ; the arthritis specific functional instrument womac (western ontario and mcmaster universities osteoarthritis index), and patient reference disability questionnaire mactar (mcmaster toronto arthritis). harris hip rating scale was also filled for all the hips, (where 90 - 100 points would be excellent, 80 - 89 good, 70 - 79 fair, and below 70 is assumed a poor result). the radiographic assessment was by measurement of the cup and stem alignment in the immediate post - operative anteroposterior and frog - lateral views. the same views in the final follow - ups were specifically evaluated for any possible change in cup orientation, loosening in cup or stem (based on gruen classification for zones of the femoral stem and martell et.al. osseous integration of the acetabular and femoral components were assessed using the proposed criteria of moore. considering the five criteria of absence of radiolucent lines, superolateral and inferomedial buttressing, medial stress - shielding, and radial trabeculae for acetabular shell and stem bony ingrowth according to engh. osseo - integration in stem was evaluated by the presence of spot welds, cortical hypertrophy ; and absence of radiolucent lines, and pedestal formation. harris - galante ii (hg ii) porous - coated prosthesis (hgp, zimmer, warsaw, indiana) was implanted in 15 and versys - trilogy (v - t) system (zimmer, warsaw, indiana) in the remaining 37 hips. the straight stem is tivanium ti-6 al-4v alloy with a proximal pure titanium fiber metal mesh coating ; with a collar and a modula.r morse tapered neck, which is available in three lengths. there are porous pads, made of commercially pure titanium wire, proximally on the anterior and posterior surfaces and a small medial pad immediately distal to the collar. the shell is a partial hemisphere made of titanium alloy with fiber metal mesh coating with variable number of holes for screw fixation. versys femoral stem (zimmer, warsaw, indiana) is a collarless proximally and circumferentially coated prosthesis for cementless use. trilogy shells are coated with commercially pure titanium fiber metal, which is clinically proven to enhance fixation through bone ingrowths. infection prophylaxis was with cephalosporin and gentamicin at the time of surgery and 48 hours post - surgery. thromboembolic prophylaxis was mostly by warfarin for 6 weeks with the intended inr (international normalized ratio) of 1.7 to 2. early mobilization and first post - surgery day ambulation and crutch walking for 6 weeks were the uniform care received by all the patients. the prosthesis survivorship limit was defined as implant life span and revision - ready state with progressive symptoms. definite when subsidence, varus, or valgus orientation change was observed in femoral component and angle change, or migration of two millimeter or more seen in two views for the acetabular component. the 42 patients (52 hips) included fourteen males (33.3%) and twenty - eight females (66.7%), with the mean age of 48.83 years (13 years, range 22 - 75) at surgery. harris - galante ii prosthesis was used in 15 cases and versys - trilogy prosthesis in 37 hips. the average duration of follow - up was 65 months (range 26 - 136). the hg ii group of prostheses had a longer follow - up of 105 months (range 52 - 136). this figure was 49 months for versys - trilogy group (range 26 - 78). the overall mean follow - up was 65 months (32, range 26 - 136). the overall arthroplasty survival (i.e., well - functioning prosthesis with no clinical or radiographic evidence of wear, loosening, infection, etc.), which would suggest the need for revision was 65 months. therefore, 43 hips in 34 patients were in good and functional status by the time of last follow - up. post - operatively, hips had a mean flexion arc of 114 degrees and 9 degrees of flexion contracture. the overall hhs with a mean of 85 (15, range 24 - 100) was excellent in 65.9%, good in 27.3%, fair in 4.5% and poor in 2.3% of cases. the womac score had a mean of 22.7 (13, range 3 - 62), with 3 being the best and 62 the worst case scenario. the pain subscore of womac was 2.87, joint stiffness 2.21 and functional subscore 17.62. the items in the function, which were of most concern to the patients were, in a descending order : inability in stair climbing ; sitting or getting up from the floor or from flat - top toilets ; picking up objects from the floor ; and putting on or taking off socks. sf 36 measurement had a total mean score of 61.33 (range 18 - 95). out of the 8 items in sf 36, the patient expectation questionnaire of mactar had the following findings : pain relief was achieved in 41 cases (97.6%), improvement in walking in 39 (92.8%), and improved ability in performing daily living activities in 37 (88%). the correlation of the above scoring system in this group of patients was evaluated. a close correlation between harris hip score and total womac score pain and function items, but not as much with stiffness item in womac (p values 0.002, 0.001 and 0.45, respectively). sf 36 and harris hip score were closely correlated and value of r was 0.67. sf 36 was more closely correlated with pain and function subscores of womac (r=-0.77 and 0.78, respectively). there was no infection, and no thromboembolic event in any of the 52 hips. in the last follow - up assessments, 44 hips (84.6%) were functional and well fixed ; 8 cases had undergone revision and one patient is suspected of the early stage of loosening and is being followed. pedestal was seen in 3 (7%) stems, and 1 - 2 millimeter non - progressive radiolucent lines in 20% of femurs and 4.5% of acetabular components. heterotopic ossification as a late complication was found in 35 hips (67.3%), 29 (82.7%) of which were brookers i and ii, 5 brookers iii and one brookers iv. since there were two groups of prostheses from the same company used in this study, they also were separately evaluated : among the 15 cases of harris - galante ii prosthesis, with average follow - up of 105 months (range 52 - 136), 8 cases had developed problems, all of which had been already revised. all of the revised cases had problems in the acetabulum with cup wear, loosening, and polyethylene fracture and two of them had a simultaneous femoral loosening and osteolysis secondary to polyethylene wear debris. the etiology of hip disease in these 8 revisions, included five acetabular dysplasia, one avascular necrosis and systemic lupus erythematosus, one multiple epiphyseal dysplasia, and one primary osteoarthritis. in reviewing the original radiographs, no initial radiographic malposition was present and the stems were in normal orientation and mean shell inclination angle was 47 degrees (range 40 - 57 degrees), which was not statistically different from the versys - trilogy group (p=0.51). the primary etiology of hip disease, in terms of distribution in these two groups, was different (table 1). comparison of etiology of hip replacement in two groups broken tines of fiber metal - coated acetabular shells were seen in 5 patients, all in the failed acetabular components (figure 1). broken tine of the cup the harris hip score, womac and sf36 in the 15 hg ii cases were significantly poorer than the 37 cases with versys - trilogy prosthesis : harris hip score of 66 versus 92, womac 30 versus 20 ; and sf36 of 49 versus 66 (p value 0.009). the versys - trilogy prostheses are all surviving in a mean follow - up of 49 months (range 26 - 78) with no radiographic or clinical evidence of loosening or wear. the five early complications, mentioned above, were all in this group of prostheses. this is a small group of cases with a midterm follow - up on porous - coated hip arthroplasty in a society with unique social habits and customs. the charnley prosthesis reported by ranawat had 90% survival of the femoral component, while harris had about 80% survival with revision mainly on the acetabular side. porous - coated implants were used with the idea of removing the so - called weak link in the hip replacement from 1971. this has survived as a very good hip arthroplasty option for young active individuals with good bone stock. the results with non - circumferential proximal porous - coating prosthesis like porous - coated anatomic, pca (howmedica, rutherford, new jersey) and harris - galante i (zimmer, warsaw, indiana) were not satisfactory : failures of 43% and only 57% survival in 8 years. kim recently reported that pca prosthesis (howmedica) had 21% revision in 20 years for the acetabular component and 9% for femoral component. after generally satisfactory short and midterm results of the second generation of cementless implants (with proximal circumferential porous - coating), clohisy and harris reported a 96% 10-year survival rate for acetabular component and archibeck. in a study of 92 patients with the same follow - up had a 96.4% survival rate for acetabular and 100% for femoral components. most studies have evaluated the functional results with hhs with 83 - 95 points on average. reported 100% ten years survival in a hydroxyapatite - coated, proximally and circumferentially coated prosthesis. engh., using an extensively coated prosthesis reported on 5-year, 10-year, and 15-year follow - ups. the acetabular component in most reports is the one with more problems and the responsible section for loosening (kim, engh, archibeck). the number of holes for temporary screw fixation has also been a point of concern, as more holes might provide better access for migration of polyethylene debris behind the shell and into the femoral canal. the locking of the polyethylene cup into the metal shell is variable in different designs of prostheses. poor locking mechanism can cause micromotion between the liner and the shell, causing more wear and subsequent dislodgment of the liner. the survival rate in the present series with 96.2% for the femoral component and 84.6% for the acetabular component in 5.5 years is not a very promising result. the high revision rate of 15.4% was primarily in the hg ii components and all were related to the acetabular side with wear, breakage, and dislodgment of polyethylene liner. louwerse. in 1999 reported 26 cases of liner failure, 13 of which belonged to hg cups. curry. in a 10-year follow - up reported 271 cases of hg ii prosthesis in 2008. our hg ii group of arthroplasty in 9 years average follow - up had 46.7% overall prosthesis survival. the femoral stems were revised in only two cases that had severe bone lyses secondary to the acetabular liner problem, and the remaining 50 (96.2%) stems are stable and functioning well. although the revised cups, except one, did not have primary osteoarthritis or inflammatory arthritis, the numbers are too few to draw any conclusion as to whether the primary etiology could have had any bearing on the high rate of liner problem in hg ii cups. the appearance of broken tines was visible on radiographs, one to two years before the hips became symptomatic. broken tines are probably early warning signs of instability. excessive motion will cause wear of the liner and material debris will initiate retro acetabular and proximal femoral osteolysis. this would eventually lead to failure. at the same time, the trilogy cups (zimmer, warsaw, indiana) with versys circumferentially coated stems have 100% survival in 4 years average follow - up. the locking mechanism in the trilogy is split - ring mechanism, which has been used in several other designs with a good track record. the harris hip score in the v - t group and surviving hg ii (not revised) were excellent or good in 93.2% and good in, but in the total group including the revised hips were 85. the adjusted general health measures (sf36) and disease specific outcome measures (womac) and patients expectations have been previously studied for knee arthroplasty in this region, but not for hip arthroplasty. the hg ii group had, understandably, a significant drop in their womac and sf36 scores due to the inclusion of the 15.6% revision. expectations of the patients, that were mainly relief of pain and ability to walk comfortably, were fulfilled in nearly all the patients (97.6% and 92.8%, respectively). some preoperative problems relatively unique to our culture, flattop toilet and cross - legged sitting on the floor, were not the expectations of the patients and seem to be modified after surgery by the patients. the radiographic evaluation in the present paper showed good positioning of cups and stems in accordance with established standards. the radiolucent lines and pedestal formation in those few cases were not indicative of loosening. there were only 4 cases (9.3%) of thigh pain in this series that had no correlation with the size of the femoral stem. the incidence of thigh pain, which is related to the stability of the prosthesis, is reported between 0 and 28% in different articles. in spite of the literature report of 0.28 - 4% infection and 2.2 - 14.7% thromboembolic events the ulnar nerve injuries were in the contralateral upper limbs from arm malpositioning during anesthesia. the tibial and peroneal nerve injuries from the traction effect of lengthening observed in this report is a recognized problem, and has been reported in the literature with an incidence of 0.3 - 3.7% in hip arthroplasty, usually associated with lengthening of over 1.7 centimeters. the main limitations of the present study are its retrospective nature and small numbers of cases, however, the merits are that it is a single surgeon s experience with uniform technique and post - operative care and being a unique study in iran with the special cultural and daily living habits. the generally satisfactory results of hip arthroplasty as demonstrated by harris hip scores and functional assessments with womac, sf 36 and mactar are shown in iranian society in spite of some cultural and social differences. the outcome of cementless tha is satisfactory and comparable with the literature based on the results of function and survival of this small comparative group. the use of hgii acetabular component should be abandoned, because of the poor locking mechanism of the shell with the liner. | background : cementless hip prosthesis was designed to provide biologic fixation, without the use of cement. the second generation components have shown more reliable bone ingrowths and survival rates. we are reporting a midterm result of two designs of cementless prosthesis in a unique culture with different social habits and expectations.methods:52 primary cementless total hip arthroplasty in 42 patients with the mean age of 48.8 years were retrospectively studied. two groups of prosthesis had been implanted : harris - galante ii (hgii) in 15 and versys - trilogy (v - t) in 37 hips, both from zimmer company. the patients were assessed clinically, radiographically and with harris hip score, sf36, womac, and mactar questionnaires, with 65 months (26 - 136) mean follow-up.results:all the v - t prostheses had survived well. eight of hg ii were revised by the last follow - up in 19 - 102 months. all had undergone acetabular revision and 2 combined with femoral revision. broken tines of hgii cups were seen in 4 radiographs. the 65 months overall survival was 96.2% for femoral and 84.6% for acetabular components. 90% had good or excellent harris hip scores. the functional scores were poorer in the hg ii group. pain relief and improved walking were the two main patients expectations fulfilled in 97.6% and 92.8%, respectively.conclusions:the outcome of cementless total hip arthroplasty (tha) is satisfactory and comparable with the literature based on the results of function and survival of this small comparative group. the use of hgii acetabular component should be abandoned. |
noninvasive prenatal testing (nipt) is becoming increasingly popular in the united states, with increasing advocacy in the medical community. some obstetricians one recent study concluded that cfdna testing had significantly lower false positive rates and higher positive predictive values for trisomy 21 and 18 than standard screening with serum analytes 2. the quadruple serum screen (quad screen) has been favored for reasons that it is far less expensive than cfdna. we present a case where the quad screen was very helpful in management of a highrisk pregnancy in a patient with negative cfdna, allowing timely introduction of thromboprophylaxis and fetal surveillance. our case also suggests a possible therapeutic role for prophylactic doses of enoxaparin on placental function. a 30yearold gravida 5 para 0040 at 11 + 0 weeks gestational age (ga) presented with a history of recurrent spontaneous abortions at 7, 9, and 12 weeks ga. our patient 's antiphospholipid antibody workup was negative, but did demonstrate factor v leiden (fvl) heterozygosity. she denied smoking, alcohol, or drug abuse. due to her past obstetrical history and fvl heterozygosity, a decision was made to start prophylactic enoxaparin 40 mg subcutaneously daily and aspirin 81 mg oral daily at 7 weeks ga. at 14 + 2 weeks a repeat ultrasound exam at 17 + 5 weeks ga was significant for a 2week lag in fetal growth. at 19 + 5 fetal anatomical examination was limited due to intrauterine growth restriction, but no obvious structural anomalies were seen and amniotic fluid volume and placental echotexture both appeared normal. doppler flow studies in both umbilical arteries revealed elevated resistance with s / d ratios of 8.7 and 9.3. first trimester screen was normal with a down syndrome risk of 1/490 (pappa : 0.45 mom, thcg : 1.68 mom, and nuchal translucency : 0.60 mom). however, due to early onset fetal growth restriction, the patient was recommended the 2nd trimester serum screen for assessment of placental function. the results of the quad screen at 17 + 5 weeks ga revealed a msafp of 2.61 mom, thcg of 3.52 mom, inhibina of 3.73 mom and ue3 of 0.40 mom. as anticipated, the down syndrome risk based on the quad screen was 1 in 3. due to the results of the serum analytes and lagging fetal growth, placental dysfunction was suspected and close maternal and fetal surveillance was initiated with weekly visits. prophylactic doses of enoxaparin 40 mg daily and aspirin 81 mg daily were restarted at 19 + 5 weeks ga. a repeat ultrasound examination at 23 + 5 weeks ga revealed worsening fetal growth restriction with an efw of 272 g and ua doppler studies revealed absent end diastolic flow (aedf). surprisingly, at 25 + 5 weeks ga normal end diastolic flow was documented in the umbilical arteries with s / d ratios of 3.0 and 3.5. at 27 + 6 weeks ga ua doppler flow studies continued to be normal with s / d ratios of 2.49 and 3.08 and an efw of 504 g. intense fetal monitoring was initiated to assess fetal wellbeing in view of the interval fetal growth and fetal weight. due to a biophysical profile (bpp) of 6/10 with nonrepetitive variable decelerations the patient was observed on labor and delivery and a course of betamethasone was administered for fetal lung maturation. the nonstress test was subsequently reactive with a reassuring bpp of 8/8 and ua s / d ratio of 3.0. at 29 + 2 weeks ga the patient was admitted with preterm premature rupture of membranes and the betamethasone course was repeated. two days after admission the patient went into spontaneous labor and due to a nonreassuring fetal heart rate tracing, delivery by a cesarean section was performed. apgar scores were 5, 7, and 9 at 1, 5, and 10 min and neonatal weight was 720 g. on the pathology report the amniotic membranes were described as dull, thin, delicate, and green tinged. the placenta weighed 170 g with signs of acute vasculitis, funisitis, and chorioamnionitis. in recent years nipt is becoming increasingly popular. even in patients not at high risk of fetal aneuploidy some providers are offering nipt 1. however, nipt is unable to evaluate placental dysfunction, and can not be used for determination of risk for preterm delivery, fetal growth restriction, preeclampsia, placental abruption, intrauterine fetal demise, and perinatal death. additionally, prohibitive cost has been identified as a major hurdle in the more routine use of nipt in low risk patients 3. in a costconsequence analysis in belgium, the authors concluded that the price of nipt needs to be lowered substantially due to the government 's limited resources for universal reimbursement 4. in our patient due to a normal first trimester serum screen, the second trimester screen was not obtained initially. however, due to the ultrasound findings, we obtained the second trimester serum screen and all four analytes on the quad screen were abnormal indicating that iugr was probably related to placental pathology. due to the abnormal quad screen results intensive maternal and fetal monitoring was initiated which allowed us to deliver a live fetus in a timely fashion. while nipt may be the most accurate screening for certain aneuploidies, the american college of obstetricians and gynecologists and the society of maternalfetal medicine caution that its use is solely for fetal aneuploidy and that nipt should not be part of routine laboratory assessment, but instead should be an informed patient choice for women at high risk of fetal aneuploidy 5. in patients who are at risk of placentalbased pregnancy complications the integrated screen may be a better option for assessment of fetal chromosomal abnormalities, placental function, and fetal risks associated with placental dysfunction. in our case, the quad screen results helped us in initiating an extensive surveillance of the pregnancy which may have allowed the pregnancy to reach viability. the suspected placental dysfunction based on the serum analytes led us to restart prophylactic enoxaparin and aspirin which appears to have helped in our case. the previously abnormal ua blood flow with aedf improved markedly following the initiation of the above medications with normalization of blood flow in the umbilical arteries. normal ua s / d ratios were maintained until delivery and the authors believe this reversal may have been due to a therapeutic effect on placental blood flow from thromboprophylaxis of enoxaparin. previous authors have recognized that aedf may rarely temporarily improve only to have subsequent worsening 7. further there is no evidence in the literature that once placental function has deteriorated that prophylactic doses of enoxaparin and aspirin can reverse the pathologic process. in our case, the enoxaparin and aspirin were restarted due to the finding of the abnormal serum analytes. even though prior reports do not support beneficial effects of enoxaparin once umbilical arteries reveal aedf, in our case it appears to have had a therapeutic effect. it is our belief that if prophylactic enoxaparin had not been restarted in a timely fashion, downstream resistance of the placental circulation would have continued with a high likelihood of a fetal demise. we believe this case may also demonstrate a therapeutic role of prophylactic doses of enoxaparin. while nipt is a far more accurate screening tool for certain fetal aneuploidies, our case illustrates the role of the serum analytes of the quad screen as markers of placental function in addition to aneuploidy screening. abnormalities in these markers can identify patients at highest risk for poor pregnancy outcomes due to placental dysfunction with consequent modification of management of care during pregnancy 8. in patients that undergo nipt only more recently, authors have argued in favor of offering nipt universally to all pregnant women in place of the serum screen. since more women have placentalbased pregnancy complications, especially in the younger age group, we strongly favor the integrated serum screen over nipt which will help identify these highrisk women and allow timely intervention. written informed consent was obtained from the patient for publication of this case report and accompanying images. | key clinical messagenoninvasive prenatal testing (nipt) is becoming increasingly popular with some offering it as a primary screen option in all patients in place of serum screening. serum screening offers insight into placental function, which nipt does not. abnormal levels of analytes in the serum screen have been associated with pregnancy complications. |
common placebos are usually inert tablets or sham surgery based on false information. since the publication of henry k. beecher 's in 1955, the phenomenon has been considered to be clinically important. placebo effects were shown to be genuine psychobiological events attributed to the overall therapeutic context. from a psychological point of view, many mechanisms might contribute to placebo effects, including expectations, conditioning, learning, motivation, memory, reward and anxiety reduction, etc. apparent benefits of a new drug may derive from the placebo effect but not from the drug per se. therefore, modern clinical trials control for this effect by using a placebo, in which the subjects are blinded as to whether they receive a drug treatment or a placebo. in this way, placebo - controlled trials might provide information about the real effectiveness of a drug. the view of placebo effect was notably challenged when a systematic review of clinical trials in 2001 concluded that there was no evidence of clinically important effects, except perhaps in the treatment of pain and other continuous subjective outcomes. since evidence - based medicine (ebm) is increasingly emphasized, placebo is still widely used in clinical trials other than in the treatment of pain and continuous subjective outcomes. currently, international journals as well as clinical researchers are inclined to avoid or even ignore the ethical aspects of placebo. however, accumulating ethical concern has arisen from the worldwide use of placebo in randomized control trials (rcts), because the investigators in a certain trial may render its participants without early and optimal treatment. where for compelling and scientifically sound methodological reasons the use of placebo is necessary to determine the efficacy or safety of an intervention and the patients who receive placebo or no treatment will not be subject to any risk of serious or irreversible harm. (extracted from the declaration of helsinki) a recent study, the european cooperative acute stroke study iii (ecass iii) reported by bluhmki and by hacke supported the use of alteplase three hours after the onset of stroke symptoms, while together with other studies, ecass iii raised some concerns about thrombolytic studies. despite repeated emphases of early treatment on acute ischemia, the control group in ecass iii failed to receive any treatment except the placebo. the lack of evidence - based therapies should not justify the exclusion of empirical use of anti - platelet agents, or others. in this regard, it is almost always the case in china, since traditional chinese medicine has been a routine for patients suffering from cerebrovascular ischemia. another example of clinical trial that may raise ethical concerns is the one conducted by kappos,, about oral fingolimod in treating relapsing multiple sclerosis (ms). this placebo - controlled trial revealed that fingolimod improved the relapse rate, the risk of disability progression, and end points on mri. however, the longer the trial lasts, the longer the patients in the placebo group must go without treatment. in this context, the well - acknowledged and widely used therapeutic approaches should be considered first and foremost as controls, when rcts are designed. although placebos may be helpful in treating ms, the better - acknowledged ways such as interferon - beta and glatiramer acetate can potently alleviate the sufferings of patients with ms and can readily serve as positive controls in that study. as a clinician, the sanctification of placebo is partly due to the repeated emphasis of rcts as the gold standard of clinical trials. dichotomy seems necessary in evaluating the role of placebo in scientific research ; the need for placebo - controlled design can not be denied at certain stage of drug development, whereas for larger phase iv studies, better alternatives should be considered. ethically, the clinicians are always expected to provide their patients with the best choices of treatment other than placebo, and the best available treatment as a positive control other than placebos is preferable to the patients. in neurological field, for example, aspirin is widely accepted as a control in trials with regard to prevention or treatment of cerebral ischemic disorders, because its prophylactic as well as therapeutic effects on cardiovascular events are well established. although the control group is a must in rcts, an alternative way such as a delayed - start approach, can be utilized to avoid ethical issues that may arise. delayed - start studies are designed primarily to test the disease - modifying effect of a drug, whereas they can reduce the ethical concerns that patients in the placebo group must go without treatment until the end of the study. the control subjects enrolled in the aforementioned thrombolytic study should at least start to accept conventional medication therapies immediately after the time - window for thrombolytic studies. similarly, clinicians should always bear in mind that no treatment equals killing for their patients. clinicians / researchers need to understand the implications of offering no treatment in certain situations before enrolling experimental subjects. more importantly, a thorough explanation about the aforementioned possible implications should be performed by the clinicians. the placebo effect is produced on the premise that the subjects believe the effectiveness of placebo. this is different from the practice in rcts in which the subjects are informed of the possibility of accepting only a sham medical intervention. informed consent for a study is usually required to be considered ethical, including the disclosure that some patients are to receive placebos only. in this case, the subjects do not know whether they might be getting a real treatment or a sham one, and thus might suspect the efficacy of the medical interventions even before the trials. as a result, placebos do not work as strongly in clinical trials, as in basic research. what 's worse, the deception involved in the use of placebos creates conflicts between the hippocratic oath and the honesty of the doctor the purpose of the clinical trials should be explained in detail in the process of obtaining informed consent from the participants, while it is not always the case, especially in low - income countries. for patients in low - income countries, the participation in clinical trials is intriguing simply because poor patients have the chance of getting free treatment, or they can get rewards for attending the research. this may lead to selection bias as well as ethical debates because the poor participants have no better choice than attending the trials. the honest relationship between doctors and patients is further worsened because of the notion of the patients that they are utilized. in summary, although the ethics of placebos have been debated frequently in history, even in the revision process of the declaration of helsinki, placebos have given rise to ethical debates much more often than ever before. as the pilgrimage of placebo is still on the way, refinement of controls in rcts | a placebo is a sham medical intervention that can produce a placebo effect. laboratory evidence supports the existence of several mechanisms of placebo effects in both healthy population and patients with a variety of medical conditions. the ethics of placebos have long been debated. however, accumulating ethical concern has arisen from the worldwide use of placebo in randomized control trials (rcts), which may render their participants without early and optimal treatment. although the pilgrimage of placebo is still on the way, refinement of controls in rcts is worth paying new attention to. |
prior to the genome era, compositional signatures (1) or sequence alignments (2) were used to delineate the phylogenetic patterns across organisms. the availability of entire genome sequences has sparked a further development of methods for phylogenetic reconstructions on a genome - wide scale. following this long tradition in molecular evolution, similar methods were expanded to encompass complete genome information, based on either compositional patterns (3,4) or concatenated alignments of orthologs (5,6). more recently, methods that exploit the entire gene complement of completely sequenced genomes have been developed. these include phylogenies based on patterns of conservation of gene order (7), gene fusion events (8), gene content (7,913), protein folds (12), and average ortholog similarity (14). only a few of these approaches were successful in resolving difficult cases, such as correctly grouping pathogens having highly reduced genomes with their free - living relatives and clustering proteobacteria into a monophyletic group (7,9,14). in addition, most of the above mentioned methods are (i) not scalable to hundreds of species (e.g. concatenated alignments), (ii) unable to place correctly species with reduced genomes (13) and (iii) strongly affected by the number and phylogenetic proximity of species (e.g. gene order) (15). herein, we define a new composite measure termed genome conservation, which expresses both the conservation of sequence and gene content between two genomes. this value is derived from the sum of alignment scores between all proteins for every pair of organisms. larger genomes tend to share more genes, irrespective of their phylogenetic distance (9). thus, a higher conservation score can result from a higher number of shared genes, rather than from phylogenetic proximity. to counterbalance this effect the genome conservation method is naturally adjusted for missing genes and for gene lengths. if a gene is absent in one of the compared genomes, its contribution to the similarity is zero. however, if this absence is a direct result of reductive evolution and difference in genome sizes, absence of this gene would be calibrated by the normalization scheme described below. the gene length is taken into account in the summation of the blast scores : longer genes generate greater alignment scores and thus, would be more important contributors than shorter genes. thus, the final similarity is based on both gene content and average sequence similarity of genes, with the adjustment for gene length. to obtain a similarity measure between any pair of genomes, we have compared all proteins using blastp (16), with an e - value cut - off of e-10, and used the bit - score as the measure of similarities between two sequences. the bit - score has the advantages of being independent of the searched database size. moreover, it does not calibrate for protein length, thus, longer proteins have a greater impact than shorter ones. to eliminate noise created by paralogy in the cases when multiple hits were observed, only the best hit was used (i.e. the most significant sequence similarity). the total number of sequence similarities thus obtained exceeds 25 million pairs for 153 genomes. we will denote the sum of all best hits between genomes a and b as (a, b). the usage of best blastp hits, when compared with orthologs, abolishes the issue of ortholog identification, which is still an unresolved problem for datasets of this size. however, it results in non - reciprocal genomic similarities, that is (a, b) (b, a). to calculate the conservation between genome a and genome b, we used the minimum of the two values, i.e. min((a, b), (b, a)). to normalize for differences in genome sizes, we calculated the genome conservation distance measure d in two ways (d1 and d2) : d1 = 1-s / min((a, a), (b, b)), or d2 = ln(s/(2 (a, a) (b, b)/(((a, a)) + ((b, b))))). d1 (9) and d2 (7) correspond to strategies for the transformation and normalization of self - similarity and adjustment for genome sizes, proposed previously. the tree discussed in the text was produced using d2, for consistency with gene content trees, reported to be optimal with this normalization (7). gene content trees were generated as described elsewhere (7), using identical data as for the genome conservation tree. average ortholog similarity was computed as the pairwise score divided by the smallest number of hits between the genomes, divided by 1000 as a scaling factor, or (a, b)/(min(n(a, b), n(b, a)) 1000), where n(a, b) is the number of hits between genomes a and b. these values were also normalized between 0 and 100, for comparison with other measures (figure 2). the values of pairwise distances were used to construct a distance matrix ; trees were calculated using quicktree (17). bootstrap values were generated by resampling the pool of alignment scores between pairs of genomes for 1000 times. this procedure was not applied to the gene content tree, as it requires a jack knife approach rather than genuine bootstrapping (7). in order to evaluate the conservation within taxonomic units, we computed an average of the conservation values, while eliminating taxonomic over - representation. within each species, we averaged the pairwise conservation values of its strains. for higher taxonomic ranks (e.g. genus, family), the conservation between each pair of its sub - ranking taxa was averaged, thereby avoiding bias of unequal taxonomic sampling of sequenced species. ideally, a species distance metric should enable the reliable inference of phylogeny across variable evolutionary time scales and taxonomic ranges. to assess the performance of genome conservation, we used standard neighbor - joining procedures (17,18) and produced a phylogenetic tree for 153 species with known genomes (19). the obtained tree (supplement 1) clusters all the major clades consistently with current taxonomic knowledge (20) and is similar to the gene content - based tree (7,9), with a number of important exceptions, described below. overall, compared to gene content phylogeny (7), a previously proposed and widely accepted method for whole - genome - based phylogenetic reconstruction, genome conservation produces significantly improved results (supplement 2). in the genome conservation tree, the alpha-, gamma-, delta- and epsilon proteobacteria form highly supported clades and consistently form a monophyletic proteobacterial clade (figure 1a). beta proteobacteria form a clade inside gamma proteobacteria, and there are two inconsistencies with the accepted taxonomy. first, aquifex aeolicus is placed within proteobacteria. second, the pseudomonas clade and nitrosomonas europaea seem to be flipped with respect to each other. both are coupled to tree nodes with low bootstrap values (686 and 585 out of 1000, respectively). in comparison, the gene content method fails to produce a plausible phylogenetic reconstruction of proteobacteria (figure 1b), or other taxa (supplement 2). furthermore, the average ortholog similarity approach generates a plausible scenario for evolution of proteobacteria in the shallow branches of the tree, but fails to group both delta and epsilon subdivisions with other proteobacteria, defining them as separate deeply branching groups (data not shown). the recognition of abundance of horizontal gene transfer (hgt) among archaea and bacteria led to the questioning whether a reliable bacterial phylogeny can possibly be reconstructed (22). yet, the overall agreement of whole - genome based methods, such as genome conservation, average pairwise sequence similarity, gene content and 16s rrna phylogenies clearly demonstrates the existence of a consistent bacterial phylogeny (23). in comparative genomics, it is crucial to accurately measure the evolutionary distance between organisms. levels of conservation of 16s rrna sequence may not be sufficient to estimate the evolutionary distance and guarantee species identity, especially in the case of recently divergent organisms (24). presently, species distances are often estimated in millions of years of divergence (2527). however, the time of divergence estimated from the molecular clock is extremely imprecise (28) and only organisms with fossil records can be dated with some accuracy (29). another popular form of estimating evolutionary distance is measuring the number of neutral substitutions per site. this technique is appropriate for higher eukaryotes or closely related bacteria ; however, saturation in mutations hinders reliable estimations for highly divergent bacterial species (30). we propose the use of pairwise genome conservation metric as a stable whole - genome based evolutionary measurement to assess conservation between organisms. a pictorial representation of the genome conservation matrix across all presently sequenced organisms readily demonstrates the ability of this species distance metric to define evolutionary relationships at variable taxonomic ranges, from strain variants up to the three domains of life (figure 2a ; the complete set of values is available as supplement 3). we have compared the similarity matrices derived from the three principal genome - based phylogeny methods, namely genome conservation, gene content and average ortholog similarity. all matrices evidently contain a strong phylogenetic signal, represented both by the diagonal (self - hits) and various groupings of related taxa (figure 2). all matrices are also able to clearly separate the three domains of life and delineate closely related groups. these similarity matrices are transformed to distance matrices for the construction of phylogenetic trees (see materials and methods), which produce different results. massive gene loss in some intracellular parasites, such as buchnera and wolbachia, creates an effect where these species share their entire gene content with multiple, distantly related lineages. the similarity estimated from gene content, normalized by the size of the minimal genome, fails to accurately estimate species distances (figure 2b). the genome conservation approach also suffers from the same effect, however on a significantly lower level (figure 2a). finally, average ortholog similarity (figure 2c) is independent of genome size, and thus resistant to the problem of drastically reduced genome sizes. however, it is evident that genome conservation allows the detection of phylogenetic groupings at variable taxonomy ranges, from stains up to entire domains of life (figure 2a). despite the fact that some of these patterns are also present in the gene content and average ortholog similarity matrices (figure 2b and c, respectively), their resolution is less pronounced, reflected by a blurred distribution of color - encoded similarity across taxa. having demonstrated that the genome conservation metric reflects meaningful evolutionary relationships, we subsequently explored its ability to resolve long - standing arguments in defining the concept of bacterial taxa (31). using genome conservation as a measure of evolutionary divergence, we investigated how the levels of taxonomical classification in bacteria correspond to evolutionary distances (figure 3). overall, there is a clear decrease in genome conservation at the higher taxonomic ranks. however, the definition of some taxonomic units is not precise, and the ranges of genome conservation for various ranks often overlap (figure 3). the other two measures, namely, gene content and average ortholog similarity, also exhibit a gradual reduction across increasing taxonomic distances, yet within a narrower value range, which renders them less effective in detecting taxonomic ranks (data not shown). it is worth noting that in all cases, the ranks of genus and family are the least well - defined, according to any genomic similarity measure. in particular, we found that the broadest distribution of genome conservation scores is observed within the genus rank. for example, mycobacterium tuberculosis and m.bovis are 96% similar whereas mycoplasma gallisepticum, m.pneumoniae and m.penetrans are only 16% similar. another example of questionable classification involves prochlorococcus marinus strains mit9313 and med4, which present a challenging case for rrna - based taxonomy. these two strains are 97% similar in their 16s rrna sequence (32), while representing distinct genetic populations, with a 2-fold difference in genome size and content (33), as well as specific phenotypic properties. genome conservation between these strains is only 49%, which is more typical for distances between genera within a family, rather than strains within a species. the genome conservation measurement of classification provides a possibility of a precise and quantitative definition of each taxonomic unit and a guide for future taxonomic classification. in summary, the genome conservation method uses a genomic perspective of gene content and couples it with sequence divergence at the whole - genome level. despite the limitation that an entire genome is required in order to place a species in its taxonomic context, this approach can delineate poorly resolved taxa and potentially be coupled with local rrna - based phylogenies. however, with the new approaches to whole - genome sequencing in environmental genomics (34), the genome conservation approach can provide an unambiguous and consistent classification system for the newly discovered species. the proposed species distance metric provides a clear measure based on sequence divergence for use in comparative genomics and taxonomy. part of the complete tree of life containing the proteobacteria generated by genome conservation (a) and gene content (b) methods. classes are color - coded, and the spirochaetum leptospira interrogans and deeply branching aquifex aeolicus are shown in black. trees were generated using d2 normalization as described in materials and methods ; the complete tree is available in supplement 1. similarity matrices across all completely sequenced organisms, derived from genome conservation (a), gene content (b) and average ortholog similarity (c). genome conservation and gene content were computed using d1 normalization (see materials and methods). species are ordered consistently across the different matrices, sorted according to their position on the genome conservation tree (supplement 1), and major clades are indicated in (a). the conservation levels in percentages are color - coded, and the values for individual pairwise scores for genome conservation are available (supplement 3). it is evident that there are three fields of values, seen as lighter blue sub - matrices representing eukarya, archaea and bacteria, from top left to bottom right in (a). highly similar groups are evident, for instance escherichia coli strains (red or yellow) and enterobacteria (green), both within -proteobacteria. for see text for discussion, genome conservation computed using d1 normalization (see materials and methods). | species evolutionary relationships have traditionally been defined by sequence similarities of phylogenetic marker molecules, recently followed by whole - genome phylogenies based on gene order, average ortholog similarity or gene content. here, we introduce genome conservation a novel metric of evolutionary distances between species that simultaneously takes into account, both gene content and sequence similarity at the whole - genome level. genome conservation represents a robust distance measure, as demonstrated by accurate phylogenetic reconstructions. the genome conservation matrix for all presently sequenced organisms exhibits a remarkable ability to define evolutionary relationships across all taxonomic ranges. an assessment of taxonomic ranks with genome conservation shows that certain ranks are inadequately described and raises the possibility for a more precise and quantitative taxonomy in the future. all phylogenetic reconstructions are available at the genome phylogeny server :. |
hyperplasia refers to tissue growth into the oral cavity, located over the alveolar ridges or the soft tissues of the vestibular sulcus. its etiology is multifactorial, but some irritative factors are more commonly associated, such as periodontal disease, poor oral hygiene, smoking, and poor denture fitting. the treatment of this kind of lesion includes elimination of the causing factors and surgical removal of the lesion. if the causal factor persists, the tissue becomes more fibrous over time [2, 3 ]. the most common techniques used for removing the hyperplastic lesion are surgical scalpel, electrical scalpel, carbon dioxide laser, erbium : yag laser, neodymium : yag laser, and diode laser. the co2 laser is an appropriate option for surgical procedures in soft tissues since it operates at a wavelength of 10.6 n, which is within the medium range of the electromagnetic infrared spectrum. this wavelength is absorbed by tissues with high water content [4, 5 ]. this energy is transformed into heat, causing cell rupture from water boiling ; therefore, tissues with high water content suffer less damage. many advantages of the co2 laser include the possibility of minimal bleeding, decreasing edemas, flexibility of the wound healing tissue, reduced postoperative pain, and no need of a conventional suture [610 ]. these positive aspects of the use of a co2 laser has allowed an improvement in maxillofacial surgeries. despite some disadvantages of co2 laser, such as the delay in the initial repair chronology due to heat necrosis [11, 12 ], this technique provides suitable repair without scar formation and constitutes an alternative to the conventional incision and suture method. a sixty - three - year caucasian old female presented at the dental clinic of the university (so jose dos campos dental school unesp) looking for dental treatment. the clinical exam showed completely edentulous and presented a hyperplastic lesion over the alveolar ridge extending to the vestibular sulcus in the lower anterior region (figures 1 and 2). according to the patient, two previous surgeries were performed to remove the excess of mucosa, but it relapsed twice. this lesion could impair the retention and stability of a future prosthesis. considering this clinical situation and the relapse history, the procedures for the confection of a complete denture consisted of a single impression of the upper and lower arch with an irreversible hydrocolloid to record the ridge and lesion area. casts were obtained (figure 3), and experimental bases (figure 4) with wax ridges were made, outlining the lesion area. the teeth were mounted following the wax ridges (figure 5) and aesthetic approval was received from the patient. after an adequate aesthetic result was obtained, the lesion area outlined at the lower cast was removed with a burr (figure 6) to the extension of the denture base. this area of the cast was covered with wax (figure 7), and both the upper and lower dentures were cured (figure 8). the lesion removal was made using a carbon dioxide laser (sharplan 15 f, israel - fapesp 97/07645 - 2), and to carry out the vaporization the co2 laser was used in continuous mode with a focused beam with diameter of 4 mm and 8 watt (figure 9) the vaporized area was clinically evaluated to verify the absence of bleeding (figure 10). safety glasses, masks, and procedure gloves protected the professionals who participated in the surgery, and vaporization was done under constant aspiration of the plume. dentures were positioned, and the lower denture was immediately rebased with a soft tissue conditioner to stabilize the denture and facilitate wound healing (figure 11). postoperative examination was done after seven days and showed satisfactory tissue repair at the surgical area (figure 12). the postsurgical exams were done after 7, 14, 21, and 30 days (figure 13) of continual wearing of the prosthesis. the clinical results of the case report presented are in agreement with other studies utilizing co2 laser for soft tissues. in the current study, the co2 laser was used in continuous mode to control the extension of thermal damage using a focused beam to reduce its intensity.. histologically demonstrated that the co2 laser, in both the pulse and continuous modes, allowed superficial ablation with minimal thermal damage. some authors, like dobry., also affirmed that the pulse mode caused less thermal damage, but that a longer actuation time was needed with the tissue. the advantages of using a co2 laser has been clinically demonstrated in the present study, presenting minimal bleeding during the surgery with no need of sutures while also presenting a good healing response, with minimal wound contraction, less inflammatory reaction, and good re - epithelialization with no scar formation. these conditions were also found in the studies of de arruda paes and niccoli - filho and keng and loh who clinically demonstrated that co2 laser is ideal for use in these kind of surgeries, bringing comfort and aesthetics to the patient. explained that less wound contraction occurs because the co2 laser does not remove the tissue collagen.. showed that co2 laser is effective in vaporizing oral mucosa and achieving haemostasis by vessel coagulation. these findings are in agreement with niccoli - filho. who also demonstrated that good aesthetic and functional characteristics were quickly achieved in an oral surgery using co2 laser, enabling an earlier prosthetic rehabilitation. thus, the co2 laser is a useful instrument, which provides control of the surgical field and esthetic and functional results. the technique presented in this paper was easily executable and allowed a better prediction of the surgery results. based on the results of the present case report we can conclude : the use of a co2 laser radiation allowed good haemostasis at the surgical area, absence of infections and post operative comfort ; the use of co2 laser radiation to remove hyperplastic tissue make the use of prosthesis feasible and promoted an immediately condition to reestablish the aesthetic and functional aspects. | the aim of this study was to present a case report of the surgical removal of hyperplasia in the oral cavity, using carbon dioxide (co2) laser radiation and rehabilitation with a complete denture. epulis fissuratum occurs in complete denture patients, because a constant irritative action induces the mucosa to grow under poorly fitting dentures. these lesions must be removed, and to avoid a relapse, new complete dentures should be made to maintain healthy surgical tissues. the clinical sequence presented in this case shows a completely edentulous patient with epulis fissuratum on the lower alveolar ridge extending to the vestibular sulcus of the anterior region of mandible. immediate complete dentures were made prior to the lesion removal with co2 laser radiation, providing satisfactory results in oral function and tissue health. |
there is much controversy regarding the use and timing of enteral feeding support in patients with dysphagia and aspiration risk. the decision process becomes far more complex in those patients with advanced dementia, due to ethical and moral issues associated with the continuing comfort care per os (po) feeds with the knowledge that the person is aspirating, and the use of percutaneous endoscopic gastrostomy tube (peg) placement, and associated mortality rates. dementia is a leading cause of death in the us, with mortality affected by aspiration, hydration, and nutritional status. data in the year 2000 show there were approximately 4.5 million people in north america with a diagnosis of dementia, and more than half progressed to the moderate to severe stages of their disease. in 2001 there were 24.3 million people in the world with dementia, and by 2040, the number is estimated to increase to over 81 million. the prevalence of dementia is estimated to double every 5 years after 65 years old, and at age 85 years, the prevalence is approximately 50%.1,2 dementia is a terminal diagnosis. alzheimer s disease and other related illnesses causing dementia are progressive, incurable, and lead to a complete loss of cognitive function, and subsequent death. a characteristic feature of the final phase of dementia, which can last from 6 months to 2 years, is loss of interest in eating, dysphagia, or both.3 an estimated 60% of nursing home residents have dementia, with approximately half (480,000) being in the last stages of their disease. the prognosis and progressive clinical course affects the important decision about peg insertion.4 mean survival after dementia diagnosis varies between 1 and 16 years, but one - third of demented individuals live to advanced stages.4 the ability to perform activities of daily living are typically lost in a hierarchical fashion, with eating and bed mobility lost last. the most severe phase of dementia is characterized by loss of capacity to provide self - care in basic activities of daily living, such as eating, bathing, and walking independently. in this stage there are also many behavioral symptoms that compromise quality of life for both patients and caregivers, and are sources of great stress to the latter, with institutionalization being the ultimate consequence. many patients and their families have limited understanding of the terminal nature of a dementia diagnosis. due to the complexity that characterizes advanced dementia, many moral, ethical, religious, and medical decisions arise ; these involve appreciating the risks, benefits, and alternatives to adjunctive enteral (peg) feeding, non per os (npo) status, and the role of comfort care or compassionate po which is defined as continuing to feed a patient by mouth for quality of life and or comfort, in spite of and with information that the patient is aspirating and at risk for aspiration pneumonia or worse. surprisingly, there is little discussion of these issues in the literature and in routine medical care. in this paper, we reviewed the existing literature on peg placement in patients with dementia, in terms of subsequent mortality rates, beginning with the hypothesis that peg does not prolong life. we also explored the impact of peg in specific dementia groups to identify refined prognostic indicators that may be further used to develop comprehensive guidelines for the placement of pegs in this population. a systematic literature search was performed using pubmed, to identify studies and their levels of evidence. key words were identified as search terms : aspiration, dysphagia, swallowing difficulty, dementia, alzheimer, peg, and enteral and feeding tube. three searches gleaned the most results. the search combining dementia and (dysphagia or aspiration) and (endoscopic gastrostomy or enteral) yielded 97 articles. the search combining (dementia or alzheimer) and (dysphagia or aspiration) and (percutaneous endoscopic gastrostomy or enteral or feeding tube) yielded 99 articles, and the search gleaning the most results, 100 articles, and therefore used for this study combined the following terms (dementia or alzheimer) and (dysphagia or aspiration or swallowing difficulty) and (percutaneous endoscopic gastrostomy or enteral or feeding tube). our inclusion criteria required that the article 1) must have been a scientific research paper ; 2) must have addressed dementia, dysphagia or aspiration risk, or peg tube placement ; and 3) must have been originally written in or translated into the english language. the exclusion criteria comprised the following : 1) studies focused solely on the pediatric population ; 2) studies focused on peg placements in other diagnoses only (studies including cohorts with dementia patients among other neurological diagnoses were not excluded if the dementia patients were separated out in the results) ; 3) abstracts only ; and 4) nonscientific research, editorial, text book, or opinion - based papers. once the articles were selected, the reference sections were scanned for other relevant studies. it was then determined whether the study was derived from or based on a systematic review of the literature. next, we looked at the size of the study, the number of patients with dementia within the study, and specific outcomes, most importantly survival, comparing those patients with dementia who received pegs to those who did not. ten articles were highlighted (the review process is summarized in the preferred reporting items for systematic reviews and meta - analyses [prisma ] diagram as figure 1). overall results are summarized in table 1, stratified by year, study design, number of subjects, level of evidence, and study findings. the retrospective case series, which included a dementia subgroup containing 103 people with a peg, revealed 54% mortality at 1 month and 90% mortality at 1 year.1 a second large retrospective database spanning 10 years, which contained 8,688 demented patients who received pegs, broke down mortality rates between males and females.2 according to this study, at 1 year, mortality rates for males and females with dementia status post - peg insertion were 61% and 50%, respectively. at 3 years, the mortality for males and females with dementia status post - peg was 78% and 84%, respectively. a third retrospective analysis looked specifically at indications for and survival status post - peg in patients 65 years and older.3 the mean age of participants was 79 years old. the participants were classified into seven diagnoses : stroke ; dementia ; parkinson s ; other neurological diseases (mainly amyotrophic lateral sclerosis) ; malignancies with dysphagia ; malignancies without dysphagia ; and miscellaneous. results based on time frames were as follows : at 30 days, demented patients had 25% mortality ; at 3 months, 37% mortality ; and at 1 year, 58% mortality. another retrospective study, of 90 patients with dementia and dysphagia and of average age ~86 years, revealed 14.4% mortality within 30 days and 1-year survival of 54.4%.7 a well - known retrospective study from the va medical center in washington dc had 41 demented patients referred for pegs.8 after educating family members, 23 received pegs and 18 did not, secondary to family member refusal after discussion. the median survival in the group status post - peg was 59 days compared with the group without the peg, who survived 60 days. a retrospective cohort study with 311 patients with pegs, 143 with dementia, compared demented patient survival with peg to that of patients with other diagnoses (patients without dementia).9 the 12-month survival was 51% in patients with dementia, and 49% in the group without dementia. more than 20% of patients with dementia lived more than 3 years after peg. two predictors of poor survival after peg (among other medical issues) were patients who were male and older than 80 years of age. in this study, there was no evidence to support a poorer prognosis after peg in elderly people with dementia compared with elderly who are cognitively intact, unless the patients are older than 80 years of age, male, or have the other poor prognostic indicators mentioned in this study, including hypoalbuminemia. one retrospective study audited 719 patients who died within 30 days after peg insertion.10 of the 719 patients who received pegs, 135 (18%) had dementia, 82% were 70 years of age or older, and 50% were greater than 80 years of age. of note, there was a particularly low rate (29%) of written consent for the procedure among dementia patients. after their review, acknowledging the report that mortality rate of patients with dementia fed by gastrostomy is considerable, the group in this article advised against gastrostomy in dementia patients.10 a randomized prospective study looked at 99 hospitalized patients with advanced dementia.4 the median mortality at 6 months in the group with status feeding tube was 195 days, and in the group without the feeding tube, this was 189 days. overall, the peg and non - peg median mortality at 6 months was no different, at 50%. a prospective study, which included a dementia cohort of 55 patients, compared tube - fed (tf) versus (vs) non - tf patients.5 mortality for the peg patients at 6 months was 44% as compared with 26% among controls. the authors concluded that only 50% of demented patients with inadequate oral intake are likely to survive beyond 6 months after peg placement and that no improvement in performance status is likely to occur in any patient. this study specifically focused on patients with inadequate oral intake secondary to dementia, in order to determine whether the nutritional parameters at the time of peg placement would predict survival. it also recommended that prior to initiating peg feeding, the limited benefits that would be achieved should be taken into consideration. a prospective observational study of 674 patients included 280 dementia patients after peg placement.6 over half of the patients were over the age of 80 years, with an age distribution similar to that of patients undergoing peg placements nationally in the united states. the median survival was 171 days in patients over age 80 with dementia, which was worse when compared with other subgroups. the median survival reached 423 days in demented patients from nursing homes and 467 days in demented patients younger than 80 years. results from five studies reported mortality for the patients with dementia after peg placement.57,10, the studies reviewed reported on varied mortality time frames. the specific results were : 13%18%,6 14.4%, 18%,10 25%,7 and 54%.5 one study reported 3-month mortality at 37%.7 six month mortality was revealed in one study as 44%.12 the mortality at 1 year was reported in five of the studies reviewed. those specific results were as follows : 45.6%, 51%,9 50%61%,6 and 58%.7 one study reported 1-year mortality at 90%.5 one study revealed mortality at 3 years to be 78%84%.6 results of comparison studies regarding median mortality in tf patients vs non - tf patients were as follows. one study revealed dementia patient median survival with a feeding tube as 195 days (6.5 months) compared with median survival without tube feeding as 189 days (6.3 months), with no obvious difference in survival when comparing both groups.11 the following are results from individual studies. one study revealed that the group status post - peg insertion had 44% mortality vs controls who had 26% mortality. this study revealed a higher mortality for patients with dementia who had pegs in place.12 another study revealed median survival after peg to be 59 days, a low number. yet another study revealed patients with dementia and peg who were in nursing homes demonstrated higher median survival at 423 days, whereas dementia patients with peg and stroke median survival was at 181 days, a similar number to the study comparing tf vs non - tf patients.13 a large retrospective database spanning 10 years, which included 8,688 demented patients who received pegs, broke down the mortality rates between males and females.2 this study revealed females fared slightly better, though mortality rates were still high after peg. the female dementia subgroup had 50% mortality at 1 year and 78% at 3 years. the male dementia subgroup had 61% mortality at 1 year and 84% mortality at 3 years. as previously reported in retrospective studies, an analysis looked specifically at the following for patients age 65 years and older : indications for peg placement, and survival after peg.7 the mean age of participants was 79 years old. patients were classified into seven diagnoses : stroke, dementia, parkinson s, other neurological diseases (mainly amyotrophic lateral sclerosis), malignancies with or without dysphagia, and miscellaneous. mortality rates were broken down farther into specific time frames : 30 days, 3 months and 1 year. regarding consideration of age prior to placement of peg in patients with dementia, only 33% of patients over age 80 were still alive after 1 year, as compared with 73% under age 80. in this particular study, dementia was not found to be a negative prognostic factor for survival in elderly patients after peg, specifically those younger than 80 years old. another retrospective study of 90 patients with dementia and dysphagia, whose average age was 86 years, revealed 14.4% mortality within 30 days, and 1-year survival of 54.4%. the retrospective case series, which included a dementia subgroup containing 103 people with a peg, revealed 54% mortality at 1 month and 90% mortality at 1 year.1 a second large retrospective database spanning 10 years, which contained 8,688 demented patients who received pegs, broke down mortality rates between males and females.2 according to this study, at 1 year, mortality rates for males and females with dementia status post - peg insertion were 61% and 50%, respectively. at 3 years, the mortality for males and females with dementia status post - peg was 78% and 84%, respectively. a third retrospective analysis looked specifically at indications for and survival status post - peg in patients 65 years and older.3 the mean age of participants was 79 years old. the participants were classified into seven diagnoses : stroke ; dementia ; parkinson s ; other neurological diseases (mainly amyotrophic lateral sclerosis) ; malignancies with dysphagia ; malignancies without dysphagia ; and miscellaneous. results based on time frames were as follows : at 30 days, demented patients had 25% mortality ; at 3 months, 37% mortality ; and at 1 year, 58% mortality. another retrospective study, of 90 patients with dementia and dysphagia and of average age ~86 years, revealed 14.4% mortality within 30 days and 1-year survival of 54.4%.7 a well - known retrospective study from the va medical center in washington dc had 41 demented patients referred for pegs.8 after educating family members, 23 received pegs and 18 did not, secondary to family member refusal after discussion. the median survival in the group status post - peg was 59 days compared with the group without the peg, who survived 60 days. a retrospective cohort study with 311 patients with pegs, 143 with dementia, compared demented patient survival with peg to that of patients with other diagnoses (patients without dementia).9 the 12-month survival was 51% in patients with dementia, and 49% in the group without dementia. two predictors of poor survival after peg (among other medical issues) were patients who were male and older than 80 years of age. in this study, there was no evidence to support a poorer prognosis after peg in elderly people with dementia compared with elderly who are cognitively intact, unless the patients are older than 80 years of age, male, or have the other poor prognostic indicators mentioned in this study, including hypoalbuminemia. one retrospective study audited 719 patients who died within 30 days after peg insertion.10 of the 719 patients who received pegs, 135 (18%) had dementia, 82% were 70 years of age or older, and 50% were greater than 80 years of age. of note, there was a particularly low rate (29%) of written consent for the procedure among dementia patients. after their review, acknowledging the report that mortality rate of patients with dementia fed by gastrostomy is considerable, the group in this article advised against gastrostomy in dementia patients.10 a randomized prospective study looked at 99 hospitalized patients with advanced dementia.4 the median mortality at 6 months in the group with status post feeding tube was 195 days, and in the group without the feeding tube, this was 189 days. overall, the peg and non - peg median mortality at 6 months was no different, at 50%. a prospective study, which included a dementia cohort of 55 patients, compared tube - fed (tf) versus (vs) non - tf patients.5 mortality for the peg patients at 6 months was 44% as compared with 26% among controls. the authors concluded that only 50% of demented patients with inadequate oral intake are likely to survive beyond 6 months after peg placement and that no improvement in performance status is likely to occur in any patient. this study specifically focused on patients with inadequate oral intake secondary to dementia, in order to determine whether the nutritional parameters at the time of peg placement would predict survival. it also recommended that prior to initiating peg feeding, the limited benefits that would be achieved should be taken into consideration. a prospective observational study of 674 patients included 280 dementia patients after peg placement.6 over half of the patients were over the age of 80 years, with an age distribution similar to that of patients undergoing peg placements nationally in the united states. the median survival was 171 days in patients over age 80 with dementia, which was worse when compared with other subgroups. the median survival reached 423 days in demented patients from nursing homes and 467 days in demented patients younger than 80 years. results from five studies reported mortality for the patients with dementia after peg placement.57,10, the studies reviewed reported on varied mortality time frames. the specific results were : 13%18%,6 14.4%, 18%,10 25%,7 and 54%.5 one study reported 3-month mortality at 37%.7 six month mortality was revealed in one study as 44%.12 the mortality at 1 year was reported in five of the studies reviewed. those specific results were as follows : 45.6%, 51%,9 50%61%,6 and 58%.7 one study reported 1-year mortality at 90%.5 one study revealed mortality at 3 years to be 78%84%.6 results of comparison studies regarding median mortality in tf patients vs non - tf patients were as follows. one study revealed dementia patient median survival with a feeding tube as 195 days (6.5 months) compared with median survival without tube feeding as 189 days (6.3 months), with no obvious difference in survival when comparing both groups.11 the following are results from individual studies. one study revealed that the group status post - peg insertion had 44% mortality vs controls who had 26% mortality. this study revealed a higher mortality for patients with dementia who had pegs in place.12 another study revealed median survival after peg to be 59 days, a low number. yet another study revealed patients with dementia and peg who were in nursing homes demonstrated higher median survival at 423 days, whereas dementia patients with peg and stroke median survival was at 181 days, a similar number to the study comparing tf vs non - tf patients.13 a large retrospective database spanning 10 years, which included 8,688 demented patients who received pegs, broke down the mortality rates between males and females.2 this study revealed females fared slightly better, though mortality rates were still high after peg. the female dementia subgroup had 50% mortality at 1 year and 78% at 3 years. the male dementia subgroup had 61% mortality at 1 year and 84% mortality at 3 years. as previously reported in retrospective studies, an analysis looked specifically at the following for patients age 65 years and older : indications for peg placement, and survival after peg.7 the mean age of participants was 79 years old. patients were classified into seven diagnoses : stroke, dementia, parkinson s, other neurological diseases (mainly amyotrophic lateral sclerosis), malignancies with or without dysphagia, and miscellaneous. mortality rates were broken down farther into specific time frames : 30 days, 3 months and 1 year. regarding consideration of age prior to placement of peg in patients with dementia, only 33% of patients over age 80 were still alive after 1 year, as compared with 73% under age 80. in this particular study, dementia was not found to be a negative prognostic factor for survival in elderly patients after peg, specifically those younger than 80 years old. another retrospective study of 90 patients with dementia and dysphagia, whose average age was 86 years, revealed 14.4% mortality within 30 days, and 1-year survival of 54.4%. there are many controversies regarding whether or not to place a peg in a patient with dementia who is aspirating. quality of life must be considered, not only for the patient but for the caregiver as well. while an in - depth discussion of these issues goes well beyond the scope of this paper, it is important to recognize some of the major problems that health care professionals, caregivers, and the patients themselves encounter. stages 6 (moderate severe dementia) and 7 (severe dementia) are listed in table 2, with some of the hallmark signs of disease progression. voice and speech deterioration, as well as dysphagia with aspiration risk, demonstrate the cognitive effects on laryngeal function and are included in stage 6e.3 discussions regarding peg insertion do not appear to be happening during the early stages of dementia diagnosis, and many patients who receive a peg do not have health care proxies. patients diagnosed with dementia could be seen when presenting with initial signs of difficulty eating or swallowing, and could have regular follow - up so that aspiration risk can be assessed and potentially, intervention undertaken. at are they being inserted too late ? would there be a better outcome if the pegs were inserted earlier ? when is too late ? the research has shown an increased survival rate with demented patients under age 80, nearly double that of patients over age 80. other considerations are : age of onset of dysphagia, age of onset of dementia, severity of dementia, and disease duration, course, and comorbidities. every patient must still be regarded as an individual. medications should be assessed for their effects on swallowing and perhaps, medication schedules adjusted to facilitate eating, to prolong safe eating by mouth. caregivers, and often health care professionals as well, do not have all the information prior to making a decision. the data should be reviewed by medical staff and family members when deciding whether and when to insert a peg. when the discussion and or recommendation to not insert a peg is brought up with caregivers, this is often wrongly interpreted as a recommendation that will mean no food or no care for their loved one. caregivers need to be educated regarding quality of life po feeding, which may be interpreted as continuing to feed patients by mouth, with the safest diet and strict aspiration precautions in place, so they may still enjoy the taste of food. future studies that divide cohorts of retrospective studies into levels of severity of dementia would be informative. the study of the specific divisions and levels of dementia could assist in formulating future recommendations based on age, sex, nutritional status and the home environment. some benefits of peg placement that were not addressed in our review include : provision of a route for reliably administering medications ; supplementation of hydration and nutritional status while still allowing for compassionate po or comfort care ; as well as ensuring the bridging of nutritional needs during interval hospitalizations for conditions that may cause a transient decline in mental status. also, while this review does not document that peg prolongs life in this population, it is possible that a prospective study has yet to be constructed that accurately measures prolongation as well as quality of life for the patients, and their families, during their declining years. there is presently no evidence in the existing literature to suggest long - term survival rates improved in patients with advanced dementia who underwent peg placement for dysphagia and aspiration risk. however, the relevance for quality of life, need for nutrition and hydration, and ethical consideration are complex and controversial issues that warrant further study prior to achieving consensus and formalizing clinical care guidelines. | purposeover 4.5 million people in north america had a diagnosis of dementia in the year 2000, and more than half had advanced disease with potential aspiration risk. there is much controversy regarding the use and timing of enteral feeding support in these patients with dysphagia. the management of dysphagia is far more complex when considering quality of life, comfort care hand feeding, the use of percutaneous endoscopic gastrostomy tube (peg), and associated mortality rates. this study seeks to critically review the literature that evaluates peg placement in this population.methodsa systematic literature review of pubmed, from 19952012, was conducted to identify studies relating to peg placement in dementia patients with dysphagia. the principal outcomes and related survival rates for this population were compared.resultsin total, 100 articles were identified in the search. of these, ten met the search criteria and were analyzed. there was one study with a 2b level of evidence, one with 3b, and the remainder had level 4. all studies discussed long - term survival in the peg versus non - peg populations. no studies showed definitive evidence to suggest long - term survival rates improved in patients who underwent peg placement as compared to those who did not. two studies documented median survival worse in patients over age 80 with dementia and peg placement.conclusionthere is presently no evidence to suggest long - term survival rates improved in patients with advanced dementia who underwent peg placement for dysphagia. relevance to quality of life, need for nutrition and hydration, and ethical considerations in the decision process are discussed. |
bone dysplasias are characterized by the replacement of normal bone with fibrous tissue containing abnormal bone or cementum.1 familial gigantiform cementoma, which is a subgroup of osseous dysplasias, is a rare condition of the jaw (table 1).2,3,4 its true incidence is unknown, as is the gender and ethnic predispositions. the etiology is also unclear, but it is believed to have a genetic component. the familial form is reported as an autosomal dominant trait with variable expression.5,6 the condition may be asymptomatic ; in these cases, the lesions are detected radiographically as an incidental finding. the familial form presumably differs from the non - familial form clinically and pathologically.7 ehlers - danlos syndrome (eds) is a group of disorders affecting connective tissues, causing primarily dermatological and joint disorders. the prevalence of the condition varies between 1:10,0008 and 1:150,000.9 eds is an autosomal dominant inherited disorder, which can be primarily diagnosed on clinical findings and family history.10,11,12,13 the classical symptoms of eds are joint hypermobility, skin hyperextensibility, fragile and soft skin, the presence of atrophic scars, and easy bruising.10 at least 15 subtypes of the syndrome have been described to date. eds type viii (periodontitis type) is characterized with severe periodontitis leading to precocious loss of permanent teeth and alveolar bone resorption.11 the periodontal problems begin with puberty and mostly lead to loss of teeth before the age of 30.10 the facies characteristics are hypertelorism, widening of nasal bridge, and narrow face.12 the present case report is the first known describing concurrent familial gigantiform cementoma and eds in a single patient. the aim of this report is to discuss the oral management of patients diagnosed with both familial gigantiform cementoma and eds. a 34-year - old man self - referred to the istanbul university faculty of dentistry. his chief complaints were tooth loss, difficult mastication, poor esthetics, and periodontal disease. the extraoral clinical examination showed a difference between the proportions of upper and lower facial height and class iii malocclusion was also present (fig. tooth 12 was replaced with a premolar, and occlusal contact existed only between teeth 26 to 36. a reduced occlusal vertical dimension was observed by evaluating closest speaking space, proportional face measurement and interocclusal rest space. after performing dental prophylaxis, teeth 36 and 46 were extracted because of severe infection and were evaluated histologically. the mineralized material showed apposition and resorption lines, and spherical mineralized tissue (large pulp stones) were free - floating within the pulp chamber (fig., the patient was diagnosed with familial gigantiform cementoma with ehlers - danlos syndrome type viii. the patient had a previous composite restoration on tooth 11 and mottled enamel on teeth 35 and 45. the old restorations were repaired, and the defects were restored with new composite restorations. after healing, irreversible hydrocolloid impressions were made ; diagnostic casts and record bases with wax occlusion rims were also fabricated. the occlusal vertical dimension was established using visual observation of the space between the rims when the mandible is in its physiological rest position, judgment of the overall esthetic facial support and phonetic tests.13,14 the tests revealed that the occlusal vertical dimension needed to be increased approximately 9 mm to satisfy esthetic and functional requirements. orthodontic treatment was not recommended because of possible root resorption, and orthognathic surgery was not recommended due to a surgical contraindication. in addition, it was impossible to restore the mouth with fixed prostheses. a tooth - supported maxillary complete denture15 to provide lip support and a mandibular overlay removable partial denture (orpd) were selected as the treatment alternative. orpds, a subset of overdentures, are often referred to as a removable partial denture that has part of their components covering the occlusal surface of the abutment teeth to restore them into a functional occlusion.16 the mandibular framework was cast in a cr - co alloy with retention beads on the occlusal surfaces for the veneering material. after intraoral evaluation of the framework, veneering material was placed, and artificial teeth were arranged. a heat - processed silicone liner (molloplast - b) served as the retainer for the tooth supported complete denture (fig. no muscle tenderness, tooth sensitivity, or temporomandibular dysfunction was observed during this period. the 38-year - old sister of the first patient also selfreferred to the istanbul university faculty of dentistry. unlike her brother head and neck examination showed no abnormalities or facial asymmetry, but the temporomandiular joint did demonstrate hypermobility. a difference between the upper and lower facial height and class iii malocclusion were diagnosed (fig. teeth 14, 17, 18, 26, 38, 41 and 45 were missing, besides teeth 36 and 37 were impacted (fig. the incisors and canines had mottled enamel, and there was only occlusal contact between teeth 15 and 46. like her brother, she was diagnosed with familial gigantiform cementoma with eds type viii. all tooth defects were treated with composite restorations. the occlusal vertical dimension needed to be increased by approximately 12 mm. to satisfy esthetic and functional requirements. new tooth - supported complete dentures providing lip support and rehabilitating the decreased occlusal vertical dimension were fabricated (fig. acceptable retention was achieved without requiring soft liner ; the dentures were fabricated as described above. no muscle tenderness, tooth sensitivity, or temporomandibular dysfunction was observed during this period. a 34-year - old man self - referred to the istanbul university faculty of dentistry. his chief complaints were tooth loss, difficult mastication, poor esthetics, and periodontal disease. the extraoral clinical examination showed a difference between the proportions of upper and lower facial height and class iii malocclusion was also present (fig. tooth 12 was replaced with a premolar, and occlusal contact existed only between teeth 26 to 36. a reduced occlusal vertical dimension was observed by evaluating closest speaking space, proportional face measurement and interocclusal rest space. after performing dental prophylaxis, teeth 36 and 46 were extracted because of severe infection and were evaluated histologically. the mineralized material showed apposition and resorption lines, and spherical mineralized tissue (large pulp stones) were free - floating within the pulp chamber (fig., the patient was diagnosed with familial gigantiform cementoma with ehlers - danlos syndrome type viii. the patient had a previous composite restoration on tooth 11 and mottled enamel on teeth 35 and 45. the old restorations were repaired, and the defects were restored with new composite restorations. after healing, irreversible hydrocolloid impressions were made ; diagnostic casts and record bases with wax occlusion rims were also fabricated. the occlusal vertical dimension was established using visual observation of the space between the rims when the mandible is in its physiological rest position, judgment of the overall esthetic facial support and phonetic tests.13,14 the tests revealed that the occlusal vertical dimension needed to be increased approximately 9 mm to satisfy esthetic and functional requirements. orthodontic treatment was not recommended because of possible root resorption, and orthognathic surgery was not recommended due to a surgical contraindication. in addition, it was impossible to restore the mouth with fixed prostheses. a tooth - supported maxillary complete denture15 to provide lip support and a mandibular overlay removable partial denture (orpd) were selected as the treatment alternative. orpds, a subset of overdentures, are often referred to as a removable partial denture that has part of their components covering the occlusal surface of the abutment teeth to restore them into a functional occlusion.16 the mandibular framework was cast in a cr - co alloy with retention beads on the occlusal surfaces for the veneering material. after intraoral evaluation of the framework, veneering material was placed, and artificial teeth were arranged. a heat - processed silicone liner (molloplast - b) served as the retainer for the tooth supported complete denture (fig. no muscle tenderness, tooth sensitivity, or temporomandibular dysfunction was observed during this period. the 38-year - old sister of the first patient also selfreferred to the istanbul university faculty of dentistry. unlike her brother head and neck examination showed no abnormalities or facial asymmetry, but the temporomandiular joint did demonstrate hypermobility. a difference between the upper and lower facial height and class iii malocclusion teeth 14, 17, 18, 26, 38, 41 and 45 were missing, besides teeth 36 and 37 were impacted (fig. the incisors and canines had mottled enamel, and there was only occlusal contact between teeth 15 and 46. panoramic radiography showed radio - opaque masses scattered throughout the mandible and maxilla. like her brother all tooth defects were treated with composite restorations. the occlusal vertical dimension needed to be increased by approximately 12 mm. to satisfy esthetic and functional requirements. new tooth - supported complete dentures providing lip support and rehabilitating the decreased occlusal vertical dimension were fabricated (fig. acceptable retention was achieved without requiring soft liner ; the dentures were fabricated as described above. no muscle tenderness, tooth sensitivity, or temporomandibular dysfunction was observed during this period. few cases have been reported because eds type viii is a rare disease17 ; among these reports, only diagnosis and routine dental treatment were discussed, thus, dental treatment options for patients with eds remain unestablished. in eds type viii patients, loss of the occlusal vertical dimension (ovd) has not been reported, but this may reflect the sparse literature, particularly describing the oral manifestations. notably, our patients ' father also had a class iii malocclusion, and the findings may only be coincidental ; more cases are needed to confirm our suspicions. also notable, the clinical manifestations overlap between the different eds types, and it is a highly variable clinical entity, presenting a broad clinical spectrum that may also include an increased risk for malocclusion.18 one of the most significant oral features of the syndrome is early - onset periodontitis1,9 which results in premature loss of primary and permanent teeth.8,10 histopathology suggests that this may be caused by reactive or dysplastic changes in the periodontal ligament7, but radiographically, the lesions adjacent to the teeth appear to have little possibility of originating from the periodontal ligament.7 although antibiotic prophylaxis was administered in present cases, post - surgical hyperpigmentation and fibrous nodules were detected after the extractions. in the absence of clinical symptoms, re - evaluation with panoramic radiographs every 2 or 3 years is adequate.5 in these cases, the reduced occlusal vertical dimension and negative horizontal overlap were restored with overdentures and overlay removable partial dentures. this is a simple, reversible, non - invasive, and cost - effective solution that resolves the esthetic and functional concerns. however, there are several disadvantages associated with them such as increased risk of framework and veneer material fracture.19 overall, a non - invasive treatment approach was considered the best and most effective treatment option because it resolved the patients ' esthetic concerns, improved mastication, and improved speech function. this is a very rarely encountered condition in clinical practice ; however, the diagnosis is simple, relying on adequate clinical, histological and radiographic examination. the dentist should be able to easily diagnose in order to manage treatment satisfactorily. | ehlers - danlos syndrome is an autosomal dominant hereditary disorder of connective tissue, while familial gigantiform cementoma is a condition that usually manifests as multiple radiopaque cementum - like masses throughout the jaws. this case report discusses the oral management and prosthetic rehabilitation of two patients presenting familial gigantiform cementoma with ehlers - danlos syndrome. |
cervical cancer is a major health challenge with approximately 530,000 news cases and 270,000 deaths annually worldwide despite remarkable advances in screening and prevention through the development of human papillomavirus (hpv) vaccine. while the majority of cervical cancer cases can be potentially cured with surgery, chemoradiation or a combination of these strategies, treatment options for recurrent or metastatic disease are limited to pelvic exenteration or palliative chemotherapy. a recent phase iii trial evaluating the combination of cisplatin, paclitaxel and bevacizumab (monoclonal antibody against vascular endothelial growth factor receptor) in the first - line treatment of metastatic disease elicited a 50% response rate and median overall survival (os) of approximately 17 months (tewari., 2014). despite these relatively positive data that led to the approval of the first targeted therapy for this disease (bevacizumab), the median progression - free survival of 8 months demonstrates the aggressive behavior of this disease. hence, there is an urgent need to advance the understanding of the molecular abnormalities driving cervical cancer pathogenesis that could lead to novel targeted therapies. comprehensive genomic profiling of metastatic tumors is an increasingly relevant tool to identify somatic alterations leading to additional therapeutic options and a better understanding of tumor molecular pathogenesis. herein, we describe the first three cases of cervical carcinoma harboring fgfr3tacc3 fusions revealed by a next - generation sequencing assay able to detect all classes of genomic alterations including gene fusions. the fusion of the fibroblast growth factor receptor gene 3 (fgfr3) with the transforming acidic coiled - coil containing gene (tacc3) has been described in glioblastoma multiforme, bladder urothelial carcinoma, and non - small cell lung cancer (wu., 2013). while fgfr mutations have been described in cervical carcinomas, the fgfr3tacc3 fusion has not been reported previously (cappellen., 1999). this fusion resulting in fgfr pathway activation provided the rationale for treating one of the patients with a fgfr tyrosine kinase inhibitor (tki) in a clinical study setting and other molecular alterations involving the pi3k / akt / mtor pathway hold the potential to inform treatment decisions. the patient was diagnosed in 1997 at age 36 with stage ib1 adenocarcinoma of the cervix and underwent a modified radical hysterectomy, left salpingo - oophorectomy and bilateral pelvic lymphadenectomy. twelve years later she developed sudden, significant hemoptysis, and work - up revealed bilateral upper and lower lobe lung masses with left hilar adenopathy. she developed respiratory failure requiring intubation, two arterial embolizations and eventually palliative right middle lobectomy to ameliorate the persistent bleeding. histopathologic examination of the resected lung mass revealed a carcinoma with mixed glandular and squamous features (adenosquamous carcinoma). the tumor cells were diffusely immunoreactive for p16 and were positive for hpv by pcr, consistent with recurrent cervical cancer. the patient received multiple palliative chemotherapy regimens (i.e., paclitaxel / carboplatin, cisplatin / topotecan, pemetrexed) as well as stereotactic body radiation therapy. following two years of active surveillance, her pet / ct scans showed an enlarging left upper lobe mass (5.4 cm with suv 12.6) causing destruction of the left third rib, and a pleural - based lesion in the right lung (suv 2.9). transbronchial lung biopsy of the left upper lobe mass revealed a tumor with both squamous and focal glandular differentiation (fig. the tumor cells were diffusely positive for p16, pax8, and p63 by immunohistochemistry and hpv 16 by pcr. the morphology, immunohistochemical staining pattern, and hpv results were consistent with those of the right lung metastatic lesion resected 5 years previously. comprehensive genomic profiling of the left upper lobe lung tumor was performed to identify additional therapeutic options. hybridization capture of 236 cancer - related genes and 19 genes commonly rearranged in cancer (foundationone) was applied to 50 ng of dna extracted from archival formalin - fixed, paraffin embedded left upper lung tumor tissue and sequenced to high, uniform coverage. all classes of genomic alterations (base substitutions, small indels, rearrangements, copy number alterations) were determined and revealed the following : fgfr3tacc3 fusion (breakpoints at fgfr3 intron 17 and tacc3 intron 10), akt1 missense mutation (e17k), mtor point mutation (p1312l), and atrx truncating nonsense mutation (w1883). based on the genomic profiling results, the patient was enrolled in a clinical study evaluating a multi - kinase tki targeting fgfr (nct1831726). the patient was treated with the study drug for four cycles with best response of stable disease suggesting expected target (fgfr) inhibition (fig. a 47 year - old female underwent investigation of abnormal uterine bleeding and a cervical biopsy showed an invasive well - differentiated keratinizing squamous cell carcinoma of the cervix. at the time of diagnosis pelvic soft tissue and pelvic lymph node involvement were demonstrated radiographically (figo stage iiib), and she was treated with primary chemoradiation achieving remission. the patient developed recurrent disease in the pelvis and adnexa 20 months later and was treated with carboplatin and paclitaxel with partial response after three cycles, receiving a total of five cycles. in july 2014, ct scans showed disease progression, and the patient was started on topotecan and bevacizumab, which was administered for 4 cycles before disease progression causing sigmoid colon involvement and ureteral obstruction. pemetrexed was started resulting in relief of urinary tract obstruction, but also led to development of a colorectal fistula requiring a colostomy. the patient had excellent performance status and was evaluated for debulking surgery but was deemed not to be a candidate for pelvic exenteration. comprehensive genomic profiling of the original cervical biopsy was pursued to identify additional therapeutic options and revealed the following : fgfr3tacc3 fusion (breakpoints at fgfr3 intron 18 and tacc3 intron 7), braf 3 tandem duplication (breakpoint in intron 9 with duplication of exons 1018), activating pik3ca missense mutation (e545k), cdnk2a loss, and subclonal activating missense mutations in kras (g12c), and hras (g13r). recently, ct scans showed disease progression and the patient enrolled on a clinical trial evaluating a pan - fgfr inhibitor. a 33-year - old woman was diagnosed with stage ii poorly differentiated non - keratinizing carcinoma of the cervix and she underwent a radical hysterectomy and bilateral salpingo - oophorectomy. the primary tumor measured 5.5 cm and invaded through the inner two - thirds of the cervix. the immunostain profile (positive : p16 and p63 ; negative : ck20, er, pr, chromogranin and synaptophysin) was consistent with cervical squamous cell carcinoma. the patient developed rapid disease progression with intra - abdominal and lung metastases leading to palliative treatment with carboplatin / paclitaxel followed by gemcitabine. comprehensive genomic profiling of the tumor from the hysterectomy specimen was pursued to identify additional therapeutic options, which revealed the following : fgfr3tacc3 fusion (breakpoints at fgfr3 intron 17 and tacc3 intron 10), partial loss of stk11 (loss of exons 29), and rb1 loss (breakpoints in introns 6 and 12). the patient died of uterine hemorrhage 10 months after diagnosis before targeted therapy could be initiated. the fgfr family consists of four structurally related subtypes of tyrosine kinase receptors that play an important role in cell growth, differentiation and angiogenesis via binding of twenty different fgf family ligands (wu., 2013). ligand binding triggers activation of downstream pathways implicated in carcinogenesis, including the pi3k / akt, ras / mapk, phospholipase c, and stat pathways. anomalous signaling through fgfr can occur through overexpression of receptors, activating mutations, amplification of the wild type gene, or by fgfr - containing translocations. fgfr3 amplification or activating mutation may lead to activation of fgfr3 and may therefore confer sensitivity to fgfr family inhibitors. fgfr3tacc3 fusions have been reported to transform rat fibroblasts and to induce tumors in mice. fgfr3 mutations have been described in approximately 25% of cervical carcinomas in large studies (cappellen., 1999, rosty., 2005, wu., 2000). in the cosmic database, fgfr3 mutation has been found in 2 out of 120 cervical squamous cell carcinoma specimens. in the cervical squamous cell carcinoma and endocervical adenocarcinoma tcga dataset, putative high - level amplification of fgfr3 was reported in < 1% (1/212) of samples analyzed (cbioportal, dec 2014). while transcriptome sequencing revealed rare fgfr3 fusions in glioblastoma, cholangiocarcinoma, breast, prostate, lung, bladder, head and neck, and thyroid cancers (wu., 2013), fgfr3 fusions are formed by both interchromosomal and intrachromosomal rearrangements, the latter typically involving the tacc3 locus, which is located within 150 kb of the fgfr3 gene on chromosome 4p16 (still., 1999). similar to the chromosomal proximity of fgfr3 and tacc3, tacc1 and tacc2 genes are also located near their respective fgfr1 and fgfr2 genes, leading to the suggestion that these gene families arose through duplication of a common ancestral fgfr / tacc gene pair. tacc proteins have been implicated in regulation of mitosis and epithelial mesenchymal transition (emt). tacc interacts with histone acetyltransferases, and drives proteins to the mitotic spindle via its distinctive coiled - coil domain at the c terminus. tacc3 transcript is overexpressed in cervical cancer human specimens compared to normal cervical tissue and mediates the epidermal growth factor (egf)-induced emt in cervical cancer cell lines through upregulation of pi3k / akt and erk pathways (ha., 2013). in contrast, fgfr3tacc3 fusion proteins appear to localize to spindle poles and cause disruption of chromosome segregation and aneuploidy by a mechanism dependent on fgfr tyrosine kinase activity. these preclinical results suggest a role for fgfr3tacc3 fusion as an oncogenic - driver in multiple tumor types, worthy of target inhibition by small molecule kinase inhibitors. these preclinical results are supported by the observable clinical response in case 1 to fgfr signaling blockade. in each of these three cases, in addition to putative activation of fgfr signaling, each of the three cases displays co - occurring mutations predicted to activate the pi3k / akt / mtor pathway, which has been implicated in cervical tumorigenesis. pi3k / akt / mtor pathway activation can be mediated by activating mutations in pik3ca, akt1, or mtor or loss of function of stk11. pik3ca mutations are common in cervical carcinoma (up to 38% ; 11% of cosmic [jan 2015 ] and 25% of tcga [cbioportal, jan 2015 ] cervical cancer datasets). less commonly, homozygous loss of stk11 (10% of cosmic [july 2014 ] and 3% of tcga [cbioportal, feb 2015 ]) and akt1 activating mutations (not reported in cosmic [march 2015 ] ; observed in 2% (3/194) of tcga [cbioportal, may 2015 ]) have been reported in the cervical carcinoma datasets. base substitutions in the e545 hotspot codon located in the helical domain, such as observed in case 2, have been shown to be oncogenic (kang., 2005). activated pi3k converts phosphatidylinositol 4,5-biphosphate (pip2) to phosphatidylinositol 3,4,5-triphosphate (pip3), which in turn recruits akt to the cell membrane for activation through phosphorylation. akt is an intracellular serine / threonine kinase that promotes cell proliferation and survival by activating the mtor pathway, interacting with cell - cycle proteins and apoptotic proteins. akt e17k, observed in case 1, is a constitutively activating missense mutation within the akt1 pleckstrin homology domain that causes pi3k - independent recruitment to the cell membrane. stk11 encodes serine / threonine - protein kinase 11, also known as lkb1, that activates ampk, and negatively regulates the mtor pathway in response to cellular energy levels (shaw., 2004). stk11 alterations that result in the partial or complete loss of the kinase domain, such as observed in case 3, are predicted to result in a loss of function. studies have shown an association of mtor signaling with prognosis and response to chemotherapy and radiation therapy and the frequency of mutations activating this pathway provided the rationale for clinical studies evaluating the efficacy of mtor inhibitors for the treatment of cervical cancer. a phase 2 study of single agent temsirolimus in patients with recurrent or metastatic cervical carcinoma reported partial response and stable disease rates of 3% (1/33) and 57% (19/33), respectively (tinker., 2013). in a study of 23 patients with pik3ca mutated breast or female gynecological cancers, treatment with pi3k / akt / mtor pathway inhibitors in combination with other drugs resulted in a partial response of 40% among five patients with advanced cervical scc (janku. despite the small number of patients on these studies, the results suggest a higher efficacy when mtor inhibitors are combined with inhibitors of other pathways that can circumvent feedback loops causing resistance. the contribution of the additional co - occurring alterations in the raf / mek pathway and in tumor suppressor genes also deserves consideration, as they may be impacting the clinical course or be expected to alter responsiveness to single - agent therapy. in case 2, the braf 3 tandem duplication may result in expression of the constitutively active braf kinase domain in the absence of the braf n - terminal autoinhibitory domain, and subsequent hyperactivation of the mapk pathway (sievert., 2013). similarly, the subclonal missense mutations (codons 12 and 13) in the g box domains of the ras - family gtpases, kras and hras, also observed in case 2 are predicted to create proteins incapable of gtp hydrolysis and therefore constitutively activate mek signaling. finally, p53 and rb somatic mutation is generally uncommon in hpv - associated cervical carcinomas as the high risk hpv early gene 6 and 7 proteins cause a functional loss of these master tumor suppressors. it seems reasonable to speculate that the unusual rb1 somatic loss observed in case 3 could have also contributed to the especially aggressive clinical course in this patient. the identification of fgfr3tacc3 fusion, in addition to previously observed genomic drivers, in these three cases of cervical carcinoma, supports preclinical evidence of the role of fgfr signaling in a subset of hpv - driven carcinogenesis. specimens from cervical cancer xenograft models exhibit increased expression of fgf2 and fgfr1 genes, while hpv16 e6/e7 oncoproteins reduce their ligand - induced proliferation (cheng., in addition, hpv e6 oncoprotein has been shown to induce the expression of fibroblast growth factor binding protein (fgf - bp), which regulates the bioavailability of fgf-1 and fgf-2 in the extracellular matrix, and thereby modulates tumor angiogenesis (stoppler., 2001). these observations coupled with the clinical response observed in case 1 support fgfr signaling pathway as a potential therapeutic target in the treatment of cervical carcinoma. results of ongoing clinical studies investigating potent and specific inhibitors of fgfr could provide a signal of efficacy in this disease. the potential utility of fda - approved multikinase inhibitors such as pazopanib, lenvatinib and regorafenib in cervical carcinoma carrying fgfr molecular alterations also deserves investigation since these agents inhibit fgfr family members with distinct specificity. pazopanib, fda - approved for advanced soft tissue sarcoma and renal cell carcinoma, inhibits fgfr1 and 3 ; lenvatinib, fda - approved for advanced radioactive iodine - refractory differentiated thyroid cancer, inhibits fgfr1, 2, 3 and 4 ; regorafenib, fda - approved for metastatic colorectal cancer, inhibits fgfr1 and 2. however, at this time, there is no approved indication to use these agents for treatment of solid tumors harboring fgfr molecular aberrations. this case series highlights the importance of a comprehensive genomic profiling approach able to detect all classes of genomic alterations including uncommon gene fusions to reveal potentially targetable somatic alterations for mutation - matched therapy selection. case 1 provides proof of concept that treating with an fgfr inhibitor can result in clinical benefit in metastatic cervical cancer carrying fgfr3tacc3 translocation in agreement with results observed in other malignancies. in a preliminary report, a patient with refractory bladder cancer and lung metastasis harboring a fgfr3tacc3 fusion demonstrated a partial response to treatment with a pan - fgfr inhibitor, supporting this strategy in patients with similar translocations, irrespective of tumor histology (bahleda. cases 1 and 2 also highlight the importance of utilizing a molecular profiling approach able to detect all classes of genomic alterations (base substitutions, insertion / deletions, copy number alterations, and gene fusions) simultaneously on a small tissue sample. based on the small amount of material available for testing, it is certain that this fusion would not have been detected by a sequential testing approach. these three cases also demonstrate a co - occurrence of more common genomic alterations in pi3k / akt / mtor pathway that might require combinatorial treatment strategies given the challenge in identifying the primary driver | cervical cancer epitomizes the success of cancer prevention through the human papillomavirus (hpv) vaccine, but significant challenges remain in the treatment of advanced disease. we report the first three cases of cervical carcinoma harboring an fgfr3tacc3 fusion, which serves as a novel therapeutic target. the fusion, identified by comprehensive genomic profiling, activates the fgfr pathway that has been implicated in hpv - driven carcinogenesis. one of the patients whose tumor contained the fgfr3tacc3 fusion was treated with an investigational fgfr tyrosine kinase inhibitor. concomitant molecular alterations involving the pi3k / akt / mtor and raf / mek pathways were also identified and suggest other treatment strategies that deserve investigation. this case series highlights the role of comprehensive genomic profiling in the identification of new therapeutic targets and in targeted therapy selection for patients with cervical cancer. |
anca - associated vasculitides (aav) are autoimmune diseases characterized by inflammation of the blood vessel wall and include microscopic polyangiitis (mpa), granulomatosis with polyangiitis (gpa, formerly wegener 's), and eosinophilic granulomatosis with polyangiitis (egpa, formerly churg - strauss syndrome). antineutrophil cytoplasmic antibodies (anca) can usually be detected in the blood of aav patients. cytoplasmic anca (canca), mainly directed against proteinase 3 (pr3-anca), predominate in gpa, and perinuclear anca (panca), directed against myeloperoxidase (mpo - anca), in mpa. during the past few decades, significant advances have been made in understanding the disease pathogenesis and clinical management of aav, with substantial decrease in mortality. during long - term follow - up, the clinical status does not always correlate with the serum levels of anca. as there is a need to individualize the treatment, the emphasis has been placed on discovering new potential biomarkers of disease activity, which may help us to tailor the treatment better. interleukin-1 (il-1) cytokines play an important role in immune regulation and inflammatory processes. il-33 is a recently identified cytokine that is a member of the il-1 family that also consists of il-1 and il-18. the extracellular form of il-33 activates a target cell by binding to the t1/st2 receptor and induces th2 associated cytokines but il-33 also functions at intracellular level as a nuclear factor regulating transcription [35 ]. alarmins, endogenous danger signals activating the immune system, released by necrotic but not apoptotic cells [5, 6 ]. st2, a member of the interleukin-1 receptor family, is selectively expressed on th2 (t helper type 2) cells and mediates important th2 functions. there are two different forms of st2 : transmembrane form (st2l) and secreted soluble form (sst2). the il-33/st2 pathway has been the subject of a number of recent studies [815 ]. elevated sst2 levels have been found in patients with several cardiovascular and/or pulmonary diseases (asthma, acute dyspnoea, and myocardial infarction), but also sepsis, and elevated sst2 levels are considered an unfavourable prognostic marker in these patients. il-33 levels were increased in patients with disease associated with chronic inflammation such as ankylosing spondylitis, rheumatoid arthritis, henoch - schonlein purpura, or multiple sclerosis. in sle patients elevated sst2 levels were recently described to correlate with disease activity. low levels of il-33 and high levels of sst2 were noted in patients with hiv infection or amyotrophic lateral sclerosis. the aim of this study was to determine whether serum levels of il-33 or st2 are markers of disease activity in patients with aav, a disease characterised by endothelial injury and chronic inflammation. the study included 165 samples from 139 aav patients (67 men, 72 women) recruited from the department of nephrology, general university hospital, prague, czech republic, during a 15-month period. the disease activity was assessed with bvas (birmingham vasculitis activity score version 3), with the use of clinical data and laboratory tests routinely performed (erythrocyturia, proteinuria, and serum levels of anca, crp, and s - creatinine). active disease was defined as bvas 1 and remission as bvas = 0. relapse was defined as a new / worse disease activity and bvas 1 after previous remission. persistent activity was defined as no new / worse activity, with persistent bvas 1. in total, 38 samples were from patients with active disease (16 active newly diagnosed, 11 active with relapse, and 11 persistent activity) and 127 samples were from patients in remission that were available for examination. twenty - one patients were sampled repeatedly but within each subgroup all patients were unique. samples from newly diagnosed patients were collected immediately after the diagnosis of aav had been confirmed but some of the patients had already started immunosuppressive treatment. the control group contained 62 patients (34 men, 28 women) : 22 healthy individuals and 40 patients with chronic kidney disease (median of creatinine 168 mol / l in nondialysis patients ; 19x hemodialysis patients, 8x iga nephropathy, 10x polycystic kidney disease, and 3x other). after obtaining blood samples, all patients were routinely followed up in the clinics and clinical activity (relapses) and deaths were recorded for the purpose of this study. serum il-33 and sst2 levels were measured with a commercially available elisa (enzyme - linked immunosorbent assay) kit. the sst2 elisa kit was obtained from mbl international, woburn, ma, usa (code number 7638). the chi square test was used for comparison of discrete variables ; if the minimum expected value was less than 5, the yates correction was applied. for continuous variables, the kruskal - wallis test with multiple comparison or the mann - whitney test in the case of two groups was used. the subgroup of patients with active newly diagnosed aav had higher median sst2 levels than healthy and renal controls (median 0.39 ng / ml versus 0.13 ng / ml and p < 0.01, table 3, figure 1). median levels of sst2 in patients with active aav tended to be higher compared to patients in remission but the difference did not reach statistical significance. subsequent analysis revealed that aav patients with active newly diagnosed disease have higher sst2 levels than aav patients in remission (p < 0.001, figure 1). on the contrary sst2 levels in patients with relapse or persistent activity nine of 12 patients, who were sampled repeatedly in different phases of disease (active newly diagnosed aav remission, or remission relapse), had lower levels of sst2 in remission than in active disease stage (table 4). levels of il-33 were significantly higher in all aav patients, in active aav, and in aav in remission than in healthy and renal control patients (p = 0.002, p = 0.007, and p = 0.007, resp.). levels of il-33 tended to be lower in patients with active aav than in patients in remission but the difference did not reach statistical significance (median il-33 levels = 0 pg / ml in both groups, mean il-33 levels are 13.16 pg / ml in active aav patients versus 41.79 pg / ml in patients with remission, p = 0.119). no relation to levels of anca, bvas, s - creatinine, crp, and proteinuria was disclosed. in the group of newly diagnosed patients there was no difference in group with serum creatinine above 300 mol / l (8 patients) and below 300 mol / l (8 patients) (median st2 levels 0.463 ng / ml and 0.221 ng / ml, resp., p = 0.667). in the group of newly diagnosed patients there was no difference in st2 levels between patients who already initiated immunosuppressive treatment (9 patients) and those without treatment (7 patients) at the time of blood sampling (median st2 levels 0.434 ng / ml and 0.350 ng / ml, resp. there was no difference in st2 levels in patients with higher bvas (bvas 15, 7 patients) and lower bvas (bvas < 15, 9 patients) (median st2 levels 0.214 ng / ml and 0.343 ng / ml, resp., all newly diagnosed patients had il-33 levels 0 ng / ml. there was no difference between pr3-anca and mpo - anca but levels of il-33 in patients who suffered from granulomatosis with polyangiitis were higher than il-33 levels in patients with microscopic polyangiitis (p = 0.012). levels of il-33 were higher in younger aav patients than older patients (p = 0.007). no significant difference in sst2 and il-33 levels was found between patients with and without lung involvement (median sst2 levels in 0.17 ng / ml and 0.13 ng / ml, resp., p = 0.338, median il-33 levels = 0 pg / ml in both groups). the median length of follow - up (fu) after blood collection was 30 (range 250) months. during the fu, 22 patients (15.8%) died and 60 patients (43.2%), 38 pr3-anca and 22 mpo - anca, suffered from a relapse. we did not observe any difference in st2 or il-33 levels between patients who relapsed and those without relapse (median 0.14 ng / ml versus 0.17 ng / ml and p = 0.181) and between patients who died and the surviving patients (median sst2 0.16 ng / ml versus 0.16 ng / ml, p = 0.650 ; median il-33 levels = 0 pg / ml in both groups). elevated levels of sst2 are present in the circulation of patients with various inflammatory diseases. the proinflammatory cytokine il-33 is the ligand for st2. to our knowledge, this is the first study to examine the association between serum levels of il-33 and sst2 in aav patients and disease activity. similar to a previous study in sle patients, we demonstrated higher sst2 levels in active newly diagnosed aav patients compared to healthy controls. elevated sst2 levels in patients with active vasculitis may suggest a relationship of sst2 with the inflammatory process in aav. however, while the study in sle showed a correlation of sst2 levels with disease activity, we were not able to demonstrate association of sst2 with any other markers of disease activity, including anca levels, crp, or bvas. contrary to the levels of serum st2, there was no difference in il-33 in patients with active aav compared to patients in remission. in theory, this might be caused by the lower production and/or secretion of il-33 or by its rapid clearance. there are a growing number of publications describing the precise function of this intriguing novel cytokine that has attracted a lot of attention lately but the cellular source of il-33 is less clear. nevertheless, it seems that il-33 is mainly expressed in quiescent endothelial cells and the expression is lost when contact inhibition is disrupted or cells are exposed to proinflammatory il1- or tnf- [5, 7 ] which are processes known to participate in neutrophil priming in early pathogenesis phases of aav. subsequently, when remission is achieved and endothelial damage repaired, il-33 levels might increase, which was also noted in this study even though the results did not reach statistical significance. whether endothelial cells are the dominant il-33 secreting cells in vivo is, however, uncertain. interestingly, the situation in an immune - complex vasculitis, henoch - schonlein purpura (hsp), was just the opposite ; the authors described elevated il-33 levels in acute hsp that decreased in remission. we were not able to demonstrate any statistically significant difference in different disease activity subgroups of aav patients in this study, but this might be caused by low sensitivity of the assays used. another explanation for lack of increase in il-33 levels in active aav might be that il-33 is subject to degradation by caspases released from the apoptotic cells in aav, in accordance with the hypothesis that was suggested for amyotrophic lateral sclerosis. primed neutrophils undergo apoptosis more readily, at which times they express pr3 and mpo on their surface. it is possible that degradation of il-33 by proapoptotic caspase-3 released from primed neutrophils in active aav may explain the decreased levels of il-33. as the serum receptor for il-33, sst2 molecule, was increased in active aav patients, it is conceivable that sst2 acts as a contraregulating agent, regulating inflammatory process. levels of st2 and il-33 may behave differently in patients with extrarenal involvement, which would require further studies. a few of the newly diagnosed patients had received immunosuppressive treatment prior to blood collection, which may, in general, influence levels of circulating biomarkers in aav. however, we were not able to find any difference in il-33 levels and st2 levels between the already treated and untreated newly diagnosed patients at the time of blood sampling. the serum levels of sst2 and particularly il-33 were low and we were not able to detect measurable il-33 levels in any of the control patients. thus, a more sensitive assay might be needed for routine detection. alternately, intracellular il-33 expression and production may not result in a robust increase in serum il-33 levels and il-33 as with some other cytokines may have a very short half - life. analysis of tissue il-33 expression, for example, in kidney biopsies might provide important clues to reveal the role of il-33/st2 pathway in the pathogenesis of aav, which was, however, not an objective in this study. it might be useful to confirm the results in a longitudinal prospective study but the limited number of repeated measurements performed in patients in this study is in accordance with the cross - sectional results. in conclusion, results of this pilot study suggest that sst2 may be a marker of activity in active newly diagnosed patients with aav. further studies with serial samples and a prospective follow - up are needed to elucidate the precise role of sst2/il-33 in aav. | objective. st2, a member of the interleukin-1 receptor family, is selectively expressed on th2 cells and mediates important th2 functions. il-33 is a specific ligand of st2. the aim of the study was to determine whether serum levels of soluble st2 (sst2) or il-33 predict activity of the disease in patients with anca - associated vasculitides (aav). methods. 139 aav patients and 62 controls were studied. il-33 and sst2 in the blood were measured with a commercially available elisa. results. newly diagnosed aav patients had higher sst2 levels than controls (p < 0.01). levels of sst2 were significantly higher in active newly diagnosed aav patients than in patients with remission (p < 0.001). il-33 levels were higher in aav patients than in the control groups (p = 0.002). however, serum il-33 levels were not increased in patients with active aav compared to patients in remission. il-33 levels were higher in patients with granulomatosis with polyangiitis than in patients with microscopic polyangiitis (p = 0.012). conclusions. serum sst2, but not serum il-33, may be a marker of activity in aav patients. |
atherosclerotic disease of the coronary arteries is one of the most common causes of mortality and morbidity. despite all the methods of diagnosis and the new strategies in the treatment of patients, cardiovascular diseases (cvds) are considered as the most important cause of mortality in iran over the last decade.1 a number of known risk factors have been recognised for this disease, but less known factors such as trace elements may have a role in the progress of atherosclerosis.2 the serum level of copper (cu) and oxidants like free radicals have also been considered in coronary artery disease (cad).34 several studies show that atherosclerosis is characterised with fatty fibrous plaques, localised in intima. endothelial damage is the most important factor in the atherosclerotic process.5 the main effect of these factors on the endothelium is oxidative stress, lipid peroxidation and the formation of free radicals. cu and cvd may be associated directly, through a direct effect on the vascular endothelium, or indirectly through lipoprotein metabolism. copper ions can convert the superoxide and the hydrogen peroxide into the highly harmful hydroxyl radical that can damage to endothelium.6 the aim of this study is investigation about relationship between serum levels of cu and the severity of atherosclerosis measured by syntax score. this is a cross - sectional study conducted on patients hospitalised in sari heart centre, mazandaran university of medical sciences from february 2010 to july 2012. written informed consent was obtained from all enrollees, according to the criteria of the ethical committee of mazandaran university of medical sciences. the sample consisted in 337 patients with chronic stable angina that each of them had been admitted for diagnostic coronary angiography into typical indications, such as evaluation of stable exertional angina. the patients who had history of infectious disease in recent 2 months, collagen vascular disease and recent cardiac events were ineligible. coronary angiography was performed by the judkins technique through femoral artery access and the angiograms evaluated by two cardiologists who were blinded to the study plan. all patients divided into four groups to evaluate severity of cad according to syntax scoring system. the diagnosis of cad was based on the presence of 50% luminal diameter stenosis of the major epicardial coronary artery which determined in a standard manner during the coronary angiograms. distinction has been made only between occlusive (100% diameter stenosis) and non - occlusive (50 - 99% diameter stenosis) disease. a multiplication factor of 2 is used for non - occlusive lesions and 5 for occlusive lesions. cad was categorised to mild for scores 1 - 22, moderate for 23 - 32 and severe for scores 33and higher.78 cardiovascular risk factors including age, sex, systolic and diastolic blood pressure, smoking status, history of dyslipidaemia, diabetes, etc. were assessed for each subject. dyslipidaemia was defined as total cholesterol (tc) to high - density lipoprotein (hdl) more than 4. hypertension (ht) is defined as a systolic blood pressure above 140 mmhg, or diastolic blood pressure above 90 mmhg, respectively, or current use of antihypertensive medication. diabetes mellitus (dm) is defined as a known history of dm (fasting blood glucose 126 mg / dl or glucose tolerance test (gtt) higher than 200 mg / dl or treatment with insulin or oral hypoglycaemic agents. different categories of cigarette smoking status are defined according to world health organisation (who) guidelines.9 daily smoker is defined as who smokes cigarettes at least once a day ; occasional smoker is who smokes cigarettes but not every day ; ex - smoker is formerly daily or occasional smoker who currently does not smoke and never smoked defined as who never smoked before or smoked too little in the past. blood samples were collected after 14-hours fasting for measurement of total cholesterol, hdl- cholesterol, glucose immediately before the coronary angiography was started. then, the concentrations were determined following preparation of calibration curves and evaluation of line equation. baseline demographic and laboratory data are presented for continuous variables as mean sd and discrete variables as frequencies. parametric and non - parametric data analysed with t - test and chi - square between normal and total atherosclerotic groups. the mean difference of cu level between four groups was analysed using a one - way analysis of variance (anova). baseline demographic and laboratory data are presented for continuous variables as mean sd and discrete variables as frequencies. parametric and non - parametric data analysed with t - test and chi - square between normal and total atherosclerotic groups. the mean difference of cu level between four groups was analysed using a one - way analysis of variance (anova). there was no significant difference in smoking habits, occurrence of dm and ht, serum tc / hdl and blood glucose levels between four groups. the serum level of cu in normal coronary group was significantly lower than the total atherosclerotic groups (p value = 0.001). the serum level of cu was significantly increased with severity of atherosclerosis and significantly upper in sever atherosclerotic patients (33 syntax scores and above it) than mild and moderate cad groups. (p value = 0.001). the serum level of cu in four groups therefore, there were significant differences in serum level of cu between normal coronary group and atherosclerotic patients and between cad groups. there was no significant difference in smoking habits, occurrence of dm and ht, serum tc / hdl and blood glucose levels between four groups. the serum level of cu in normal coronary group was significantly lower than the total atherosclerotic groups (p value = 0.001). the serum level of cu was significantly increased with severity of atherosclerosis and significantly upper in sever atherosclerotic patients (33 syntax scores and above it) than mild and moderate cad groups. (p value = 0.001). the serum level of cu in four groups therefore, there were significant differences in serum level of cu between normal coronary group and atherosclerotic patients and between cad groups. cad has been associated with several risk factors including sex, age, dyslipidaemia, diabetes mellitus, cigarette smoking and hypertension.10 oxidation is an important pathway in the pathogenesis of cad through oxidation of low - density lipoprotein (ldl) and free radical formation1112 and cu is essential micronutrient for enzymes that catalyse oxidation reduction reactions.12 in this study, higher serum level of copper in patients with atherosclerosis compare of the normal group was detected. on the other hand, the serum level of cu increased dependent to degree of atherosclerosis. these findings suggest that high level of this element may play a role in atherosclerotic process. investigated that serum cu level were significantly upper in patients with cad than in healthy subjects.13 shokrzadeh. determined the levels of cu in 30 patients with ischemic cardiomyopathy diagnosed with coronary angiography they found higher serum cu levels in the patient groups than in the healthy subjects.14 in a study performed in patients with acute coronary syndrome by altekin., there was the positive correlation between serum cu levels with elevated troponin t, troponin i and ck - mb values (0.9, 1.0 and 30 ng / ml, respectively).15 the same results were declared by cikim. in patients with acute coronary syndrome.16 our study was performed in patients with stable angina and we found that serum level of copper is higher in cad patients than normal group. the relationship between serum cu levels and cad is controversial in some studies.1718 alissa. proposed that serum cu level had not significant difference in cad group and healthy subjects6 but in his study, case and control groups were not completely matched. in the present study, all groups were matched in regards of cardiovascular risk factors and we found that higher serum cu level is an important cad risk factor. this study showed that, there is basic relationship between serum cu level and atherosclerosis, and revealed an association between cu level and the severity of atherosclerosis. larger prospective cohort studies are needed to confirm our observations, and experimental data may further elucidate the biological mechanisms of the associations. | background : atherosclerotic disease is the most important cause of mortality in the world. oxidation is an important pathway in the pathogenesis of coronary artery disease (cad) through oxidation of low - density lipoprotein (ldl) and free radical formation. copper (cu) is an essential micronutrient for enzymes that catalyse ldl oxidation reactions. therefore, an evaluation of cu in the atherosclerotic disease is important.materials and methods : in this study, 334 subjects without recent cardiac event and history of collagen vascular or infectious disease were investigated. all patients divided into four groups to evaluate severity of cad according to syntax scoring system. all groups were matched in cardiovascular risk factors.results:the serum level of cu was significantly higher in total atherosclerotic groups than normal group (p value = 0.001) and significantly increased with severity of atherosclerosis.conclusion:the finding indicated that the serum level of cu is higher in atherosclerotic patients and it increases with severity of atherosclerosis. therefore, it may be possible that the basic relationship exist between serum cu level and atherosclerosis and an association between cu level and severity of atherosclerosis. |
coccygodynia, first described by simpson in 1859, is disabling pain in the coccyx that is usually provoked by sitting or changing from a sitting to a standing position. this tail bone pain may radiate rostrally to the sacrum or lumbar spine or laterally to the buttocks. patients may rarely present with associated rectal pain or radicular symptoms one third of patients have associated back pain, contributing to misdiagnosis1,2,23,24). due to unfamiliarity with this condition by spine specialist compounding the problem, most neurosurgeons and orthopedic spine surgeons are uncomfortable treating coccygodynia due to lack of surgical training with coccygectomy. fortunately, the knowledge to properly diagnose and surgically treat chronic refractory coccygodynia may be easily acquired. diagnosis is based on the history and physical exam, supplemented by imaging findings and local injection of the coccyx. to raise awareness among our neurosurgical colleagues on coccygodynia and surgical treatment, we review the literature on coccygectomy and describe the surgical technique based on our extensive experience at university of california, davis medical center (ucdmc). relevant articles were retrieved with pubmed using the key words, " coccygodynia " and/or " coccygectomy ". we limited the search to the english - language literature published from 1980 to january 2012. selected manuscripts were analyzed for the number of patients, age, gender, symptom duration, etiology, radiographic classification, type of surgery, use of antibiotics or drain, complications, and followup period. statistical analysis of the data was performed using the statistical package for the social sciences (spss), version 16.0 (spss, inc., continuous variables were expressed as the meanstandard deviation, and categorical variables were expressed as frequencies and percentages. the chi - square test was used to evaluate any potential association of the two different surgical procedures with the postoperative outcome and complication rate. odds ratios (or) and 95% confidence intervals (ci), were calculated by means of simple logistic regression analysis. after the uc davis institutional review board approval was obtained, 61 patients who had coccygectomy for chronic refractory coccygodynia between 1997 and 2009 were identified. for a variety of reasons, only 26 patients participated in a telephone survey. x - rays of responders were classified according to the postacchini and masobrio methods (table 1). outcomes were categorized as excellent (complete pain relief), good (relief of most pain but mild discomfort after prolonged sitting), fair (minimal or no pain relief), or poor (pain worse after the surgery). all patients received a bowel preparation preoperatively to help prevent fecal contamination of the wound and to minimize complications in the unlikely event of a rectal perforation. after the induction of general anesthesia, the patient was positioned prone on a wilson frame. the coccygeal region and anus are prepared with iodine or chlorhexidine. following the skin preparation, the perianal area is isolated with a 3 m 1010 steri - drape, and the incision site is then draped with sterile towels. a midline vertical incision is made over the coccyx followed by an exposure from proximal to distal direction. after removal of the sacrococcygeal disc the coccyx is elevated and separated from the surrounding tissues circumferentially in a subperiosteal plane using monopolar electrocautery, proceeding with an en bloc resection in a rostral to caudal direction in the manner of key at al. (table 2). dissection from a proximal to distal direction avoids the risk of rectal injury, especially in the case of an anteverted coccyx. in some cases of posterior intercoccygeal subluxation the co-1 is identified anatomically by the cornua at the articulation with the caudal sacral segment, and is usually twice as wide as the co2 segment. it is often helpful to resect the cornu with a kerrison rongeur to facilitate mobilization of the coccygeal segment. in the case of a fused s5-co1 disc, the resection proceeds distal to the first mobile segment, or total coccygectomy is preferred for thinner patients, where the co1 segment may present a symptomatic bony prominence if it is not removed. cutting monopolar electrocautery current on a low setting is favored over coagulating current to limit damage to surrounding tissues that could result in wound infection or rectal injury. a clamp may be appliedat the lateral aspects of the coccyx to aid in posterior elevation and retraction. a complete resection may be ascertained by examining the resected specimen (fig. 1) and by comparing a lateral c - arm fluoroscopic radiograph with preoperative imaging to ensure complete resection. all remaining sharp prominences on the caudal sacrumare smoothed using a rongeur, and bone wax is applied. after hemostasis is ensured, the overlying fascia and skin are closed in layers, with subcutaneous sutures for the skin. a liquid skin adhesive (dermabond) is applied to help protect the wound from contamination. broad - spectrum antibiotics are continued for 48 - 72 hours, and the patient receives nursing care in a lateral position or supine on a specialized sacral cutout cushion to avoid direct pressure on the surgical wound. relevant articles were retrieved with pubmed using the key words, " coccygodynia " and/or " coccygectomy ". we limited the search to the english - language literature published from 1980 to january 2012. selected manuscripts were analyzed for the number of patients, age, gender, symptom duration, etiology, radiographic classification, type of surgery, use of antibiotics or drain, complications, and followup period. statistical analysis of the data was performed using the statistical package for the social sciences (spss), version 16.0 (spss, inc., continuous variables were expressed as the meanstandard deviation, and categorical variables were expressed as frequencies and percentages. the chi - square test was used to evaluate any potential association of the two different surgical procedures with the postoperative outcome and complication rate. odds ratios (or) and 95% confidence intervals (ci), were calculated by means of simple logistic regression analysis. after the uc davis institutional review board approval was obtained, 61 patients who had coccygectomy for chronic refractory coccygodynia between 1997 and 2009 were identified. for a variety of reasons, only 26 patients participated in a telephone survey. x - rays of responders were classified according to the postacchini and masobrio methods (table 1). outcomes were categorized as excellent (complete pain relief), good (relief of most pain but mild discomfort after prolonged sitting), fair (minimal or no pain relief), or poor (pain worse after the surgery). all patients received a bowel preparation preoperatively to help prevent fecal contamination of the wound and to minimize complications in the unlikely event of a rectal perforation. after the induction of general anesthesia, the patient was positioned prone on a wilson frame. the coccygeal region and anus are prepared with iodine or chlorhexidine. following the skin preparation, the perianal area is isolated with a 3 m 1010 steri - drape, and the incision site is then draped with sterile towels. a midline vertical incision is made over the coccyx followed by an exposure from proximal to distal direction. after removal of the sacrococcygeal disc the coccyx is elevated and separated from the surrounding tissues circumferentially in a subperiosteal plane using monopolar electrocautery, proceeding with an en bloc resection in a rostral to caudal direction in the manner of key at al. dissection from a proximal to distal direction avoids the risk of rectal injury, especially in the case of an anteverted coccyx. in some cases of posterior intercoccygeal subluxation the co-1 is identified anatomically by the cornua at the articulation with the caudal sacral segment, and is usually twice as wide as the co2 segment. it is often helpful to resect the cornu with a kerrison rongeur to facilitate mobilization of the coccygeal segment. in the case of a fused s5-co1 disc, the resection proceeds distal to the first mobile segment, or total coccygectomy is preferred for thinner patients, where the co1 segment may present a symptomatic bony prominence if it is not removed. cutting monopolar electrocautery current on a low setting is favored over coagulating current to limit damage to surrounding tissues that could result in wound infection or rectal injury. a clamp may be appliedat the lateral aspects of the coccyx to aid in posterior elevation and retraction. a complete resection may be ascertained by examining the resected specimen (fig. 1) and by comparing a lateral c - arm fluoroscopic radiograph with preoperative imaging to ensure complete resection. all remaining sharp prominences on the caudal sacrumare smoothed using a rongeur, and bone wax is applied. after hemostasis is ensured, the overlying fascia and skin are closed in layers, with subcutaneous sutures for the skin. a liquid skin adhesive (dermabond) is applied to help protect the wound from contamination. broad - spectrum antibiotics are continued for 48 - 72 hours, and the patient receives nursing care in a lateral position or supine on a specialized sacral cutout cushion to avoid direct pressure on the surgical wound. total of 28 manuscripts met the inclusion criteria and were analyzed (table 3). only two of them were prospective series19,32). the majority were retrospective uncontrolled case series. in total, the reviewed series included 742 patients with coccygodynia that had coccygectomy as definitive pain management. the youngest patient was 11 and the oldest was 78 with the mean age ranging from 26.4 to 52.8 years3,8). out of 706 patients with their sex identified, 592 were females (83.9%) and 114 were males (16.1%), with the male to female ratio of 1:5.2. the etiology of the coccygodynia was reported in all except for five articles, totalling 556 patients6,17,19,26,31). the most common causeof coccygodynia was direct trauma, reported in 325 patients (58.5%). idiopathic coccygodynia, childbirth, and recent lumbar spinal surgery or rectal surgery or epidural injection comprised of 174 cases (31.3%). abnomal imaging characteristics have been described, most often using a classification system introduced by postacchini and massobrio which characterizes kyphotic angulation and/or subluxation (table 1)4,8,11,15,16,24,30). out of 176 x - raysanalyzed, there were 64 type i (36.4%), 55 type ii (31.3%), 31 type iii (17.6%) and 26 type iv(14.8%). abnormal imaging on plain and dynamic x - rays, including hypermobility, ventral angulation, and subluxation, are associated with coccygodynia but are not sine qua non characteristics. there were four (one prospective) comparative studies assessing the efficiency of injections to coccygectomy13,25,30,32). their findings showed that injection with manipulation is effective in providing pain relief, but they also suggested that coccygectomy is a reasonable choice for those in whom the conservative management failed. prior to coccygectomy, the majority of patients had undergone non - operative treatment modalities for a variable period of time, ranging from 3 months to 15 years. in four papers3,6,13,30) a second generation cephalosporin was administered for 48 hours postoperatively, while the rest of the studies vary significantly as to the type and duration of their chemoprophylaxis. have recommended the use of a drain for avoiding the void space which could compromise the results6,19,22). on the other hand, the usage of drain in close proximity to rectum may be considered as a cause for increased infection rate, but this systematic review could not confirm any direct relation between the usage of drain and rate of infections. following surgery, laxative or enema together with a low residual diet patient follow - up ranged from 4 months to 16 years after the surgery, with the vast majority of the studies having a mean follow - up period of more than 2 years. our literature review revealed that 623 out of 742 patients treated with coccygectomy had excellent or good outcome (84.0%). some of the authors used visual analogue scale (vas) and the oswestry disability index (odi) for evaluating their results3,5,6,13,16,22,23,27). perkins.23) and hodges.13) presented a decrease of vas from 8.3 to 4.5 and 7.3 to 3.6, respectively. similarly the reduction of odi was from 35.6 to 12.5 and 55 to 36, respectively. cebesoy.5) also commented that there was a progression of vas reduction through time from 5.18 pre - operatively to 3.18 at 6 months and 2.94 and 2.76 at 12 and 24 months, respectively. kerr.16) reported that the outcome appeared to be durable over time and not dependent on the cause of pain. perkins.23) reported only 54% with a good outcome (7/13) probably due to the fact that most of their patients also had associated lumbar spinal disorders (10/13). bayne.2) reported 40% fair to poor outcome (19/48) and advocated against the gardner surgical technique (table 3) and for the use of perioperative antibiotics. key 's proximal to distal surgical technique was reported in seven studies3,4,6,16, 23,25,26,30), whereas in only one study was gardner 's distal to proximal exposure used2). statistical analysis of the incidence of complications between the two different techniques revealed a trend toward a higher complication rate in patients treated with the gardner method (10 of 48 patients ; 20.8%) compared with key 's operation (30 of 232 patients ; 12.9%), but this difference did not reach statistical signi. overall, the risk of complications was increased by almost 80% among patients who underwent gardner'ssurgical exposure compared with patients managed with key 's technique (or, 1.77 ; 95% ci, 0.80 - 3.93). patients who underwent key 's operation were more likely to have better results compared with those who were operatedwith the gardner method ; the results of key 's operation were excellent in 203 (87.5%) patients, fair in 11 (4.7%) patients and poor in only 18 (7.8%) patients, while 29 (60.4%), 11 (22.9%) and 8 (16.7%) of the patients who were managed withthe gardner method had excellent, fair and poor results, respectively. patients who underwent key 's operation were almost five times more likely to experience an excellent result than those who were managed with the gardner method (or, 4.59 ; 95% ci, 2.28 - 9.21). escherichia coli and staphylococcus aureus were the most frequently recorded bacteria in those series with reported microbiology results. moreover, there were 2 hematomas, 6 wounds dehiscence that lead to further surgical management, 3 persistent drainage and 6 delayed healings. there were 4 cases (0.6%) of re - do operation to excise to re - operation for excision of remnant coccyx or the distal cornua of the sacrum. the average patient age was 42 years (range 25 - 78 years), and the male to female ratio was 1:4. follow - up telephone survey was able to be obtained only from 26 patients from the entire series (42.6%). the most common cause of coccygodynia was direct trauma, recorded in 15 patients (57.7%). of note, three cases (11.5%) developed after esophagectomy, lumbar fusion and lumbar diskectomy (1 case each). the median duration of follow up from the time of surgery was 37 months (range 2 - 133 months). of the 26 respondents, we could classifythe coccyx according to the schema described by postacchini and massobrio in 24 patients (table 2). of these coccyges, seven were type i (29.2%), seven were type ii (29.2%), two were type iii (8.3%), and seven were type iv(29.2%). the number of patients with outcomes rated as " excellent, " " good, " " fair, " and " poor " were 13, 9, 2, and 2, respectively. 31% of respondents claimed they had been misdiagnosed as having some other pathological condition explaining their coccygodynia. 85% of respondents stated they would undergo the operation again if faced with the same situation. 96% of respondents would have had the procedure sooner if they had been given the option, and 85% would recommendthe surgery to others. the mean vas score preoperatively was 9.60.8, and postoperatively it was 3.13.1 (p 20 of sacrococcygeal or intercoccygeal angulation) is seen in approximately 70% of patients with coccygodynia (fig. other lesions include anterior subluxation and a bony spicule on the dorsal tip of the coccyx (seen in 5% and 14% of coccygodynia, respectively). lumbosacral mri with contrast is recommended in all patients to define normal and abnormal bony anatomy and to rule out less common causes of coccygodynia, such as abscess or tumors. ct should be ordered in cases of acute pelvic trauma, and as an adjunct to mri in evaluating neoplastic disease. the goal of treatment is to eliminate or significantly reduce coccygeal pain and allow the patient to resume a premorbid lifestyle. incidentally discovered tumors or other pathologies are referred immediately to appropriate specialists. all patients with acute coccygodynia (symptoms 2 months) should receive the same therapies recommended for acute coccygodynia before trying more invasive measures. patients who have failed acute management should obtain dynamic sacrococcygeal x - rays and mri to rule out tumor or other pathology. favorable results are reported after corticosteroid and local anesthetic injections given on an as - required basis20,23). they may be given alone or in combination with invasive manipulation (i.e., transrectal flexion and extension) under general anesthesia, the latter being considered more successful32). the corticosteroid plus local anesthetic should either be injected into the soft tissues around the sides and tip of the coccyx (using methylprednisolone) or the sacrococcygeal (sc) junction and dorsalperiosteum of the coccyx (using triamcinolone). however, percutaneous sacrococcygeal junction injection is sometimes recommended if local injection and/or manipulation fails, and may be accomplished fluoroscopically, or with digital rectal localization ofthe sc junction. if corticosteroid injections have no effect after 2 successive monthly injections, physical therapy combined with corticosteroid injection is an effective second - line option. physical - therapy measures include transrectal pelvic floor massage and coccygeal mobilization32). in cases of persistent symptoms unresponsive to conservative treatment, different types of surgical treatment have been described but key 's method is most popular (table 2)3,4,6,7,15,16,19,21,22,23,24,25,26,30). some have even advised against coccygectomy because they found that coccygectomy frequently failedto relieve the symptoms9). statistical analysis revealed that patients who underwent coccygectomy according to key had five times more chances to havea better outcome. according to postacchini and massobrio, the coccyx can be removed either totally or partially with comparablegood results24). in most papers, however, total resection had better outcomes than partial resection and was recommended to reduce the chance of re - do operation11,12,15,16,21,25). a periosteal resection instead of a subperiosteal resrection has also been reported as an option3,22). we feel that the subperiosteal resection, which preserves the ligamentous attachments and anococcygeal ligament, is a safer and more straightforward than a periosteal and ligamentous resection, and could theoretically decrease the rare complication of rectal hernia. in our surgical case series, we encountered no cases of intraoperative rectal perforation or delayed rectal hernia associated with internal wound dehiscience. kerr. reported that no significant difference in outcome could be detected based on traumatic versus nontraumatic causes (p=0.33)16). on the other hand, coccygectomy is more successful in cases of traumatic and postpartum coccygodynia than in idiopathic coccygodynia (75% success versus 58% success)2,22). bayne. reported that coccygectomy is seldom successful for pain associated with lumbar disc disease requiring lumbar laminectomy and spine fusion2). confining coccygectomy to patients with radiographic instability on dynamic x - rays can result in good or excellent outcomes in 92% of cases (fig. wound infection is the most common complication after surgery, with rates ranging from 2% to 22%9). wound infection is facilitated by the location of the skin incision in the inter - lineal fold in proximity to the anus. infection rates as high as 50% have been reported, although in recent series the rates are much lower (0 - 3%)1,5). overall infection from 28 papers was 64 cases (10.0%) (table 3). it is notable that smallcase series had the highest complication rates, ranging from 26.7 to 50%5,13,25,30). bayne. reported also a large number of complications (20.6%) that was attributed to no antibiotic prescription in contrast to the rest of the authors and protocols2). escherichia coli and staphylococcus aureus were the most frequently recorded bacteria in those series with reported microbiology results. there were several options to decrease the wound infection. among the surgical approaches, key 's surgical exposure showed lower incidence of complications compared with gardner 's technique. patients who underwent key 's operation were almost five times more likely to experience an excellent result than those who were managed with the gardner method. it is of note that some authors reported that sub - periosteal resection gives better results and reduce the risk of infection compared with total coccygectomy3,22). the use of a drain along with postoperative antibiotics may reduce the incidence of postoperative wound problems including infection. bayne. reported a large number of complications (20.6%), but also mentioned that the lack of preoperative antibiotics, which is standard now2). there is no consensus how long antibiotics should be used postoperatively. in most papers, antibiotics were used for 72 hours after the surgery and covered bothaerobic and anaerobic organisms. patient may be given a low residual constipating diet to avoid the need for bowel movements inthe immediate post - operative period. there were 2 hematomas, 6 wounds dehiscence that lead to further surgical management, 3 persistent drainage, and 6 delayed healings. there were 4 cases (0.6%) of re - do operation to excise remnant coccyx or the distal cornua of the sacrum. none of the published case series have reported the complication of rectal hernia or rectal injury but two case reports exist.10, 18 the overall total complication rate was 13.3%. it is notable that there were 4 case of re - do operation to resection the remnant of the coccyx. several authors reported the advantages of performing total coccygectomy compared with partial one, which appears to be associated with an increased incidence of recurrent pain and revision surgery11,12,15,16,21,25). residual coccygeal fragments or a prominent sacral edge in a thin patient may lead to poor outcomes and necessitate reoperation for redo coccygectomy, or rongeuring of the edge of the sacrum. we recommend total coccygectomy instead of partial coccygectomy to reduce the need for re - do operation. it can be helpful to confirm for any protrusion or remnant with c - arm just before closing the wound. all patients should receive a dynamic lateral sacrococcygeal x - ray in both the standing and sitting positions. four types of coccygeal configurations have been described by postacchini and massobrio : type i (normal, curved slightly forward, seen in 68% of the general population), type ii (more curved, straight forward), type iii (sharply angulated), and type iv(subluxed)24). in coccygodynia, patients are less likely to have type i (31%) and more likely to have type iii andiv configurations. fusion of the sacrococcygeal joint appears to be a predisposing factor in coccygeal pain (51% versus 37% in people without coccygodynia) 24). radiographic instability as evidenced by posterior subluxation and hypermobility (> 20 of sacrococcygeal or intercoccygeal angulation) is seen in approximately 70% of patients with coccygodynia (fig. other lesions include anterior subluxation and a bony spicule on the dorsal tip of the coccyx (seen in 5% and 14% of coccygodynia, respectively). lumbosacral mri with contrast is recommended in all patients to define normal and abnormal bony anatomy and to rule out less common causes of coccygodynia, such as abscess or tumors. ct should be ordered in cases of acute pelvic trauma, and as an adjunct to mri in evaluating neoplastic disease. the goal of treatment is to eliminate or significantly reduce coccygeal pain and allow the patient to resume a premorbid lifestyle. incidentally discovered tumors or other pathologies are referred immediately to appropriate specialists. all patients with acute coccygodynia (symptoms 2 months) should receive the same therapies recommended for acute coccygodynia before trying more invasive measures. patients who have failed acute management should obtain dynamic sacrococcygeal x - rays and mri to rule out tumor or other pathology. favorable results are reported after corticosteroid and local anesthetic injections given on an as - required basis20,23). they may be given alone or in combination with invasive manipulation (i.e., transrectal flexion and extension) under general anesthesia, the latter being considered more successful32). the corticosteroid plus local anesthetic should either be injected into the soft tissues around the sides and tip of the coccyx (using methylprednisolone) or the sacrococcygeal (sc) junction and dorsalperiosteum of the coccyx (using triamcinolone). however, percutaneous sacrococcygeal junction injection is sometimes recommended if local injection and/or manipulation fails, and may be accomplished fluoroscopically, or with digital rectal localization ofthe sc junction. if corticosteroid injections have no effect after 2 successive monthly injections, physical therapy combined with corticosteroid injection is an effective second - line option. in cases of persistent symptoms unresponsive to conservative treatment, however, coccygectomy is offered as the definitivetreatment option24,28). different types of surgical treatment have been described but key 's method is most popular (table 2)3,4,6,7,15,16,19,21,22,23,24,25,26,30). nevertheless, controversy exists11,24). some have even advised against coccygectomy because they found that coccygectomy frequently failedto relieve the symptoms9). statistical analysis revealed that patients who underwent coccygectomy according to key had five times more chances to havea better outcome. according to postacchini and massobrio, the coccyx can be removed either totally or partially with comparablegood results24). in most papers, however, total resection had better outcomes than partial resection and was recommended to reduce the chance of re - do operation11,12,15,16,21,25). a periosteal resection instead of a subperiosteal resrection has also been reported as an option3,22). we feel that the subperiosteal resection, which preserves the ligamentous attachments and anococcygeal ligament, is a safer and more straightforward than a periosteal and ligamentous resection, and could theoretically decrease the rare complication of rectal hernia. in our surgical case series, we encountered no cases of intraoperative rectal perforation or delayed rectal hernia associated with internal wound dehiscience. kerr. reported that no significant difference in outcome could be detected based on traumatic versus nontraumatic causes (p=0.33)16). on the other hand, coccygectomy is more successful in cases of traumatic and postpartum coccygodynia than in idiopathic coccygodynia (75% success versus 58% success)2,22). bayne. reported that coccygectomy is seldom successful for pain associated with lumbar disc disease requiring lumbar laminectomy and spine fusion2). confining coccygectomy to patients with radiographic instability on dynamic x - rays can result in good or excellent outcomes in 92% of cases (fig. wound infection is the most common complication after surgery, with rates ranging from 2% to 22%9). wound infection is facilitated by the location of the skin incision in the inter - lineal fold in proximity to the anus. infection rates as high as 50% have been reported, although in recent series the rates are much lower (0 - 3%)1,5). overall infection from 28 papers was 64 cases (10.0%) (table 3). it is notable that smallcase series had the highest complication rates, ranging from 26.7 to 50%5,13,25,30). bayne. reported also a large number of complications (20.6%) that was attributed to no antibiotic prescription in contrast to the rest of the authors and protocols2). escherichia coli and staphylococcus aureus were the most frequently recorded bacteria in those series with reported microbiology results. there were several options to decrease the wound infection. among the surgical approaches, key 's surgical exposure showed lower incidence of complications compared with gardner 's technique. patients who underwent key 's operation were almost five times more likely to experience an excellent result than those who were managed with the gardner method. it is of note that some authors reported that sub - periosteal resection gives better results and reduce the risk of infection compared with total coccygectomy3,22). the use of a drain along with postoperative antibiotics may reduce the incidence of postoperative wound problems including infection. bayne. reported a large number of complications (20.6%), but also mentioned that the lack of preoperative antibiotics, which is standard now2). there is no consensus how long antibiotics should be used postoperatively. in most papers, antibiotics were used for 72 hours after the surgery and covered bothaerobic and anaerobic organisms. patient may be given a low residual constipating diet to avoid the need for bowel movements inthe immediate post - operative period. there were 2 hematomas, 6 wounds dehiscence that lead to further surgical management, 3 persistent drainage, and 6 delayed healings. there were 4 cases (0.6%) of re - do operation to excise remnant coccyx or the distal cornua of the sacrum. none of the published case series have reported the complication of rectal hernia or rectal injury but two case reports exist.10, 18 the overall total complication rate was 13.3%. it is notable that there were 4 case of re - do operation to resection the remnant of the coccyx. several authors reported the advantages of performing total coccygectomy compared with partial one, which appears to be associated with an increased incidence of recurrent pain and revision surgery11,12,15,16,21,25). residual coccygeal fragments or a prominent sacral edge in a thin patient may lead to poor outcomes and necessitate reoperation for redo coccygectomy, or rongeuring of the edge of the sacrum. we recommend total coccygectomy instead of partial coccygectomy to reduce the need for re - do operation. it can be helpful to confirm for any protrusion or remnant with c - arm just before closing the wound. when performed for an appropriate indication, coccygectomy provides an effective and long - lasting pain relief. | objectivea review of the literature on coccygectomy and our patients was performed to assess the effectiveness of coccygectomy for chronic refractory coccygodynia.methodsan english language pubmed search was conducted with the terms " coccygodynia " and " coccygectomy " from january 1980 to january 2012. we retrospectively reviewed the medical records and performed telephone questionnaire on 61 patients who underwent coccygectomy at ucdmc between 1997 and 2009.resultsthere were 28 case series from 1980 to 2012 for a total of 742 patients who underwent coccygectomy following failed conservative management. the mean age ranged from 26.4 to 52.8 years. the most common cause was direct trauma (58.5%) with a male : female ratio of 1:5.2. most patients (84%) had a good to excellent outcome after coccygectomy. the most common complication is wound infection (10.0%). the overall complication rate was 13.3%. similarly, 84.6% of patients from our own surgical case series reported good to excellent outcomes with 11.5% wound infection.conclusioncoccygectomy is an effective treatment for chronic refractory coccygodynia. the surgery isrelatively simple to perform but precaution must be taken to avoid wound infection. |
a 29-year - old woman with marfan 's syndrome was scheduled to undergo the elective bentall 's operation and thoracic aortic replacement with artificial graft. there were no abnormal findings or special histories on the preoperative evaluations. on patient 's arrival at the operating room, after anesthetic induction, the right subclavian vein was cannulated with mac multi access catheter (arrow international inc., pa, usa) using the infraclavicular seldinger technique. during this attempt to cannulate the subclavian vein, after ensuring the aspiration of dark and nonpulsatile blood, a guidewire was introduced without any resistance. this was followed by sliding the catheter over the guidewire, and the wire was withdrawn without apparent resistance. after that, the patient received standardized management according to institutional guidelines including mechanical ventilation and cardiopulmonary bypass during intraoperative period. the patient was transferred to the intensive care unit (icu) and was very stable in vital signs and laboratory tests. there was no evidence of hemorrhage including the volume of blood drained to chest bottles and follow - up chest x - ray (fig., the patient was transferred to the general ward and the central venous catheter was removed. ten minutes after the removal of the catheter, the patient complained of dyspnea and pleuritic pain on right side, at which time approximately 1,700 ml of blood was abruptly drained to chest bottles and the patient 's chest x - ray confirmed a hemothorax on the same side as the cannulation. although the blood pressure was stable with administration of intravenous fluid and norepinephrine, hematocrit values dropped from 32.0% to 22.6%. the patient promptly underwent emergent thoracotomy and approximately 2,500 ml of clotted blood was evacuated during operation. the patient was maintained hemodynamically stable with infusion of 0.05 mcg / kg / min of norepinephrine, 3,500 ml of crystalloid and 4 units of packed red blood cell. there was an injury site by the central venous catheterization on the right side of superior vena cava, which was 3 cm distant from the junction of the right atrium and superior vena cava without any other injuries and reinforced by hemostatic materials. after transfer to the icu, vital signs and other laboratory findings such as arterial blood gas analysis were stable without any pharmacologic supports. the patient was transferred to the general ward after a 4-day icu stay and discharged on the 15th day after the thoracotomy without any complications. central venous catheterization has many important roles in anesthesia and icu care and can be conducted in central veins such as internal jugular, subclavian and femoral veins. although subclavian vein cannulation has been chosen for several advantages such as lower rate of infectious and thrombotic complications, stability, wide diameter of vessel and constant anatomical relationship, it is more frequently associated with mechanical complications including pneumothorax and hemothorax than other methods for access to central veins. the former is mainly associated with mechanical complications and the latter results from infection of the catheter. the risk factors for mechanical complications and failures of subclavian vein catheterization include body - mass index (higher than 30 or lower than 20), women, previous catheterization, previous major surgery or radiation therapy in the region, only one year of training, and more than two needle passages. in this case, however, the patient had no risk factors except woman. also, there have been several reports about early hemorrhagic complications associated with subclavian venous catheterization. s. holt and colleagues reported a hemothorax due to pulmonary arterial bleeding within 10 minutes after supraclavicular approach to subclavian vein. two cases were reported about hemothorax after removal of subclavian vein catheter, which described hemothorax in a child (age 13 months) and hemothorax due to ipsilateral arteriovenous fistula for hemodialysis after removal of subclavian vein catheter in adult. in this case, there were no symptoms or evidence for hemothorax including laboratory test, drained blood volume and vital signs until the catheter was removed on postoperative day 3. although the reason of late hemorrhagic complications remains unclear, some reasons have been suggested including the pleural injury during catheterization, the erosion of the vessel wall due to catheter itself and the infusion of hyperosmolar solution [6 - 8 ]. however, in our case, these reasons do not seem likely, because the hemothorax was developed within 10 minutes after removal of the catheter. some cases have been reported about perforation of vena cava or heart causing hemothorax despite of adequate direction and length of needle insertion because of contraction of the heart and stiffness of catheter. in addition, it was reported that superior vena cava was displaced to the right by dissecting aorta. accordingly, we assume that although the subclavian catheter was misplaced and injured to right - shifted superior vena cava directly, the tip of catheter fit the injured hole perfectly, which made the patient asymptomatic and made physicians certain that the catheterization was conducted well. to our knowledge, it is very rare that the adult patient who was very stable hemodynamically and had no symptoms and clinical evidences for hemothorax during 72 hours after catheterization suffered massive hemothorax, developing immediately after removal of the catheter and requiring thoracotomy. after central venous catheterization, chest radiography is mandatory to confirm the proper location of catheter tip. normally, catheters should be parallel to the wall of central veins and show a gentle curvature within the superior vena cava. in this case, however, we did not find any evidence for injury of superior vena cava on chest x - ray (fig. the success rate of subclavian vein catheterization by anatomical landmark technique is associated with the experience of clinicians. also, fatal complications such as hemothorax, hemomediastinum and cardiac tamponade can occur by repeated attempts and accidental puncture of arteries. therefore, ultrasound guided central venous cannulation can be very useful in speed and patients ' safety and comfort, especially in patients where there is difficult access despite repeated attempts. reductions in the failure rate from 55% to 8% and the complication rate from 41% to 4% was reported with the use of ultrasound in subclavian vein catheterization compared to anatomic landmark - based techniques. hence, we suggest that physicians should be familiar with ultrasound - guided technique through adequate training. in conclusion, the authors re - emphasize careful monitoring even after subclavian venous catheter removal to detect this kind of potentially fatal complication and the need for ultrasound guidance during insertion of central venous catheters. | hemothorax is a possible immediate complication of central venous catheterization. we experienced a patient who suffered from massive hemothorax 72 hours after right subclavian venous catheterization. a 29-year - old female patient with marfan 's syndrome underwent the bentall 's operation and aortic arch replacement with an artificial graft, which was performed uneventfully. she recovered favorably in the intensive care unit and was transferred to the general ward on postoperative day 3. immediately after the removal of the catheter in the general ward, massive hemothorax developed and emergent thoracotomy should have been performed to control bleeding. we report this case to re - emphasize the careful monitoring even after removal of central venous catheter and the need for ultrasound guidance during insertion of central venous catheters. |
the aim of this study was to identify the definition, therapeutics, and mechanism of diabetes from the resources of itm in order to provide new recommendations for the treatment of diabetes. we searched therapeutic itm books such as exir - e - aazam, tibib - e - akbari, as well as material medical itm books such as makhzan - ol - advieh and tohfat - ol - momenin to find topics on ziabites. we also searched sciencedirect and pubmed databases with keywords herbal medicine and diabetes to confirm the effectiveness of the reported methods. we also determined that there were different mechanisms and dosage forms for the management of ziabites. the number of materials related to ziabites mechanisms in makhzan - ul - adwiah was 138. for instance, asperugo procumbens amplifies liver and cucurbita maxima diminish kidney temperature. forms of administration recommended in eksir - e - aazam include oral, inhalation, rectal and topical ; among which oral and inhalation have been verified by conventional medicine. we also found articles that substantiated the antidiabetic effect of some medicinal herbs mentioned in exir - e - aazam. there are several forms of administration recommended in itm for the treatment of diabetes, which have not been used in conventional medicine. therefore, further studies are required to substantiate their effects scientifically in order to develop new effective drugs. | background : diabetes mellitus is a complex disorder of carbohydrate metabolism. according to past studies, the word ziabites in iranian traditional medicine (itm) correlate with diabetes. the aim of this study was to identify the definition, therapeutics, and mechanism of diabetes from the resources of itm in order to provide new recommendations for the treatment of diabetes.methods:we searched therapeutic itm books such as exir - e - aazam, tibib - e - akbari, as well as material medical itm books such as makhzan - ol - advieh and tohfat - ol - momenin to find topics on ziabites. we also searched sciencedirect and pubmed databases with keywords herbal medicine and diabetes to confirm the effectiveness of the reported methods.results:we identified 17 itm books that referred to ziabetes. we also determined that there were different mechanisms and dosage forms for the management of ziabites. the number of materials related to ziabites mechanisms in makhzan - ul - adwiah was 138. for instance, asperugo procumbens amplifies liver and cucurbita maxima diminish kidney temperature. forms of administration recommended in eksir - e - aazam include oral, inhalation, rectal and topical ; among which oral and inhalation have been verified by conventional medicine. we also found articles that substantiated the antidiabetic effect of some medicinal herbs mentioned in exir-e-aazam.conclusion:there are several forms of administration recommended in itm for the treatment of diabetes, which have not been used in conventional medicine. hence, reported solutions can be analyzed for the management of diabetes. notably, there are many herbs mentioned in itm that remain to be studied. therefore, further studies are required to substantiate their effects scientifically in order to develop new effective drugs. |
recent advances in computer hardware and software have greatly extended the time scales that can be covered by biomolecular simulations. these longer time scales (beyond nanoseconds) one important characteristic of these force fields is the ability to accurately model the formation of salt bridges, or pairs of amino acids whose oppositely charged side - chains are within hydrogen - bonding distance in proteins. however, it has long been suspected that the forces between oppositely charged amino acids are overly attractive in molecular dynamics (md) simulations with current biomolecular force fields, and there have been a number of efforts to reduce this artifact in the improvement of various force fields. previous theoretical studies have analyzed the contribution of salt bridges to protein or protein protein complex stability, using both implicit and explicit modeling of solvation. others have studied salt bridges using amino acid analogues, often employing biasing techniques in the simulations. more recently, a comprehensive comparison of force field / water model combinations was conducted for salt bridge interactions between the amino and carboxyl groups of zwitterionic amino acids, using extensive simulations in explicit solvent on the microsecond time scale. here, we evaluated six biomolecular force fields for their ability to accurately model the strengths of salt bridges between the side - chains of oppositely charged amino acids by unbiased, microsecond - scale md simulations in explicit solvent. in particular, we directly compared current amber, charmm, and opls force fields in simulations of association between the side - chain analogues of three different pairs of amino acids, arg / asp, lys / asp, and his(+)/asp. we further tested one of the pairs, arg / asp, by simulating association of blocked amino acid dipeptides. in addition, we evaluated the influence of the solvent model on the strengths of the salt bridges by simulating the side - chain analogue pairs using a selection of different force field / water model combinations. to our knowledge, our microsecond - scale simulations provide the most extensive sampling of salt bridge formation to date, yielding thousands of association / dissociation events, permitting quantitative comparisons, both between the force fields and with experiment. our results reveal considerable variability among the current force fields in terms of the resulting strengths of salt bridge interactions, as well as differences from experimental data. we modeled the formation of salt bridges between the following pairs of oppositely charged amino acids using side - chain analogues : arg / asp (guanidinium cation / acetate anion), lys / asp (butylammonium cation / acetate anion), and his(+)/asp (imidazolium cation / acetate anion). our systems were constructed to be consistent with the experimental conditions under which the equilibrium association constants (ka) of guanidinium acetate and butylammonium acetate have been measured, i.e., using the same concentrations (0.9 m guanidinium and 0.02 m acetate, which corresponds to 100 molecules of guanidinium and two molecules of acetate in the presence of 18 000 explicit water molecules). to ensure a net charge of zero, we included 98 chloride ions (the same counterion that is present in the experiments). the same concentrations of the cation, anion, and chloride ions were also used for the model systems consisting of butylammonium / acetate and imidazolium / acetate. starting models for these simulations were constructed using the packmol software package, immersing the appropriate number of side - chain analogues in periodic, cubic boxes of explicit solvent. for the arg / asp salt bridge, we also used blocked amino acid dipeptides (acetyl arginine only a single copy of each blocked dipeptide was included, corresponding to a concentration of 0.012 m for each salt - bridging partner, with a distance of 10 between the amino acids. all force field parameters of the side - chain analogues were based on those of the complete amino acids. for the chloride ions, parameters derived specifically for the water model were used when available ; otherwise, parameters derived for a similar water model were used. nonbonded parameters of the side - chain analogues, along with those used to model chloride ions and blocked amino acid dipeptides, are provided in table s1 of the supporting information. to alleviate any unfavorable interactions, each model was subjected to energy minimization followed by a two - stage equilibration with harmonic position restraints on all heavy atoms of the side - chain analogues (force constant of 10 kcal mol) using the desmond 3.0.1.0 software package. in the first stage, the energy - minimized system was equilibrated for 20 ps at constant temperature (25 c) using a weak langevin thermostat (frictional constant of 1 ps). during the second stage, the system was equilibrated for 1 ns at constant temperature (25 c) and pressure (1 atm) using the martyna klein thermostat and barostat (coupling time constants of 1.0 and 2.0 ps, respectively). to enable a 2 fs time step, bonds to hydrogen a short - range nonbonded cutoff of 10.0 was used, and long - range electrostatics were calculated using the particle mesh ewald (pme) method. the frame from the second half of the npt equilibration with volume closest to the average was used to start the production simulation. to obtain extensive sampling of salt bridge association (and dissociation) events, 1-s md simulations were performed for each side - chain analogue system ; 10-s simulations were performed for the blocked arginine and aspartate dipeptide systems. all simulations were carried out in the nvt ensemble using a 64-node anton special - purpose supercomputer, which is able to run md simulations roughly 2 orders of magnitude faster than conventional hardware (altogether, the simulations required a total of 40 machine - days). the temperature was maintained at 25 c using the nos hoover thermostat with a weak coupling constant of 0.5 ps. van der waals and short - range electrostatic interactions were truncated at 10.0 ; long - range electrostatic interactions were calculated using the gaussian split ewald method. to enable a 2.5 fs time step, bonds to hydrogen equilibrium association constants (ka) were calculated from the populations of the bound and unbound states of the oppositely charged side - chain analogues. for example, the ka for association between guanidinium and acetate was calculated using the following:1where pbound is the population of the bound state, punbound guanidinium and punbound acetate are the populations of unbound guanidinium and acetate, respectively, and c0 is the reference concentration of guanidinium (i.e., 0.9 m). in addition to species in which a single acetate molecule is bound to a single cation molecule, forming a 1:1 complex (e.g., the guanidinium / acetate complex), species in which acetate is bound to two cation molecules, forming a 1:2 complex (e.g., the diguanidinium / acetate complex) were observed. ka values for the latter are included in table s2 of the supporting information ; the results discussed below focus on formation of the major complex, which is the 1:1 complex. standard errors in the ka values were calculated using a block averaging method. for each side - chain analogue system, the unbound and bound states were defined using the potential of mean force (pmf) as a function of the minimum distance between the nitrogen and oxygen atoms of the positively and negatively charged analogues, respectively (minimum n o distance ; see figure 1). in particular, the point of inflection between the bound state free energy minimum (2.53) and the desolvation barrier (33.5) was used as the bound state cutoff, while 4.5 was used as the unbound state cutoff. if the minimum n o distance between an analogue pair dropped below the bound state cutoff they were classified as bound until they crossed the unbound state cutoff, and vice versa. for simulations of the blocked arginine and aspartate dipeptides, the same definitions of the unbound and bound states were used as for the guanidinium / acetate system. potentials of mean force (pmf) between three different pairs of oppositely charged side - chain analogues using six biomolecular force fields with the tip4p - ew explicit water model. the dielectric constant of water in each simulation, water, was calculated using the following equation:2where mwater is the net dipole moment of water, vwater is the volume occupied by water, t is the temperature of the system, kb is the boltzmann constant, and 0 is the permittivity of free space. the net dipole moment of water was calculated using the following:3where q is the atomic charge of each water site, and r is its position vector. the dielectric constant of water, rather than that of the complete system, was used since it is impossible to calculate the contributions of molecules with a net charge to the system dipole moment from simulations with periodic boundary conditions. the appropriate volume was thus the volume of the water molecules present in the system. for each water model used, a pure water system of the same total volume as that of the side - chain analogue systems was equilibrated using the same protocol, and the molecular volume of water calculated. for each analogue system, the number of water molecules present was multiplied by the molecular volume to calculate the approximate volume of water present in the system. we compared the following six current biomolecular force fields in terms of their ability to model salt bridge interactions : amber ff99sb - ildn, amber ff03, amber ff13, charmm27, charmm22, and opls_2005. in particular, we simulated association (and dissociation) of salt bridges between the following three pairs of oppositely charged amino acids : arg / asp, lys / asp, and his(+)/asp. we focused primarily on simulating side - chain analogues (i.e., guanidinium, butylammonium, and imidazolium cations for arginine, lysine, and histidine, respectively, and acetate anion for aspartate) since these analogues are the minimal systems for studying the formation of salt bridges ; in addition, equilibrium association constant (ka) values for such systems have been experimentally measured, providing an excellent opportunity to validate the simulations. while blocked amino acid dipeptides (i.e., acetyl amino acid n - methyl) might be regarded as being more representative of the protein environment, no experimental ka values for the association of oppositely charged amino acid dipeptides are available. nonetheless, we evaluated the force fields in simulating such systems, focusing on just one of the three salt bridges, arg / asp. finally, in addition to the above simulations, in which each biomolecular force field was paired with the tip4p - ew explicit water model, which reproduces the liquid properties of water at the temperatures and pressures relevant to biology, we also evaluated the influence of the water model on the strength of the salt bridges by testing a selection of force field / water model combinations for all three pairs of side - chain analogues. for each force field a selection of water models drawn from tip3p, mtip3p, tip4p, tip4p/2005, and spc / e were tested, including the water model with which each force field was originally derived. to validate our simulations of association between oppositely charged side - chain analogues, we computed ka values and compared these to those measured by experiments. experimental ka values have been measured for guanidinium / acetate and butylammonium / acetate association by monitoring changes in the pka of acetate in the presence or absence of either the guanidinium or butylammonium cation. our microsecond - long simulations yielded thousands of independent binding events, permitting the extraction of extremely precise ka values, with the mean lifetimes of the bound state ranging from 10300 ps and the mean lifetimes of the unbound state ranging from 20120 ps (table s2, supporting information). in general, the ka values computed from our side - chain analogue simulations are overestimated in comparison to experimentally measured values, with the amber ff03 force field overestimating the strengths of the salt bridges to the least extent and the opls_2005 force field to the greatest extent, when using the same water model (table 1). for example, when using the tip4p - ew water model, the computed ka values for the three types of salt bridges vary by as much as 4-fold, 3-fold, and 4-fold for the associations of guanidinium, butylammonium, and imidazolium with acetate, respectively, which amounts to 1.4-fold, 1.8-fold, and 1.9-fold differences in the probabilities of binding (pbound) (see figure 2). we note that our definition of the bound state is very conservative and that the use of less conservative definitions (e.g., use of the desolvation barrier as a cutoff) yields even stronger association constants, without affecting our overall conclusions. one potential factor that could influence the degree of salt bridge formation in our simulations is the choice of force field parameters for the chloride ions. to verify that these parameters are not the cause for overestimating salt bridge strength, we carried out simulations of a single guanidinium / acetate pair (corresponding to concentrations of 0.1 m) with no chloride ions present. the resulting ka values are even higher than those measured in the presence of chloride ions, indicating that the chloride ion parameters do not cause disproportionate salt bridge stability (table s3, supporting information). results are from 1-s simulations and standard errors were calculated using a block averaging method. experimental ka values of guanidinium and butylammonium acetate permit only a qualitative estimate of the associated error. taking two experimentally measured ka values of guanidinium acetate using different protocols into account, we estimate an error of 0.05, although the true uncertainty is not known. using this estimate, we have back - calculated the range of simulated pbound values that would be expected in our simulation, based on the experimental ka. probabilities of binding (pbound) between three different pairs of oppositely charged side - chain analogues using six biomolecular force fields with the tip4p - ew explicit water model. the pbound values that correspond to the experimentally determined ka values of guanidinium acetate and butylammonium acetate are depicted as horizontal gray bars ; no experimentally measured ka is available for the imidazolium acetate system. two obvious features of a force field that influence the strength of the salt bridges are the atomic charges and radii. as expected, the charmm22 force field yields ka values that are closer to experiment than those from the parent charmm27 force field since the atomic charges for the arginine, aspartate, and glutamate residues were parametrized specifically to reproduce the experimental association of guanidinium acetate. however, the charmm22 force field does not produce as close agreement with experiment as the amber ff03 force field, which shows good agreement for butylammonium acetate. given these results, it appears that the general strategy used to derive atomic charges for the amber ff03 force field is reasonably effective for modeling electrostatic interactions. this strategy involved the derivation of atomic charges in the presence of a continuum solvent model with a dielectric constant of 4 to mimic an organic solvent (protein - like) environment. the resulting atomic charges in the amber ff03 force field are notably less polarized than those in the amber ff9x family (including the amber ff99sb - ildn force field tested here), which were derived in vacuum and share the same set of atomic radii. the amber ff03 atomic charges are also less polarized than those in the amber ff13 force field, with atomic charges possessing increased polarity relative to previous amber charge models. in the amber ff13 charge model, nonpolarizable point charges have been fit to implicitly account for solvent polarization, using iterative cycles of classical md simulations with explicit water (i.e., tip4p - ew) to estimate the water charge density around the solute, followed by quantum mechanical calculations to determine updated solute charges. interestingly, although certain critical atomic radii (e.g., the nitrogen in butylammonium and the oxygen in acetate) in the amber ff13 force field were adjusted from their original values in the amber ff99 force field to reproduce experimental hydration free energies of the relevant amino acid analogues, the resulting strengths of the salt bridges are more overestimated, relative to the other tested amber force fields. notably, the amber ff13 force field results in a free energy landscape for salt bridge formation that is significantly different from those of the other force fields. in particular, as shown by the pmfs as a function of the minimum n o distance between the oppositely charged analogues for the three types of salt bridges (figure 1), the free energy minima for the bound states are consistently shifted to the right in the amber ff13 force field relative to the other force fields. when we substituted the atomic radii in the amber ff13 force field with the original radii from the amber ff99 force field, the free energy minima for the bound states shifted back toward those of the other force fields and yielded significantly deeper minima as well as more pronounced desolvation barriers, particularly for the guanidinium / acetate and imidazolium / acetate systems (figure s2, supporting information). since the atomic charges of the opls_2005 force field are not significantly different from those of the other force fields, the most likely reason for the fact that this force field overestimates the ka values to the greatest extent is that the atomic radii of the nitrogen - attached hydrogen atoms are smaller than those used by the other force fields, potentially allowing the pairs to associate more closely and increasing their electrostatic attraction (table s1, supporting information). consistent with this notion, simulations using the opls - aa force field, which differs from the opls_2005 force field only in that it omits atomic radii for these hydrogen atoms, resulted in slightly more strongly associated salt bridges (table s2, supporting information). we note that the ranking of the strengths of the three types of side - chain salt bridges in our study by the amber ff99sb - ildn, charmm27, and opls - aa force fields is consistent with that observed for their oppositely charged termini in a recent study by others. as mentioned above, we additionally tested salt bridge formation of the arg / asp pair by simulating association / dissociation of blocked amino acid dipeptides, testing six different force fields in conjunction with the tip4p - ew explicit water model. as shown in table 2, the relative ranking of the force fields in terms of the ka is generally consistent with our results from the corresponding side - chain analogue system (guanidinium / acetate). the only exception is the amber ff13 force field, which yields the weakest ka for the association of the amino acid dipeptides, as opposed to an intermediate ka value for the association of guanidinium / acetate. as indicated by the pmf between the arginine and aspartate dipeptides (figure 3), the bound state free energy minimum of the amber ff13 force field is the most shallow among the tested force fields, corresponding to the lowest frequency of salt bridge formation. the inclusion of the backbone groups, therefore, appears to alter its propensity for salt bridge formation, likely through the competition of side - chain / backbone interactions with the side - chain / side - chain interactions between the two amino acids. this result emphasizes the benefit of using unbiased simulations ; had the relative orientations of the amino acids been fixed as in previous studies, any effects of significant side - chain / backbone interactions on the frequency of salt bridge formation would not have been apparent. results are from 10-s simulations and standard errors were calculated using a block averaging method. potentials of mean force (pmf) between blocked arginine and aspartate dipeptides using six biomolecular force fields with the tip4p - ew water model. the larger noise level compared to the data presented in figure 1 is caused by simulating a single pair of binding partners, rather than a concentrated solution. to monitor side - chain / backbone association, we used the same minimum n o distance coordinate and bound and unbound state definitions as used for the side - chain / side - chain interactions. a comparison of the relative probabilities of side - chain / side - chain versus side - chain / backbone association (figure 4) reveals that the force fields generally prefer side - chain / side - chain association by a factor of 2 or more over side - chain / backbone association. the exception is amber ff13, which shows a lower preference for side - chain / side - chain association of 1.3. this slight preference over side - chain / backbone association is likely due to the substantially more polarized backbone amide and carbonyl groups of the amber ff13 force field relative to previous amber force fields (including the amber ff99sb - ildn and amber ff03 force fields). thus, as a result of the delicate balance of side - chain / side - chain and side - chain / backbone interactions, the strength of the arg / asp salt bridge appears to be most accurately modeled (least overstabilized) by the amber ff13 force field in a model system that is representative of a protein environment. the probability of side - chain / side - chain association (pboundsc / sc) over the probability of side - chain / backbone association (pboundsc / bb) for blocked arginine and aspartate dipeptides using six biomolecular force fields with the tip4p - ew water model. in addition to the force field, the choice of water model can affect the strength of salt bridges. to evaluate the influence of the water model, we tested the above three side - chain analogue systems with a selection of force field / water model combinations in addition to the force field / tip4p - ew combinations. regardless of the water model, the relative ranking of the force fields is unchanged in terms of the ka values, with pbound varying by 510% between the water models (table 1). we also evaluated the dependence of salt bridge interactions on the dielectric constant of the employed water model (water). interestingly, despite the fact that the spc / e water model yields a computed dielectric constant (water = 70 ; table s4, supporting information) that is closest to the experimental value (water = 78.4) among all of the tested water models, the use of the spc / e water model results in stronger salt bridge interactions than seen with the tip4p - ew water model, in which the water value is underestimated (water = 56 ; table s4, supporting information). in fact, as shown in figure 5, there appears to be no clear correlation between water of the water model and the strength of the salt bridges. as an aside, the charmm27 and charmm22 force fields were tested with both the standard tip3p and the charmm - modified tip3p (mtip3p) water models with which they were developed. the mtip3p water model includes atomic radii on hydrogen as well as oxygen atoms, whereas standard tip3p includes only the oxygen atom. using the mtip3p water model consistently results in a lower ka values, in better agreement with experiment. this suggests that it may be advisible to use of the charmm - modified tip3p, rather than the standard tip3p water model with any charmm force field. relationship between the probabilities of binding (pbound) and the dielectric constant of the water model (water) for three different pairs of oppositely charged side - chain analogues. the displayed data are simulation results using six biomolecular force fields and six explicit water models. the pbound values that correspond to the experimental association constants (ka) for the guanidinium acetate and butylammonium acetate systems are depicted as horizontal gray bars ; no experimentally measured ka is available for the imidazolium acetate system. the water values were calculated from the first 100 ns of each simulation. for each model, the presence of the solutes lowers the water for each system by 1015 relative to that of pure water (table s4, supporting information). note that the error bars are not visible since 95% confidence intervals for both pbound and water lie within in the symbols area in the graph. we compared the modeling of salt bridge interactions using six current biomolecular force fields. three different salt bridges (arg / asp, lys / asp, and his(+)/asp) were simulated and considerable differences in their strengths were noted, both between the force fields and with experiment. given the availability of experimentally measured ka values for the association of oppositely charged side - chain analogues, we have focused primarily on modeling salt bridge formation using these systems. we also tested the applicability of our results to amino acids by simulating blocked amino acid dipeptides for one of the salt bridges, arg / asp. our side - chain analogue simulations reveal that the computed ka values are generally overestimated, relative to experimental values, with the amber ff03 force field overestimating the strengths of the salt bridges to the least extent and the opls_2005 force field to the greatest extent when using the same water model (tip4p - ew). for the blocked arginine and aspartate dipeptides, we observed general agreement in the relative ranking of the force fields with that obtained from simulations with the corresponding side - chain analogues. the only exception is the amber ff13 force field, which resulted in the lowest probability of salt bridge formation, likely due to the presence of competing side - chain / backbone interactions. thus, while the amber ff03 force field overestimates the strength of salt bridges to the least extent for the side - chain analogue systems, the amber ff13 force field results in an even lower frequency of salt bridge formation than the amber ff03 force field for the complete amino acids. finally, we examined the influence of the water model on the strengths of the salt bridges. irrespective of the water model, the relative ranking of the force fields remained unchanged, with no clear correlation between the probability of binding (salt bridge formation) and the dielectric constant of the solvent (water). in conclusion, when running md simulations in which salt bridge formation may be of interest, careful attention should be paid to the specific force field and water model in simulations of protein systems. several current force fields yield considerably higher ka values than those experimentally determined, a discrepancy that may lead to erroneous conclusions. our encouraging results with the amber ff13 force field suggest that charge derivation strategies that implicitly incorporate solvent polarization from explicit water may significantly extend the lifetime of fixed - charge force fields, which include all of the force fields tested in this study. for example, using polarizable force fields that permit varying the charge distribution within a molecule based on both its conformation and environment may alleviate such shortcomings. in the past, the solvation of ions and charged small molecules, have been modeled using polarizable force fields, resulting in improved agreement with experiment, compared to charmm27 and amber ff99 force fields (equivalent to the amber ff99sb - ildn force field used here). future work will determine whether or not this also holds for protein salt bridges. | recent advances in computer hardware and software have made rigorous evaluation of current biomolecular force fields using microsecond - scale simulations possible. force fields differ in their treatment of electrostatic interactions, including the formation of salt bridges in proteins. here we conducted an extensive evaluation of salt bridge interactions in the latest amber, charmm, and opls force fields, using microsecond - scale molecular dynamics simulations of amino acid analogues in explicit solvent. we focused on salt bridges between three different pairs of oppositely charged amino acids : arg / asp, lys / asp, and his(+)/asp. our results reveal considerable variability in the predicted ka values of the salt bridges for these force fields, as well as differences from experimental data : almost all of the force fields overestimate the strengths of the salt bridges. when amino acids are represented by side - chain analogues, the amber ff03 force field overestimates the ka values the least, while for complete amino acids, the amber ff13 force field yields the lowest ka value, most likely caused by an altered balance of side - chain / side - chain and side - chain / backbone contacts. these findings confirm the notion that the implicit incorporation of solvent polarization improves the accuracy of modeling salt bridge interactions. |
depression is a common mental disorder, with an estimated global burden of 350 million. lifetime prevalence rates range from approximately 3% to 16.9%, with most countries falling somewhere between 8% and 12%. depressive disorder is diagnosed when the patient suffers from depressed mood, loss of interest and enjoyment, and reduced energy leading to increased fatigability and diminished activity for at least 2 weeks along with other common symptoms such as reduced concentration and attention, reduced self - esteem and self - confidence, ideas of guilt and unworthiness, blank and pessimistic views of future, ideas or acts of self - harm or suicide, disturbed sleep, and diminished appetite. if there is a history of at least one hypomanic, manic, or mixed affective episode in the past, then the depressed person is diagnosed to have bipolar depression. unipolar depressive disorders were ranked fourth in 2004 and will rise to the first place by 2030 in terms of the global burden of all diseases. bipolar disorder affected an estimated 29.5 million individuals worldwide in 2004, according to the world health organization. if the current trends for demographic and epidemiologic transition continue, it is estimated that by the year 2020, the burden of depression will increase to 5.7% of the total burden of disease and it would be the second leading cause of disability - adjusted life years. the distinction between unipolar and bipolar depression remains a challenging clinical problem, particularly when bipolar individuals present in the depressive phase and they may easily be mistaken for unipolar depression. patients with bipolar depression who are assumed to have unipolar depression will receive inappropriate therapy that can increase the risk of manic switch or cycle acceleration. measures to clinically recognize or at least to suspect the kind of disorder in the early stage can greatly improve diagnosis and management of such disorders, with more appropriate treatment selection which will help in long - term care of these groups of people. we are yet to know the details of the biological processes involved in these clinical conditions and the different factors associated. this study was undertaken with the objective of comparing the sociodemographic variables associated with unipolar and bipolar depression groups. it also aimed at assessing the degree of depression among the unipolar and bipolar groups. this study is a cross - sectional study conducted in a tertiary care psychiatry hospital in north - east india. patients with unipolar and bipolar depression from outpatient department fulfilling the inclusion criteria were selected by purposive sampling. a total of 330 depression patients, 164 unipolar and 166 bipolar cases, were included in the study. patients of the age group of 1865 years, irrespective of their genders, diagnosed with depression both unipolar and bipolar as per the international statistical classification of diseases and related health problem tenth revision (icd-10), were included. patients without medication for at least 1 month before the onset of the current episode who gave written informed consent were considered. patients with comorbid medical illness or any other psychiatric disorder including substance dependence and personality disorders were excluded from the study. a semi - structured pro forma was used for the assessment of the sociodemographic and clinical variables of the patients. the severity of depression among the depressive patients was assessed by 21-item beck depression inventory (bdi). results were described using descriptive and analytical statistics with the help of spss version 23.0 (spss south asia pvt ltd., bangaluru, karnataka, india). patients of the age group of 1865 years, irrespective of their genders, diagnosed with depression both unipolar and bipolar as per the international statistical classification of diseases and related health problem tenth revision (icd-10), were included. patients without medication for at least 1 month before the onset of the current episode who gave written informed consent were considered. patients with comorbid medical illness or any other psychiatric disorder including substance dependence and personality disorders were excluded from the study. a semi - structured pro forma was used for the assessment of the sociodemographic and clinical variables of the patients. the severity of depression among the depressive patients was assessed by 21-item beck depression inventory (bdi). results were described using descriptive and analytical statistics with the help of spss version 23.0 (spss south asia pvt ltd., bangaluru, karnataka, india). the sociodemographic data, bdi score, and the severity of the depression have been summarized in tables 1 and 2. sociodemographic and illness profile of patients with unipolar and bipolar depression (for continuous variables) sociodemographic profile of patients with unipolar and bipolar depression (for discrete variables) of total 330 subjects, 197 (59.7%) were male. in the whole sample 59.4% of the male and 36.8% of the females are found to have bipolar depression and this difference was statistically significant. the mean age at onset of unipolar depression was 36.83 11.57 years which is significantly higher than the mean age at onset of bipolar depression [table 1 ]. of 330 patients, 192 (58.2%) were in the age group of 2140 years and 56.8% of them had bipolar depression. only 15 (4.5%) of the total sample were younger than 20 years of age and had depression both in unipolar and bipolar [table 2 ]. thus, the risk of depression rises with age peaking in the middle age [figure 1 ]. table 3 shows that the age at onset of illness after 40 years is significantly associated with unipolar depression. frequency distribution based on age group illness profile of patients with unipolar and bipolar depression (for discrete variables) marital status, socioeconomic status, education, family type, and locality of the patient did not have any significant relationship with the type of depression [table 2 ]. interestingly, hindu depressed patients were likely to have bipolar depression as compared to their muslim and christian counterparts. of 330 patients, 141 were employed and they had more likelihood of having bipolar depression (57.4%). figure 2 shows that the duration of illness in both types of depression follows a similar curve with majority of them having < 48 months of illness. however, the distribution is seen to be more uniformly distributed among the bipolar depression group [figure 3 ]. unipolar depression had a relatively significant lesser duration of total illness as compared to that of bipolar illness. the mean bdi score in unipolar depression is 24.57 6.52, which is significantly higher than that of the bipolar depression group [table 1 ]. frequency distribution based on illness duration normality curve of illness duration in unipolar and bipolar groups the average age of the unipolar group at the time of interview was found to be higher nonsignificantly compared to the bipolar group which is consistent with the previous studies. like other studies, the mean age of illness onset and the illness onset age group is significantly earlier among the bipolar patients than among those with unipolar depression, with the median of the duration of illness more in bipolar group compared to unipolar. bipolar group may have earlier onset with a more chronic course. although the prevalence of unipolar depression is more in female but in bipolar depression, male we have found male are equally exposed in unipolar group which is contrary to many other studies which were population based. as the sample comprised both unipolar and bipolar depression cases and also the sampling was done on purposive method from a psychiatric hospital, we may explain over - representation of males in the sample. married group comprised the majority in both unipolar and bipolar groups, similar to the findings of berlim. in contrast to their result, we found married patients were more prone to unipolar depression compared to bipolar depression, similar to the study of motovsky and pecenak, 2013. employment status of the patient and depression were found to be strongly associated, most cases being unemployed, comparatively more among unipolar group. this is in consistent with the findings from the study conducted in university hospital, trencin, slovakia. economic status of the patients did not show any marked difference between unipolar and bipolar groups. however, most of them represented the economically healthy class, in contradiction to other studies where socially disadvantaged people are seen more prone to depression, either unipolar or bipolar. our finding may be explained on the basis of site of the study and the relative ability of the patients to bear travel expenses. hindu community comprised the majority in both unipolar and bipolar groups, with significantly more subjects in bipolar group. a deep look into religio - ethno - cultural phenomenological aspects of depression may give interesting finding. there is no significant association observed between education level of the subjects and the character of diagnosis ; however, the majority of the unipolar cases are found above matric education, but bipolar groups are found to have lower level of education as seen in other studies. this may be due to the fact of preferences to services by the highly educated group. family type of the patients did not show any significant difference between unipolar and bipolar groups ; however, majority in both were from nuclear type of family. this could be explained by the fact that emergence of nuclear family from the breakdown of joint family resulting in reduced family support. the rural population comprised the majority as the study is based on the setting with rural majority, with no significant difference between unipolar and bipolar groups. the severity of depression is more in unipolar group compared to bipolar group, which is in concordance with the findings of katz., 1982, majority of the cases were in the moderate class of severity, with significantly higher mean bdi scores in unipolar group, as seen in the study of kessler. most of the patients had no family history of illness earlier, with nonsignificant association of family history and character of diagnosis. this finding suggests that either these ailments are associated with other nonfamilial factors or most of the cases in the earlier days might be subclinical that went unnoticed or undiagnosed. our study is an effort, to find out the differences among the unipolar and bipolar groups of patients on the basis of certain sociodemographic variables along with clinical findings. male gender, employment status, hindu religion, onset of illness before 40 years, and a long chronic course of illness are the risk factors associated with bipolar depression. as this study is a hospital - based study done using purposive sampling technique, generalizability of the findings should be done with caution. we are thankful to the financial support provided by the department of biotechnology, government of india. we are thankful to the financial support provided by the department of biotechnology, government of india. | introduction : early diagnosis and management of depression is important for better therapeutic outcome. strategies for distinguishing between unipolar and bipolar depression are yet to be defined, resulting improper management. this study aims at comparing the socio - demographic and other variables between patients with unipolar and bipolar depression, along with assessment of severity of depression.materials and methods : this cross sectional study was conducted in a tertiary care psychiatry hospital in north - east india. the study included total of 330 subjects selected through purposive sampling technique from outpatient department after obtaining due informed consent. mini - international neuropsychiatric interview (m.i.n.i.) version 6.0 and beck depression inventory (bdi) were applied. statistical package for social sciences (spss) version 16.0 was applied for analysis.results:bipolar group had onset of illness at significantly younger age with more chronicity (32.85 11.084). mean bdi score was significantly higher in the unipolar depressive group.conclusion:careful approach in eliciting symptom severity and associated socio demographic profiles in depressed patients may be helpful in early diagnosis of bipolar depression. |
stroke is defined as rapidly developing symptoms and/or signs of focal and global loss of cerebral function lasting for at least 24 hours with no apparent cause other than of vascular origin. therefore, effective risk factor intervention represents the most appropriate to reduce stroke morbidity and mortality. while some risk factors such as hypertension and atrial fibrillation have been recognized as independently related to stroke occurrence, the predictive role of lipid profile has not yet been well established, similar to that reported in myocardial infarction [25 ]. many of the previous clinical investigations have suggested that increased serum cholesterol is a risk factor for ischemic stroke. the present study was designed to evaluate the lipid profile levels of patients who had experienced an acute stroke during the first 24-hour and to compare these levels in different patients suffering from the stroke, either hemorrhagic or ischemic, and healthy individuals. in this cross - sectional study, 258 consecutive patients with acute stroke admitted to the neurology department of sina hospital affiliated to tehran university of medical sciences during september 2006 and september 2007, were studied. the main goal was to determine whether hypocholesterolemia is a risk factor for primary ich. the second goal was to compare the serum cholesterol and triglyceride (tg) levels in the two types of stroke. patients between 40 - 80 years of age of either sex with clinical findings, brain ct - scan or mri indicative of cerebral infraction or intra - cerebral hemorrhage were enrolled in this study. the patients with any underlying diseases especially liver disease, familial hypercholesterolemia and hypothyroidism, taking anti lipid and sympathomimetic drugs, and the patients in whom the cerebral hemorrhage was secondary to cerebral tumor, trauma or previous coagulation disorders were excluded from the study. lipid profile was measured by collecting the patients blood after fasting for 9 to 12 hours. the lipid profile of 187 apparently healthy subjects living in the same community was provided for comparison. the subjects were matched for age and sex within the stroke patients and were selected not to have any underlying disease or a positive history of stroke. significance of differences between patients and controls and among different stroke subtypes was assessed with t - tests or factorial anova and post - hoc for nominal variables. logistic regression was used to compare the significance of different factors including the lipid profile of stroke patients and the controls. two hundred fifty eight patients and 187 controls were enrolled in this study. in the patients group, 65 subjects had suffered from hemorrhagic stroke (group 1) and the other 193 had ischemic stroke (group 2). demographic data of the patients enrolled in this study in regard with the stroke subtype the patients lipid profile in regard with the etiology of the stroke is outlined in table 2. there was no significant difference among the ischemic and hemorrhagic lipid profile, except for the tg values. table 3 demonstrates the relation between the site of hemorrhage / infarction and the measured lipid profile. there was no significant relation between the location of the infarction or hemorrhage and the reported total cholesterol, ldl, tg and hdl levels. lipid profile of the patients enrolled in the study in regard with the stroke 's subtype. lipid profile of the patients enrolled in the study in regard with the site of stroke in each subtype. data showing the influence of different variables on lipid profile is shown in table 4. high levels of ldl increase the risk of hemorrhagic stroke. while the comparison of data retrieved from patients suffering from hemorrhagic strokes with the controls, revealed ldl as the risk factor contributing to the development of ich, tg was reported as a protective factor. in the ischemic group, age and high levels of ldl the logistic regression showing each variable in each stroke subtype compared with the control group similar comparison between ischemic stroke and controls showed that any increase in the age and cholesterol was associated with a greater risk of developing the condition (cholesterol : or= 1.011, p - value < 0.001 ; age : or= 1.040, p - value < 0.001). on the other hand, comparing hemorrhagic stroke with the control group revealed that cholesterol influences the risk of developing the condition significantly (or= 1.008, p - value= 0.021), while tg was a protective factor (or= 0.978, p - value < 0.001). stroke makes a considerable contribution to morbidity and mortality and is one of the top four causes of death worldwide. lipid profile changes are thought to be a risk factor in the occurrence of stroke. on the other hand, stroke itself is also associated with changes in the lipid levels probably because of the accompanying stress and catecholamine overproduction that occurs during an acute stroke. in fact, the available reports have pointed out that stress is associated with considerable decrease in the lipid profile. the present study showed higher levels of total cholesterol in patients with ischemic stroke compared with the control group ; it should be noted that there was no significant relations between the site of infarction and the measured level. cholesterol can be differently involved in stroke, depending on the etiologic subtype. in this view, a large part of the inconsistency of observational data from large studies on the relation between cholesterol and stroke can be due to gathering data by having both types of stroke together in a same group. this can explain the absence of any detectable association between cholesterol and stroke when all types of the stroke, irrespective of the cause, are considered as outcomes. indeed, the lack of association might conceal the positive association with ischemic stroke together with a negative association with hemorrhagic stroke, as resulted in this study. denti reported that ldl - c concentrations over 100 mg / dl along with low hdl - c levels were associated with higher stroke risk. in the present study, we compared the data on the lipid profile of the patients with different types of stroke (hemorrhagic and ischemic) and the control group. findings revealed a significant relation between lipid profile and the occurrence of the ischemic stroke. it also reported that increased cholesterol and ldl levels are associated with higher risk of developing ischemic stroke. tg, however, was not reported to have a considerable role in the development of ischemic stroke. findings of the present study indicating no role for tg in the ischemic strokes are on the contrary to that of certain previous studies [3, 13 ] ; the variable, however, showed a protective effect in patients with ich. as non - lipid risk factors, patients showed, as expected, significant differences in the prevalence of diabetes (13.8 vs. 21.4%) and hyperlipidemia (15.4 vs. 16.3%) in hemorrhagic and ischemic strokes. our study showed that hypertension was significantly associated with group 1 in this study (p= 0.006), in other words a positive history of hypertension was accompanied by 4.5 times higher risk of intra - cerebral hemorrhage in comparison with ischemic infarction. on the other hand, it was revealed that each year increase in age was independently predictive of 4% higher risk of ischemic infarction compared with ich. although we measured lipid profile in acute phase of stroke and ich and realized lower levels of cholesterol compared with the pre - stroke phase, we did not figured out hypocholesterolemia as a risk factor for intracranial hemorrhage. hemorrhage. because of possible temporal changes in lipid profile after acute stroke, it would be appropriated to have a 3-month follow up for comparing with the control subjects. in addition, further studies to reveal the level of effect on different types of stroke is recommended. it could be concluded that ldl levels can be considered as a risk factor for both ischemic and hemorrhagic cerebral events. in view of our study results, total cholesterol was a risk factor in ischemic stroke whereas high tg levels had a protective role against hemorrhagic events. as a result, treating high ldl levels can be a helpful option to reduce these events and eventually decrease the related morbidity and mortality rates. performing a larger study would be helpful to figure out the definite role of hdl and tg levels in cerebral vascular insults, as well. | backgroundchanges in the lipid profile have been suggested as a risk factor for developing ischemic stroke. their role in intra - cerebral hemorrhage, however, is not clear. the present study was designed to evaluate the lipid profile levels of patients who had experienced an acute stroke during the first 24-hour and to compare these levels in different patients suffering from the stroke, either hemorrhagic or ischemic, and healthy individuals.methodsin this cross - sectional study, 258 consecutive patients with acute stroke admitted to the neurology department of our center during september 2006 and september 2007 were studied. as for the control group, 187 apparently healthy subjects living in the same community and matched for age and sex were selected. lipid profile was measured and compared between the three groups.resultsin the patients group, 65 suffered from hemorrhagic stroke (group 1) and the other 193 had ischemic stroke (group 2). except for tg values, there was no significant difference among the ischemic and hemorrhagic lipid profile. age, cholesterol, and ldl influenced the risk of developing an ischemic stroke ; tg was not reported as a risk factor or a protective one. while the comparison of data retrieved from patients suffering from hemorrhagic strokes with the controls, revealed ldl as the risk factor contributing to the development of ich whereas tg was reported as a protective factor.conclusionit could be concluded that ldl level can be considered as a risk factor for both ischemic and hemorrhagic cerebral events. |
attendances to emergency departments due to accidental digital auto - injection of adrenaline seem to be on the rise. for every 50000 epipen units administered correctly, one is accidentally performed into a digit, usually the thumb (1). we present the case of a 32 year old gp who unwittingly deployed an epinephrine pen into the palmar aspect of her distal phalanx of her right thumb. she bathed the affected digit in hot water with no relief and attended the accident and emergency department an hour later where the medical staff were unsure how to proceed with treatment. 120 minutes post - incident, she was referred to the vascular surgery unit and after examination, the distal phalanx of the thumb was still ischaemic, lacked sensation and was difficult to move. capillary refill was absent. in the first instance, a gtn patch was applied to the radial artery just below the thenar eminence. there was no benefit after 30 minutes or two hours post incident. following a google search for similar cases, various case reports and a series looking at digital epinephrine injection instructions regarding phentolamine administration were followed and 2 millilitres of 0.5% phentolamine were administered subcutaneously along the same tract taken by the epinephrine pen. as per advice online, the patient was continually monitored for hypotension or cardiac arrhythmias on the ward. within two minutes, blood supply to the digit was fully restored, with normal movement, sensation and capillary refill and the patient was discharged home. epinephrine is indicated in the emergency treatment of severe anaphylactic reactions to insect stings, bites, food, drugs and other allergens as well as idiopathic or exercise - induced anaphylaxis. the epipen (1:1000 or 0.3mgs in 0.3mls) and epipen jr (1:2000 or 0.15mgs in 0.3mls) auto - injectors administer a single dose of epinephrine intended to be delivered intramuscularly in cases of severe anaphylaxis. for stability purposes, approximately 1.7 mls remains in the auto - injector after activation and can not be used. since preliminary work by zucker in the 1950s into adrenaline analogue reversal (3) and later a case report by jordan in 1969 whereby a dental assistant had self administered epinephrine to a cut finger to reduce bleeding (10), phentolamine reversal of epinephrine has been suggested in the literature repeatedly as the most suitable method of reversal for accidental digital epinephrine injection. phentolamine is a competitive -receptor antagonist that has similar affinities for -1 and -2 receptors. it can be used in large doses for the short term control of hypertension in patients with phaeochromocytoma or in the treatment of hypertensive crisis following the abrupt withdrawal of clonidine. warm water immersions, the administration of systemic or topical nitroglycerin preparations and even topical terbutaline infiltration have been used to treat the accompanying vasospasm that occurs in the digit with limited success (4,5). despite this, using phentolamine in this manner remains poorly publicised and lacks a license for use in the uk for the reversal of end artery vasospasm caused by epipen administration. in fact it is only available as rogitine (ciba), containing 10 mg of phentolamine in 1 ml of clear solution. since administration of around 2 - 5 mls of 0.1% phentolamine appears in most reported cases to be completely effective in reversing the effects of 0.3mgs of epinephrine, the greatest danger in administering phentolamine surely comes from drug errors in calculating the correct dilution. many case reports in fact report the reversal of epinephrine after 12 hours with no harmful sequelae at all (6,7). this raises the possibility that the ischaemia induced is not complete or that the digital arteries are augmented by another blood supply. indeed, the dalhousie project clinical phase trial recorded 1340 fingers electively injected with low dose adrenaline (1:100,000) without a single case of digital skin, or fat loss, digital infarction (1,2,6 - 8). treatment with phentolamine is nevertheless worthwhile due to the distressing ischaemic pain and parasthesia suffered by the patient.(9) previously reported cases of digital infarction in the literature, dating from the pre 1950s were all associated with co - administered procaine or cocaine, both well known to cause massive arterial spasm and digital infarction on their own. of note, there have been no case reports of digital gangrene using commercial lidocaine and epinephrine since its introduction in 1948. multiple studies involving thousands of patients support the premise that the use of lidocaine with epinephrine is safe in the digits (1). hinterberger compared various methods of phentolamine dilution and administration, suggested a 0.5% solution administered by direct local infiltration to the initial puncture region is the most effective as this concentration provides sufficient phentolamine for reversal of ischaemia, without the need to present an inordinate amount of fluid to the pulp spaces of the finger, accessing the affected receptors and any residual epinephrine directly. a digital block technique was found to be less effective as it has to diffuse through tissue and a later study suggested that introduction f large amounts of diluted phentolamine itself could cause an iatrogenic hydrostatic ischaemia (9). there are no reports of systemic side effects related to the local use of small doses of subcutaneous phentolamine for the treatment of adrenaline induced digital ischaemia (4,5) nonetheless, due to the small risk of systemic absorption or accidental intravascular administration of phentolamine it has been standard practise to monitor ecg and blood pressure readings although evidence points to this being unnecessary. this case further highlights the importance of instant access to the internet and the need for available phentolamine in a&e, surgical assessment units or any department where minor surgery takes place. despite the lack of previous experience in the management of such cases, the patient was successfully treated after performing a quick search on google search, demonstrating the need for online resources to augment our practice. | after the accidental injection of epinephrine into a digit, various techniques to try and reverse the ensuing ischaemia were unsuccessful. to identify a further treatment strategy and as members of the admitting team were unfamiliar with digital injection of epinephrine a google search was performed. previous cases were described and separate sources indicated appropriate management protocols utilising phentolamine. after administration, an almost immediate reversal of ischaemic symptoms occurred. this highlights the role of the internet as an adjunct in managing unfamiliar situations and practising evidence based medicine. |
managed care is increasingly becoming a preferred administrative vehicle for states to control their medicaid budgets and assure access to and coordination of services. managed care now serves 23 percent of the medicaid population. as reported by the kaiser commission on the future of medicaid (1995), enrollment in some form of managed care grew from 4.8 million medicaid enrollees to 7.8 million in a single year1993 - 94. this rapid growth is likely to continue with some of the larger states such as california and new york forecasting large expansions in 1996. numerous states now have waivers from traditional medicaid rules (known as 1115 or 1915b waivers) that allow for such expansion. most of the statewide waivers require enrollment in some form of capitated plan, rather than the primary - care case - management form of managed care. one consequence of the shift to capitation is that the ffs billing mechanism that has generated much of the administrative data used in policy planning and research no longer exists, at least not universally nor for all medicaid services. states and hcfa have struggled with how best to replace ffs billing data with other comparable information on medicaid enrollees in capitated managed care plans. both levels of government face the tradeoffs between their need for data to manage the program efficiently and assure that beneficiaries are well served, versus a desire to reduce the administrative burden on the plans which contract with the program. some recent studies have shown that the ability of plans to produce encounter data is often limited. for example, the group health association of america (1994) found in its survey of 330 health maintenance organizations (hmos) that a substantial proportion (more than 10 percent) did not currently have the ability to report patient characteristics or diagnosis for a hospital encounter and that data capabilities for reporting ambulatory encounters were even weaker. a mathematica policy research study for the physician payment review commission (gold., 1995) problems arise most often when the data the plan collects are not automatically generated by the plan 's payment system. though most hmos that pay physicians on other than an ffs basis require encounter data, only a minority of plans with such requirements say that the required data are almost always submitted by providers. clearly a plan can not itself comply with a state 's encounter data requirements if its providers do not send in encounter records. another study of aggregate reporting showed similar problems, with some plans being unable to report aggregate measures of ambulatory care use because of the lack of such information in their administrative data base (aizer, felt, and nelson, 1996). thus the structure of an encounter data system must include, at the most basic level, the provider, then the plan, then the state, and finally the federal government (should it choose to require medicaid encounter data, which to date this article provides an overview of the types of encounter data currently being required of plans, and the problems and issues with providing and analyzing such data. it is based on information provided by hcfa and nine of the states which have had some of the earliest experience with obtaining encounter data as a substitute for ffs billing data. these states are arizona, california, hawaii, maryland, minnesota, new york, rhode island, tennessee, and washington. from them, we reviewed documentation of state data requirements, and we spoke with individuals about their experiences during the past few years as they struggled to implement useful reporting systems (table 1). we also spoke with individuals in states (e.g., massachusetts) which have chosen to require aggregate reports from plans as an alternative to encounter data. together these states had a little more than 50 percent of the medicaid beneficiaries in managed care in june 1994 (kaiser commission on the future of medicaid, 1995). some of the states have had a relatively long experience, dating back to the early 1980s, with developing reporting systems for medicaid managed care enrollees. plans understandably have been skeptical of the need for vast volumes of data to be accumulated at the state or federal level. such skepticism has increased in states which have required encounter data but have not developed the management information systems and internal analytic expertise needed to analyze the data. there is a growing recognition, expressed by almost all the states with which we spoke, that only when data are used and used regularly will the effort to produce and submit data be worthwhile. states have not often routinely used their encounter data in the past, because managed care has been a small part of the program in most states until now. also, states that did collect data lacked a clear concept of how they planned to use them, and consequently lacked concrete plans for analysis and reporting. the precursor ffs billing data have often been used for reporting expenditures, a use that is not directly relevant for encounter data, because state expenditures for capitated plans are defined by premiums. (however, utilization variables such as hospitalizations can be used to create ffs proxy measures.) we spoke with many individuals about the reasons they felt it was important or necessary to routinely collect encounter data. the data needs they expressed fell into the following categories : accountability : utilization, access, and quality analysis. studies of small, high - policy - interest populations. community - wide studies. other research and evaluation studies. developing broad utilization, access, and quality measures, plans are held accountable by the state for providing or arranging all medically necessary care funded by the benefit package for a defined population. state and federal governments need to assure that patients are getting the care that they need. though oversight of this issue occurs in a variety of ways (e.g., external reviews, onsite inspection, consumer complaints and grievances), administrative data are a useful tool for broad tracking and profiling to identify potential problem areas. these measures include, for example, rates of the use of preventive services, hospitalization (including readmissions), and selected surgical procedures. many of these measures are included in state aggregate reporting requirements and form the basis for some sections of the medicaid health plan employer data and information set (hedis), which specifies a variety of aggregate reports that states might require plans to submit (national committee for quality assurance, 1995). critics of required encounter data reporting point out that many such utilization - access - quality measures can be obtained from aggregate reports. also, many states and plans have found that administrative data lack some of the clinical detail needed for oversight, thus making medical record review and surveys necessary. thus, states and hcfa must make a compelling case regarding why they need encounter data to develop such measures. often this case is based on the federal and state need for more flexibility than is provided by routine aggregate reports. although a certain format may be useful at the time when a report is developed, it is likely that another format may be desired subsequently. for example, mental health services for young adolescents may become an important policy topic, whereas the age breakdowns for aggregate reports may group individuals 1019 years of age. plans should consider that it may be in their long - run interest to provide encounter data, rather than to repeatedly respond to modifications in state or federal aggregate reporting requirements. ffs billing data have long been used for ratesetting or for developing risk adjustors to rates. in the past, states have developed capitation rates by a relatively simple approach of developing age - sex - eligibility group cells and averaging medicaid ffs expenditures within each cell. regardless of the method, there can be problems if plans with enrollees that are in poorer - than - average health either choose not to participate or suffer large losses under medicaid managed care. plans whose networks are made up primarily of traditional safety net providers to the medicaid population (such as affiliations of community clinics or public hospitals) may be particularly vulnerable. thus it is to the advantage of both the states and the plans to have the fairest rate possible. although the state - of - the - art for developing risk - adjusted rates is still developing, there are many research projects underway to refine such approaches. some of the most promising, such as ambulatory care groups, are based on using ffs billing or encounter data to adjust payments by the diagnostic mix of a plan 's panel of patients (physician payment review commission, 1995). states which have almost all enrollees in capitated plans lose the ability to test and ultimately use such approaches unless they require encounter data. another way to reimburse plans separately for high - cost outliers is through reinsurance, which is a retrospective approach that may accomplish a similar purpose to prospective risk adjustment. a plan that enrolls a high - risk medicaid population might be allowed to retrospectively receive increased reimbursement for its high - cost population. detailed encounter data will facilitate a state 's ability to use reinsurance, because the state would not otherwise have sufficient information on the distribution of utilization of certain high - cost services (e.g., hospitalizations) in order to evaluate plans ' claims. related to this outlier issue is a more general need to frequently study small, high - policy - interest populations. these individuals, who tend to be sicker and more vulnerable, are the most likely to be affected by the health system, both positively and negatively. however, because of their small numbers, it may be hard to detect problems for these individuals from aggregate data only. more detailed encounter data may be important in allowing states to fulfill their fiduciary and oversight role for these populations. the experience of hcfa and the states with ffs billing data is illustrative of this need. early in the medicaid program, simple aggregate reports were frequently sufficient for both hcfa and the states ' needs. surveillance and utilization review subsystem (surs) and management analysis and reporting subsystem (mars) reports were required for certification of the state 's medicaid management information system and were often used by the states for internal reporting. a uniform report from the states to hcfa, the hcfa-2082 report, served many of hcfa 's management needs. however, as the program grew and as certain high - cost populations became of greater interest (examples include persons with acquired immunodeficiency syndrome, pregnant women, and the mental health and substance abuse population), these reports no longer sufficed to answer questions from state or federal legislatures or other policymakers. state and federal analysts then began using the detailed person - level data derived from ffs billing to answer questions regarding use of services and expenditures for such groups. the detailed data were needed in order to refine analyses based on specific diagnoses or specific services. another related issue that requires encounter data is the frequent need to assess the health of whole communities or other geographic areas. a state might want to compare utilization of all medicaid enrollees in a county or zip code, or for all services provided within a given geographic area. the analysis might identify geographically based access problems in order to help the states and hcfa decide whether certain hospital or health plan services are essential to a particular geographic area. to do such a study, the analyst would need the encounter data for all individuals who lived in an area (or who provided services in an area), based on patient or provider address. aggregate reports from plans would not generally be sufficient studying medicaid enrollees ' care at the community level may also facilitate cooperative identification of areas for improvement across plans. although not all - encompassing, the prior examples are illustrations of the many ways in which states, hcfa, and other policy analysts could typically use encounter data. however, such data will only be useful if they are relatively complete and an accurate representation of the care plans are providing. some of the struggle to develop successful medicaid managed care reporting systems has to do with the lack of agreement about what is the proper unit of analysis for reporting. when the unit of analysis is the plan, states might request aggregate reports on all of the medicaid enrollees a plan served in a particular reporting period. almost all states require some type of aggregate reports either on a monthly, quarterly, or annual basis. this form of analysis is particularly attractive because managed care aims to promote accountability for care to defined populations. plans could be requested to report aggregate counts of services or other summary data on each medicaid enrollee in the plan on a monthly or annual basis. we did not find any states that are requiring this type of reporting, although states potentially can construct such records from encounter data. finally, and most commonly one person gave the following definition : an encounter is a bundle of services provided to one client by one provider in one time period. using this definition, if an individual patient went to two different providers in a day, they would have two encounter records. such definitions can become difficult to operationalize when, for example, a provider is actually a hospital outpatient department with multiple clinics or a multi - specialty group practice. in such circumstances, the number of encounter records will probably depend on how the state enrolls providers and assigns unique provider identifiers. other areas that can lead to variations include, for example, such non - ambulatory services as hospital and nursing home care. we found that most states include such services and define a separate bundle of services (e.g. the hospital stay) as an encounter. also, very often pharmacy services are included with an individual prescription being called an encounter. in all cases which we examined, the definitions of encounters, as operationalized in the states ' data requirements, approximate closely the unit of analysis for their preexisting medicaid ffs billing system. there has been a period of evolution at both hcfa and in each of the states regarding the list of variables and other reporting specifications for their encounter data, and much of the experience is very recent. one state described a period early in their attempts to obtain encounter data in which they simply asked plans to submit data within the parameters of our computer system. similarly, hcfa in its original terms and conditions of approval of statewide managed care 1115 waivers has required that encounter data be collected, but has not specified the content or definition of variables. states with more experience have now learned that very precise definitions of files including lists of variables and variable definitions and precise submission specifications are needed. also, they have learned that plans need ongoing training and technical assistance, especially early in the development of the system. finally, there needs to be intensive, ongoing editing of the data as they come in, and feedback to the plans on the quality of the data. many states define and circulate the edit specifications, along with the error tolerances for each variable. working mostly independently of each other, hcfa, and other payers, each state with which we spoke had developed a variable list for what is to be included in its encounter data set. during the same recent period, 1993 - 95, a parallel effort was underway at hcfa to define a uniform encounter data set (known as mcdata) for both the medicare and medicaid programs. the draft mcdata variable list was sent on september 17, 1995, to 1,000 individuals and organizations for review and comment. they expect the final data set will be substantially similar to the draft version. in a survey sent out with the specifications, hcfa found that 93 percent of the 200 respondents felt that it was appropriate to have a national standard encounter data set. in addition to the standardization that will probably result from the mcdata effort, several states and hcfa have built on the efforts of the national uniform claim form committee, which developed the hcfa-1500 billing form for ambulatory services, and the national uniform billing committee, which developed the ub92 hospital billing form, as well as the american national standards institute (ansi), which has developed standards for electronic submission of the hcfa-1500 and ub92 forms. indeed, the push for standardization is a major impetus for the hcfa mcdata effort. table 2 shows the draft mcdata encounter data set and some other examples of variables that occur on state encounter data sets. it shows that most of the mcdata variables are required by almost all of the nine states. the variables in this typical encounter data set include the identity, and sometimes other characteristics, of the individual receiving the care (i.e., the medicaid enrollee) and the individual or institution providing the care. the reason that the characteristics of enrollees or providers are not always required is that some states plan to obtain those characteristics from their enrollment or provider reference files. (this could be more difficult at the federal level, because hcfa might not have ready access to those files, which are often complex in structure and less uniform across states. states could also have difficulty matching to the reference files if identifiers submitted by plans have errors.) the encounter data includes selected information about the service provided including the date or dates of service, the procedure code, which tells which service was provided, and the diagnosis or diagnoses, describing the condition that required the care. different types of services (for example, hospital, outpatient, pharmacy and dentistry) will require somewhat different variables, and often the states use different file formats for different types of encounter records. some states have very rigorous edits that are applied to data as it comes in. examples include edits to assure that all codes are valid, that numeric data are present where required, and that dates are in logical sequence. states generally specify submission schedules and tolerances which may vary for different categories of variables (e.g., 95 percent of primary diagnoses should be present and within valid range). states report both carrots and sticks that may or may not be included in plan contracts to enforce the submission of complete and accurate data. there may be financial penalties for late submission (e.g., a percent reduction in the capitation fee). alternatively, timely submittors of valid data may be allowed to expand medicaid enrollment more rapidly. most states and hcfa report substantial ambivalence about how tough such enforcement should be, because they are anxious to have plans participate in managed care and recognize that the data requirements are burdensome. as mentioned, many plans do not yet have well - developed internal systems that can generate encounter data completely and consistently. also, medicaid often is only one of a number of diverse parties making data requirements of a plan. if reporting requirements are poorly conceived, the state could drive away the plans that it most wishes to attract (e.g., those with substantial commercial populations). consequently, there may be interagency conflicts within state or federal government, with data and research officials being strongly in favor of encounter data, and managed care program officials being more sympathetic to plan difficulties. although most states recognize that their clout in requiring data has increased considerably over earlier years when medicaid hmo enrollments were lower, most states remain in a cooperative, rather than combative, mode and hope that this cooperative approach will lead to better data over time. it is important to note that encounter data systems are still in a developmental stage in all the states we talked to, with the exception of arizona, which has been working on their system for a decade. although some other states have a long history of contracting with hmos (e.g. california, minnesota, and new york), their major expansions into medicaid managed care have only recently forced them into serious development of their encounter data systems. the lack of internal staff capacity is one major impediment to development of an operational encounter data system. all of the steps previously outlined (e.g.. developing specifications, providing technical assistance to plans, editing the data, providing feedback, and using the data) are time consuming and require staff and resources that many financially strapped states do not currently have. states are particularly challenged now since most will at least in the short term need to continue to maintain the administrative structure for ffs billing. hcfa has provided some help through the recent development of a technical assistance document to states (mccall, 1995). there is also a technical assistance contract to assist 1115 waiver states with the development of their encounter data. however, this type of assistance is late to arrive, is limited in scope, and will not be sufficient. all levels of organizations involved in the effort to develop workable encounter data providers, the plans, the states, and hcfa will need to obtain (either directly or through contract or both) the expertise to develop such systems. in order to make the encounter data that many plans and states will be struggling to provide and analyze most useful, it is important to consider some of the lessons learned from experience with such efforts with ffs billing data, and from recent experience with encounter data. these include the following : regular communication among providers, plans, states, and hcfa is essential. states must have clear specifications for required data, and must not change those specifications very often. attempting to develop a system without the proper resources to do so at the plan, state, or federal level will lead to questionable results. putting resources into such efforts early will prevent problems later. finally, even though the system appears to function smoothly, it is important to keep examining and refining the data as they come in. similarly, a state needs to address the same issues in relating to participating plans. and finally hcfa, to the extent that it receives data from states on a mandatory or voluntary basis, will need to develop a similar approach to data cleaning and analysis. | managed care now serves 23 percent of the medicaid population. with the shift to capitation, the fee - for - service (ffs) billing mechanism that has generated much of the administrative data used in policy planning and research no longer exists. this article provides an overview of the types of encounter data currently being required of plans and the problems and issues with providing and analyzing such data. it is based on a review of documentation and interviews with representatives of nine states and the health care financing administration (hcfa). the article concludes by providing recommendations for hcfa, states, and plans in creating and improving encounter data systems. |
dna, with its building block dna bases that store the genetic information, is indispensable in protein biosynthesis. investigations on long - distance charge transfer through dna are of ongoing interest. moreover, the interaction of excited triplet states of aromatic carbonyl compounds with dna bases is also of interest for biophysical and biochemical studies. these studies may serve as a useful strategy for understanding structure and function of the bases in dna. in the present study, we want to extend the investigations on the interactions between an aromatic ketone derivative, 3,3,4,4-benzophenone tetracarboxylic acid, with selected dna bases. 3,3,4,4-benzophenone tetracarboxylic acid along with 4-benzophenone monocarboxylic acid (4-bc) are derivatives of benzophenone, and their structures are similar. to get an idea about the kinetics of triplet bptc, the previous work on 4-bc should be considered ; however, studies on the photochemical reaction of 4-bc are challenging because of an existing overlap in the spectra of triplet, ketyl radical, and ketyl radical anion between 380 and 720 nm. although the overlapped spectra of these radicals with similar molar extinction coefficients are comparable, the formation of these radicals also depends on the type of quenchers and radical quantum yields. therefore, the various absorptions of the radicals need to be considered carefully when measuring reaction rate constants. in our experiments, we have employed different approaches to analyze these kinetic behaviors. in this paper, we present the kinetics of the photoreaction of triplet bptc with various dna bases in aqueous solution. the ph - dependence of the quenching reaction rate constants is investigated thoroughly for all quenchers. in addition, from cyclic voltammetric measurements, we propose a proton - coupled electron transfer (pcet) by means of a stepwise mechanism from thymine to triplet bptc. before discussing the results, it is helpful to describe first all relevant calculations. as mentioned earlier, two kinetic problems arise when analyzing the data obtained from time - resolved laser flash photolysis : (i) the overlap in the decay of the excited triplet states and the pseudo - first - order growth of the corresponding radicals and (ii) the overlap in the decay of the excited triplet states and the pseudo - first - order growth of the corresponding radicals and their simultaneous second - order decay. to deal with these kinetics, we applied two alternative approaches. according to the low concentrations of bptc (1 10 mol l) used, triplet triplet annihilation is negligible. the interaction of triplet bptc with quencher is a pseudo - first - order reaction. thereupon, the observed quenching rate constant, kqobs, is obtained by monitoring the triplet decay of bptc at fixed wavelengths, applying the stern volmer relation, eq 1a or eq 1b:1a1bhere, kd is the decay rate constant of triplet bptc ; kqobs is the observed quenching rate constant. 0 = (1/kd) is the lifetime of triplet bptc in the absence of quencher ; = (1/kobs) is actual lifetime of triplet bptc in the presence of quencher. reaction scheme 1 illustrates the two kinetic problems, where k1 is the first - order growth of radicals,2and k2 is the second - order rate of the disappearance of radicals : (i) if the system shows a combination of parallel reactions, a decay process and the first - order growth of radicals (pathway i, scheme 1, denoted here as model i), the following equation applies:3where a, a, and a are the absorbances at time 0, t, and infinity, respectively. using kobs obtained from eq 3 and employing eq 1b with various concentrations of the quencher gives values of kqobs. (ii) if the system shows a combined decay process, a first - order growth of radicals and an overlap by their simultaneous second - order decay (pathway ii, scheme 1, denoted here as model ii), the time - resolved absorbance for the system is expressed by eq 44and56thus, taking k1 obtained from the simulation, eqs 46, and applying eq 2 with various concentrations of the quencher gives the value of kqobs. the observed quenching rate constant is expressed according to the suggestion of yurkovskaya., with the assumption that a polyprotic acid symbolized by hna, initial concentration c0 can undergo n - proton dissociation in water and form the corresponding base conjugates. each step has its own dissociation constant, kaj, j = 1,.., n. the fraction of each species is given by eq 7,7with 0 j k ; 0 k n ; ka0 = 1. for example, for triplet bptc, pka1 = 2.1 and pka2 = 4.7 (see the text), whereas for thymine, pka = 9.9. the molar fractions, depicted in figure 1, of all species in the solution are, hence, calculated by eq 7. the ph - dependence of kqobs for the reaction of triplet bptc with thymine is then divided into four regions on the basis of their pka values and molar fractions : each pair of reactants is characterized by the so - called intrinsic quenching rate constant kqi (i = 1... 4), and the kqobs can be treated as a summation of kqi multiplied by the molar fraction of the corresponding species according to eq 8. further details of this equation can be found in the supporting information.8finally, a multiple regression employed on eq 8 with known parameters (kqobs, kaj, kaj, [h ]) estimates the intrinsic values, kqi. similar expressions of the ph - dependence of the observed quenching rate constants were applied for photochemical reactions of bptc with the other quenchers. absorption spectra of bptc recorded in the absence and in the presence of thymine are shown in figure 2. it is likely that there is no association between bptc and thymine in the ground state, since the spectrum of the mixture of both shows an addition of their separate spectra. similar behaviors are observed with the other quenchers at different ph 's. by changing the ph of the solution, it is possible to control which main reactant species are present in the solution. vis spectroscopic measurements, we determined that bptc has two pka values : pka1 = 3.20 0.10 and pka2 = 5.12 0.10. the fact that only two pka values are observed implies that there are two deprotonation steps of bptc characterized by two - proton dissociation for each. base equilibria:9a9bon the basis of the pka values and contribution of species (figure 1, dashed lines), it is unambiguous that eq 9a predominates at ph > 3.2, whereas eq 9b dominates at ph > 5.1. absorption spectra of bptc (4 10 mol l) without and with thymine (1.5 10 mol l) in water at ph = 2.0. deprotonated forms of bptc show absorption spectra at longer wavelengths compared with its neutral state. bptch4 is found to have an absorption maximum at 261 nm (at ph = 2.1), whereas the spectrum of bptch2 is shifted to 269.5 nm (ph = 4.1). laser flash photolysis of bptc (1 10 mol l) in aqueous solutions at ph = 2.0 and 12.0 gives spectra as figure 3. triplet bptc shows a strong absorption peak at max = 590 nm in water at ph = 2.0, shifting to max = 550 nm at ph = 12.0. because of these results, we have chosen 590 and 550 nm as observation wavelengths for kinetic traces of all quenching experiments in the acidic media and in the basic media, respectively. the decay of triplet bptc, observed at both wavelengths max = 590 and 550 nm in water follows a first - order kinetics with kd = (57) 10 s, in agreement with reported values (7.1 10 and 6.8 10 s). transient absorption spectra of bptc (1 10 mol l) in water, monitored by transient absorbances between 360 and 690 nm obtained right after laser flash. because two pka values are observed in the ground state, it is expected that bptc also behaves in the same way in the excited triplet state. the ph - dependence of the absorbance ratio at 590 and 550 nm obtained immediately after laser flash is shown in figure 4. the pka values for triplet bptc are determined from the turning points of this t1 tn absorbance titration curve as pka1 = 2.1 0.2 and pka2 = 4.7 0.2. this indicates that triplet bptc is a stronger acid in comparison with its ground - state one. triplet absorbance titration curve of 1 10 mol l bptc in water, monitored by the ratio of transient absorbances at 590 and 550 nm, obtained right after laser flash. it is realized in figure 1 that bptch4 exits mainly in the solution of ph 4.7 the species bptc is mainly present because of the rapid equilibrium eq 11 although bptch2 could be initially excited (e.g., at ph = 5.1).11 as already pointed out, all experimental traces for triplet bptc decay were monitored at obs = 590 nm in acid solutions and at obs = 550 nm in alkaline solutions, respectively. all experimental data for the measured quenching rate constants were fitted with either model i or model ii. triplet bptc and thymine can exist in either neutral or deprotonated forms, depending on the ph of the solution (see table 1). in the presence of thymine, the lifetime of triplet bptc is decreased, and the long - lived bptc ketyl radical anion (bptc) appears, max = 630 nm (see below). the formation of the ketyl radical anion implies that the photochemical primary step is an electron transfer. in the range of ph = 2.010.0, we found that the transient kinetics for this system correspond to model ii. data analysis for this system by model ii in the solution of ph = 6.4 is presented in figure 5. it can be seen that the total radical absorbance is high (aradical/atriplet0 = 0.29) with [thy ] = 6 10 mol l (figure 5) as a result of the high radical quantum yield of bptc at ph = 6.4. plotting k1 vs [thy ] at ph = 6.4 (figure 5, inset) gives a good linearity and zero intercept, showing that the model accurately describes the kinetic behavior. the spectrum of bptc over the range of 610660 nm was obtained by laser flash irradiation of the solution of bptc (1.0 10 mol l) and thymine (1.5 10 mol l) (figure 6). bptc absorbs at max = 630 nm, and the decay monitored at obs = 630 nm obeys a second - order kinetics (k2/bptc = 3 10 cm s). in addition, inbar. reported that the pinacol yield of the 4-bc + 4-bc reaction is minor. these lead to the postulation that after an initial electron transfer followed by proton transfer, ketyl radicals (bptch) are formed, which dissociate into free radical ions and thereafter randomly encounter to undergo the pinacol reaction (bptch + bptch). decays (obs = 590 nm) of triplet bptc (1 10 mol l) + thy (concentration increases from top to bottom : 01.5 mm) in water at ph = 6.4. bptc ketyl radical anion spectrum in the presence of thymine (1.5 10 mol l) at ph = 2.0. inset : decay profile (obs = 630 nm) of bptc, which forms pinacol after the fast protonation of bptc to bptch. on the other hand, the formation of bptc could not be observed at ph = 12.0, even at high concentrations of thymine ([thy ] = 1.5 10 mol l). enol tautomerism of thy (scheme 2), which significantly changes the chemical properties of thy. in addition, it is possible that the proton transfer from the oxidized thymine is strongly hindered at ph = 12.0 and the electron reverts back to the thymine molecule instead of remaining on the bptc. therefore, no quenching reaction of triplet bptc by thy occurs (i.e., in eq 8, kq4 = 0). the ph - dependence of the observed quenching rate constants is presented in figure 7. although the quenching reaction follows a complex reaction system that shows multiple (de)protonation equilibria in the ground and excited states, the ph - dependent kqobs is adequately established. figure 7 depicts a continuous downhill and flat behavior of kqobs. the decrease in kqobs with ph is characterized by two changes (at ph 2.1 and 4.7) which coincide with the two - deprotonation stages of triplet bptc. moreover, kqobs reaches plateaus in the regions, that are attributed to the equilibria of the corresponding reactant pairs. note that in the range of ph 10 is caused by the shift of the dthyh2 equilibrium toward its anionic form. an effect of coulombic repulsion may hinder the encounter of bptc and result in dthyh ; hence, the quenching rate constants get smaller. the reactive species existing in solution at different ph 's are shown in table 1. for the whole range of ph = 2.012.0, we found that the decay of triplet bptc in the presence of ade follows model i with the formation of bptc. the appearance of transient bptc leads to the conclusion that an electron transfer reaction from ade to triplet bptc is followed by fast diffusion of the reactants. the ph - dependence of kqobs is described by eq 13 and is presented in figure 9 (see tables 1 and 2 for parameters).13it is seen that kq1 = 0, which implies no quenching reaction of triplet bptc by adeh2 under strong acidic conditions (ph 5, an explanation similar to that of the quenching reaction by thymidine for the change of kqobs with ph is used. ph - dependence of the observed quenching rate constant for the reaction of triplet bptc with dthy. ph - dependence of the observed quenching rate constant for the reaction of triplet bptc with dade. the decay profile and kinetic analysis for the quenching of triplet bptc by dade at ph = 4.0 is shown in figure 10. it can be seen that the total radical absorbance increases slightly with the concentration of dade. kinetic treatment by model i within the range of ph = 2.012.0 is applied. together with the observation of bptc at obs = 630 nm, it leads to the conclusion that electron transfer from dade to triplet bptc occurs, followed by a fast diffusion step. the ph - dependence of kqobs is expressed by eq 13 (see tables 1 and 2 for parameters). values obtained from the best fit (solid line, figure 11) are summarized in table 1. once again, an explanation similar to that of the quenching reaction by adenine for the variation of kqobs with ph is used. decay profiles (obs = 590 nm) for the bptc + dade system in water at ph = 4.0. one question remains that is related to the quenching reactions of triplet bptc by thymine at ph = 2.010 : is electron transfer followed by proton transfer ? to check the possibility, we have investigated the ph - dependence of the oxidation potential of thymine by cyclic voltammetry. whatever the reaction mechanism may be, a global nernst equation can be applied for the thermodynamics of the redox reaction:15here, e is the electrode potential ; eap0,ox / red is the apparent standard oxidation potential of the corresponding redox couple ; and [ox ] and [red ] are the total concentrations of the oxidized and reduced species in solution, respectively. therefore, the ph - dependence of eap0 for the oxidation of thymine can be described by eq 16 (see the supporting information), as shown in figure 12.16here, pka, thyh = 3.2 is the dissociation constant of thymine radical cation. the apparent oxidation potential vs ag / agcl of thymine (1 mm) as a function of ph. solid line : linear fitting with a slope of 55.2 mv / ph. between ph = 28, eap0 shows a linear variation with ph, together with the characteristic slope of approximately 59 mv per ph unit. this indicates a one - proton - coupled one - electron transfer during the oxidation of thymine. this observation and the above kinetic analysis of the quenching reaction by thymine suggest that the possible pathway of the quenching reaction of triplet bptc by thymine at ph around 28 is initially an electron transfer, followed by proton transfer (scheme 3). at ph around 810, the quenching of the triplet state of 3,3,4,4-benzophenone tetracarboxylic acid by dna bases in aqueous solution was investigated using time - resolved laser flash photolysis. the observation of the bptc ketyl radical anion (max = 630 nm) confirms that the primary photochemical step is electron transfer. although the forms of the triplet bptc and quenchers depend on the ph of the solution, we have been able to establish a relationship of the quenching rate constant of each pair of reactants with the overall quenching rate constant. particularly, the ph - dependence of the apparent standard potential of thymine in aqueous solution was investigated by cyclic voltammetry. from the kinetic analysis of the quenching reactions, the ph - dependent oxidation potential of thymine indicates that proton - coupled electron transfer by means of a stepwise mechanism from thymine to triplet bptc in the solutions of ph 28 proceeds. in the strong basic solution (ph = 12.0), no quenching reaction between triplet bptc and thymine takes place, whereas it is observed for thymidine. the abbreviations used in this article can be found in the section results and discussion. y. lin, chemistry department, chinese academy of science, beijing and was used without further purification. dna bases adenine (99%), adenosine (99%), thymine (97%), and thymidine (99%), purchased from alfa - aesar, were also used as received. all experiments were carried out at room temperature in buffered aqueous solutions. the buffer solutions (0.01 mol l) employed, covering the ph range from 3.0 to 11.0 with (a) hcl kh2po4, ph = 3.05.0 ; (b) kh2po4na2hpo4, ph = 5.09.0 ; (c) na2hpo4naoh, ph = 9.011.0, ph = 2.0 and ph = 12.0, were adjusted with hcl and naoh, respectively. transient absorption spectra were obtained by time - resolved laser spectroscopy using a lambda physik lpx-120 xecl excimer laser (308 nm, pulse energy up to 100 mj, pulse width 10 ns). the monitoring system included a 150 w xenon lamp ; a hamamatsu r928 photomultiplier tube ; a obb / pti monochromator, model 101/102 ; and a digital storage oscilloscope, 9410a lecroy. the irradiation was carried out in a 1 1 cm rectangular quartz cell. all solutions were treated identically and deoxygenated by bubbling with high - purity argon for 15 min. cyclic voltammetric measurements were performed with an autolab - pges aut 73227 potentiostat (metrohm). a three - electrode cell configuration was used : a pt counter electrode, a ag / agcl reference electrode, and a glassy carbon working electrode (0.03 cm area). the working electrode was polished with diamond paste in water after each single scan to remove possible follow - up products of the oxidative process on the electrode surface. the background current was always subtracted from the current response for further calculation. for the triplet state. | the kinetics of triplet state quenching of 3,3,4,4-benzophenone tetracarboxylic acid (bptc) by dna bases adenine, adenosine, thymine, and thymidine has been investigated in aqueous solution using time - resolved laser flash photolysis. the observation of the bptc ketyl radical anion at max = 630 nm indicates that one electron transfer is involved in the quenching reactions. the ph - dependence of the quenching rate constants is measured in detail. as a result, the chemical reactivity of the reactants is assigned. the bimolecular rate constants of the quenching reactions between triplet bptc and adenine, adenosine, thymine, and thymidine are kq = 2.3 109 (4.7 < ph < 9.9), kq = 4.0 109 (3.5 < ph < 4.7), kq = 1.0 109 (4.7 < ph < 9.9), and kq = 4.0 108 m1 s1 (4.7 < ph < 9.8), respectively. moreover, it reveals that in strong basic medium (ph = 12.0) a keto enol tautomerism of thymine inhibits its reaction with triplet bptc. such a behavior is not possible for thymidine because of its deoxyribose group. in addition, the ph - dependence of the apparent electrochemical standard potential of thymine in aqueous solution was investigated by cyclic voltammetry. the e/ph 59 mv / ph result is characteristic of proton - coupled electron transfer. this behavior, together with the kinetic analysis, leads to the conclusion that the quenching reactions between triplet bptc and thymine involve one proton - coupled electron transfer. |
when angiotensin - converting enzyme-2 (ace2) was serendipitously discovered ten years ago, neither of the two groups at the centre of its discovery [1, 2 ] could have guessed at the disproportionate number of distinct roles it plays in biology, from cardiovascular regulation to viral infection. as so often happens in modern biological research two independent approaches converged on the same discovery, to give us ace2 or angiotensin - converting enzyme homologue (aceh), back in 2000. over the past ten years our knowledge of this protein 's role in the body has increased exponentially, resulting in recombinant ace2 protein entering clinical trials back in 2009. this paper will focus on what we currently know about ace2 and its regulation, highlighting some of the gaps and discrepancies that still remain in our knowledge. ace inhibitors have been the first line of treatment against hypertension for decades, and their success has served to place ace and its biologically active product, angiotensin ii (ang ii), as central regulators of the renin - angiotensin system (ras). ang ii is produced by ace through hydrolysis of its precursor ang i. ang ii is the major vasoactive peptide in the ras, acting as a potent vasoconstrictor through its receptor at1r (figure 1). hence, inhibition of the production of ang ii and more recently its receptor - induced signalling, through the use of at1r blockers, have been highly successful treatments in hypertension. consequently there was immediate commercial interest in ace2, as another likely therapeutic target, when it was discovered as an active homologue of ace. however, as the initial publications observed to their surprise, despite high similarity to ace (figure 2), ace2 did not convert ang i to ang ii nor was it inhibited by ace inhibitors [1, 2 ]. a major difference in substrate specificity was immediately noticed, namely, that ace2 acted as a carboxypeptidase removing a single amino acid from the c - terminus of susceptible substrates whereas ace acts as a carboxy - dipeptidase (more correctly, peptidyl - dipeptidase), removing a c - terminal dipeptide. ace2 does hydrolyse the decapeptide ang i, albeit relatively poorly, but converts it to ang-(1 - 9) rather than ang ii (ang-(1 - 8)). it was initially hypothesised that ace2 counterbalanced the actions of ace as ang-(1 - 9) is also metabolised by ace and therefore competes with ang i for its active site, thus providing a novel regulatory arm to the ras (figure 1). studies revealed that ace2 hydrolyses a number of substrates and preferentially cleaves terminal amino acids from peptides ending in pro - x, where x is a hydrophobic amino acid. the hydrolysis of some ace2 substrates is chloride - dependent, as is the case for ace, and the structural basis for this selectivity has been proposed. of the biologically active peptides that ace2 cleaves, the most relevant are apelin-13 and ang ii. in order to further understand the biological relevance of ace2 an inhibitor was developed based on the c - terminal dipeptide (his - leu) of ang i. this allowed development of the potent and specific inhibitor, mln-4760, which has been used in numerous studies of ace2 action in vivo and in vitro, although the compound is not currently commercially available. the elucidation of the structure of ace2, and subsequent comparative modelling studies, explained its distinct specificity by revealing subtle differences in the active sites of ace and ace2 [710 ]. a single amino acid substitution in ace2 sterically hinders the entrance of the penultimate substrate amino acid into the active site, thereby eliminating the ace - like peptidyl - dipeptidase activity. the substrate specificity of ace2 was clarified when it was shown that ace2 had a much higher catalytic efficiency for hydrolysis of ang ii (400-fold) compared with ang i. only under conditions of elevated ang i concentrations (such as in patients on ace inhibitor therapy) is the conversion of ang i to ang-(1 - 9) by ace2 (figure 1.) the revelation that the main product of the catalytic activity of ace2 was ang-(1 - 7) (figure 1), a vasodilatory peptide, led to a complete reevaluation of its therapeutic potential. currently strategies are aimed at upregulation of ace2 expression and activity, technically more complex than enzyme inhibition. this does not rule out any potential application of ace2 inhibitors which have recently been proposed as possible anti - inflammatories, having initially but unsuccessfully been tested as potential antiobesity drugs. the main tissue sites of expression of ace2 were originally identified as testis, heart, and kidney, where it was shown to be localised on the apical membrane of polarised cells whereas ace is equally distributed between apical and basolateral membranes. the molecular basis for this differential localization has not been addressed but presumably relates to determinants in the c - terminal cytoplasmic tails of the two enzymes which are quite distinct in sequence. the tissue distribution of ace2 has now been catalogued more widely, for example, in liver, intestine, and lung [14, 15 ]. more recently ace2 has been localized in the brain, where it appears to act as a central regulator of cardiovascular function [1720 ]. ace2 is a type 1 transmembrane protein (n - terminus outside, c - terminus intracellular), predominantly localised on endothelial cells where its catalytic site, like that of ace, is exposed (so - called ectoenzyme) to circulating vasoactive peptides. the activity of ace2 can therefore be modulated via its expression on the cell surface, through its expression levels and also through its cleavage from the cell membrane. this cleavage or shedding releases the catalytically active ectodomain and when stimulated, for example, by phorbol esters, is mediated by a disintegrin and metalloprotease (adam 17). ace also undergoes constitutive and regulated shedding from the cell surface into plasma although the enzymes responsible in this case have not been identified. the success of ace inhibitors has shown that ang ii is a key mediator of hypertension, and, hence, by metabolising ang ii into ang-(1 - 7), ace2 is crucial in the modulation of blood pressure. the role of ace2 in hypertension has been clarified by its overexpression in vivo, reducing blood pressure in hypertensive models [2224 ] but not in normotensive animals. this reduction in blood pressure may be the result of increased sensitivity of the baroreflex, which has been seen upon ace2 delivery in hypertensive models, and a reduction in neuronally induced hypertension has been observed in transgenic mice. central blood pressure regulation is controlled in part by the actions of ang ii on the at1r. ang ii acts through the at1r to desensitise the baroreflex, stimulate water uptake, and increase vasopressin release and sympathetic activation, ultimately leading to increased blood pressure. the actions of ang ii are in part modulated by the increase in baroreflex sensitivity mediated by ang-(1 - 7) [28, 29 ]. comparison of hypertensive models to normotensive rodents has revealed decreased ace2 protein expression by up to 40%, in the brain, of the hypertensive models [20, 22 ]. moreover, overexpression of ace2 in the brain attenuates hypertension, via an increase in nitric oxide production and improved baroreflex. accordingly, injection of the ace2 inhibitor mln-4760 into the brain of rodents attenuates the baroreflex. site - specific overexpression of ace2 at a locus controlling sympathetic nerve activity reduces the overall hypertensive state of rats. for a review of the roles of ace2 in central blood pressure regulation, see. soon after its discovery, gene deletion studies established ace2 not only as a modulator of blood pressure but also as an essential regulator of cardiovascular function. the progressive cardiac dysfunction observed in the first ace2 mouse knockout model resembled that of tissue subjected to long - term hypoxia of the type that occurs after coronary artery disease or bypass surgery in humans. this hypothesis was strengthened by the observation that ace2-null phenotypes were reversed by concurrent knockout of the ace gene. this evidence appeared to demonstrate unequivocally that the primary role of ace2 was to counterbalance that of ace. the initial hypothesis that ace2 plays a critical role in cardiac function primarily by counterbalancing the effects of ace was not, however, entirely supported by subsequent gene deletion models. the discrepancies between the initial study and the phenotypes are described elsewhere, which saw no obvious functional or morphological changes [33, 34 ], were initially proposed to be due to differing genetic backgrounds in their models. this potential mechanism was investigated by backcrossing the hybrid model used in both studies with an initial parental line ; however, both backcrossed models showed no cardiac changes. interestingly subsequent studies using the original ace2-deficient mice described by crackower. also showed no overt cardiac changes, suggesting that the phenotype is lost over time [35, 36 ]. despite seeing no overt phenotypic change in deletion models, together these studies suggest that, rather than being a key mediator of cardiac phenotype, ace2 is essential in modulating responses to injury [33, 37 ]. in fact ace2 deletion models have a significantly higher mortality rate after myocardial infarction (mi) than wild - type mice, associated with adverse ventricular remodelling and worsening ventricular function following mi. an increase in matrix metalloproteinase2 (mmp2) and mmp9 activation, free radical production, and upregulation of proinflammatory cytokines, in the hearts of ace2-knockout mice, these events, and the adverse remodelling they cause, were reversed upon administration of an at1r blocker, and therefore the pathology of ace2 deletion in states of injury can be attributed, for the most part, to increases in the local levels of ang ii. the ability of ace2 to improve responses to injury is not only the result of clearing ang ii, thereby limiting its pathological potential, but also by producing ang-(1 - 7). the conversion of ang ii to ang-(1 - 7) by ace2 is not the only physiological route to ang-(1 - 7) production. for example, the zinc metallopeptidase neprilysin (nep) can convert ang i to ang-(1 - 7) efficiently, and both ace and nep can convert ang-(1 - 9) to ang-(1 - 7). the relative importance of these various enzymes to ang-(1 - 7) production will vary dependent on their relative expression levels in different tissues (e.g., kidney versus heart versus brain) and on physiological status. like ang ii the actions of ang-(1 - 7) extend beyond vasopressor control. infusion of ang-(1 - 7) reduces interstitial fibrosis in ang ii - independent and ang ii - dependent hypertension. interestingly in both studies there was no effect on the blood pressure of hypertensive animals when infused with chronic levels of ang-(1 - 7). there was, however, a discrepancy in the effects of ang-(1 - 7) on cardiac hypertrophy between the two studies. ang-(1 - 7) had no effect on the salt - induced hypertrophy in ang ii - independent hypertension but it significantly reduced myocyte hypertrophy in ang ii - induced hypertension. cardiac - specific overexpression of ang-(1 - 7) was observed to reduce the hypertrophic response to ang ii concurrently with a reduction in hypertrophic markers, atrial natriuretic peptide and brain natriuretic peptide, transcript levels and activation of hypertrophic signalling pathways, c - src and p38 mapk. ang-(1 - 7) inhibits myocyte cell growth in vitro through the actions of the mas receptor and accordingly prevents ventricular hypertrophy in vivo, when stimulated by myocardial infarction (mi). the reduction in myocyte diameter and ventricular weight of mice virally expressing ang-(1 - 7) was associated with a decrease in proinflammatory cytokines (tnf and il-6) compared to control. it is worth noting that ang-(1 - 7) overexpression slightly reduced exogenous ace mrna levels and ablated the approximate twofold increase in expression resulting from mi, whilst increasing ace2 expression levels in response to mi. ace2 levels have consistently been shown to alter in cardiovascular disease states. in light of the counterbalancing hypothesis it could be presumed that, since ace is consistently reported to increase in damaged cardiac tissue [4547 ], ace2 levels would also increase as a homeostatic response to offset the rise in ang ii concentration. this hypothetical upregulation is supported by evidence from human nonischaemic cardiomyopathy, which has consistently shown increased ace2 levels in the failing human heart compared to control patients [4850 ]. however, in contrast, in ischaemic cardiomyopathy, there is currently conflicting evidence for the changes in expression levels of ace2 [48, 49 ]. where ace2 upregulation has been seen in these studies, the mechanism of this damage - induced increase has been investigated using in vivo models of mi. ace2 upregulation has been repeatedly shown in rat models of mi [38, 51, 52 ]. discrepancies between the mrna and protein levels seen in the infarct zone have suggested that the increase in ace2 protein is mediated by a posttranscriptional mechanism. however, time - course investigations reveal that the increases seen at eight weeks after mi were followed by a decrease in ace2 expression in mi models compared to control after 28 weeks. although not entirely consistent these results on balance seem to indicate a compensatory role for ace2 in conditions of myocardial injury. given its role in removing ang ii, ace2 was identified as a candidate gene underlying the loci linked to hypertension, following its initial mapping to the x chromosome. comparison of ace2 expression levels in the kidneys of three rat strains showed that ace2 expression was lower in the hypertensive - prone strains and moreover that ace2 expression decreased significantly when hypertension was initiated in salt - sensitive hypertensive rats. decreased endogenous ace2 expression has been noted in spontaneously hypertensive rats compared to wistar - kyoto. the initial study did not see any genetic changes associated with the ace2 gene in these hypertensive strains, supporting subsequent data, which have, up until now, failed to show any link between ace2 polymorphisms and hypertension. ace2 is abundantly expressed in the kidneys, where its expression is inversely correlated with hypertension [55, 56 ]. the local ras within the kidneys is activated by hyperglycemic conditions, which model the environment in type 2 diabetes. studies using models of type 2 diabetes have shown at early stages, prior to diabetic nephropathy developing, that ace2 expression is reduced in the kidney, while ace expression is elevated. similarly in models of type 1 diabetes ace2 expression is elevated in early and decreased in late stage of diabetic nephropathy. additionally, studies on human samples have shown de novo expression of ace2 in the glomerular endothelium and mesangial cells of diabetic patients ; however, this expression was not seen in type 2 diabetic renal biopsies. one study carried out by taking biopsies of twenty type 2 diabetic patients and twenty healthy donors showed decreased ace2 and increased ace in tubulointerstitium and glomeruli in the diabetic patients with nephropathy indicating a pathologically important balance between the two enzymes. the hypothesis that kidney disease and the pathogenesis of diabetes are mediated by an upregulation of ace and a downregulation of ace2 was originally suggested by mizuiri.. as in the heart, loss of ace2 in the kidneys is again associated with increased susceptibility to injury. ace2-knockout mice have been shown to have enhanced susceptibility to glomerulosclerosis, coupled with increased collagen and fibronectin deposition. filtration dysfunction, evidenced by urinary albumin, was pronounced in the male mice whereas the female mice appeared to be protected. pharmacological inhibition of ace2, by mln-4760, has been shown to have similar effects, increasing urinary albumin and mesangial cell expansion and vascular thickness, in both type 1 and type 2 diabetic models [58, 64 ]. all these studies attributed the pathology seen when ace2 is lost to increases in levels of ang ii [58, 63 ]. in order to further confirm the renoprotective role of ace2, akita mice (a type 1 model of diabetes) were crossed with ace2-knockout mice and kidney function observed. this model showed an increase in urinary albumin, glomerular basement membrane thickness, fibronectin, and smooth muscle -actin compared to diabetic mice expressing ace2. surprisingly they did not see any change in ang ii in ace2-knockouts, or in the diabetic model ; despite this, they did show that use of an ang ii receptor blocker was able to attenuate some of the markers of glomerular injury and urinary albumin seen in the ace2 knockout diabetic mice. conversely, ace2 deletion disrupted the benefits of ace inhibition on diabetic nephropathy in streptozotocin - induced diabetes suggesting that ace inhibition may enhance ace2 activity. interestingly, in the same diabetic model, ang-(1 - 7) infusion resulted in pronounced renal injury. this may not be as contradictory as it first appears as they also saw a downregulation in the mas receptor, the proposed receptor for ang-(1 - 7). these current findings suggest that ace2 may participate in a compensatory mechanism in the diabetic kidney prior to the onset of diabetic nephropathy. more direct involvement of ace2 in diabetes, through its pancreatic expression, has been investigated. ace2 expression is elevated in the islets of type 2 diabetic rats, which correlates with an increase in ace, collagen iv, and tgf-1 levels. ace2-null mice have significantly increased fasting blood glucose compared to their wild - type littermates. no direct role for ace2 in the pancreas has yet been identified ; in contrast its homologue collectrin is heavily implicated in insulin exocytosis. when discovered, collectrin excited interest due to its high homology to the cytoplasmic tail of ace2. sirna knockdown of collectrin results in a reduction of insulin exocytosis in insulin - secreting ins-1 cells. in vivo, collectrin was implicated in the insulin secretory pathway through an association between collectrin and snapin, part of the snare complex [7072 ]. however, collectrin - knockout mice revealed no difference in insulin secretion from wild - type, only a decrease in insulin sensitivity. ace2 is not only homologous to ace but is a chimaera of ace, with which it has close homology in the catalytic domains of the n - terminus, and of collectrin, which closely resembles the transmembrane and intracellular c - terminal domains of ace2 (figure 2). collectrin was first identified as an unknown protein upregulated in a model of partial nephrectomy, its function remaining elusive for four years until crystals of tyrosine and phenylalanine were detected in the urine of collectrin - null mice. further investigation revealed that the levels of the neutral amino acid transporter, bat1, which reached the plasma membrane were significantly decreased in collectrin - null mice. this suggested that collectrin may act as a molecular chaperone for bat1 in the kidney, implicating ace2 in a similar role, because of their close homology. an elegant set of studies subsequently revealed that ace2 did in fact act as the molecular chaperone for bat1 in the small intestine, where collectrin is not expressed. this interaction was shown to underlie the pathology of the aminoaciduria seen in hartnup disorder. hartnup disorder is caused by a mutation on the outer edge of bat1 resulting in its failure to reach the plasma membrane. it was revealed that this mutation disrupts the ace2/bat1 complex and therefore prevents ace2 from acting as a molecular chaperone delivering the transporter to the intestinal brush border membrane. outside the cardiovascular system another noncatalytic function of ace2 had previously been shown. in 2003 a new disease termed severe acute respiratory syndrome (sars) caused by a novel coronavirus (sars - cov) spread quickly around the world, causing more than 800 deaths. ace2 was identified as the receptor for sars virus in vitro [77, 78 ] and also acts as receptor for the nl63 virus. soon after, studies confirmed that ace2 was essential for sars infection in vivo using ace2-knockout mice. concurrently it was discovered that ace2 protects murine lungs from severe acute injury and subsequently that sars - cov infections and the sars spike protein itself downregulate ace2 expression (figure 3). as mentioned above there is circumstantial but not entirely consistent evidence in the literature that ace and ace2 are coregulated. in human hypertensive patients, ace2 levels are lower in both kidney and heart compared to normotensive volunteers. a growing body of in vitro evidence suggests that this decrease is mediated at least in part by ang ii [8284 ]. the proposed mechanism for this involves at1r signalling via erk / p38 map and/or by elevated erk1/2 and jnk phosphorylation. furthermore, administration of an at1r blocker has been shown to result in an increase in ace2 levels [84, 86 ]. as such there is linked regulation of both ace2 and ace, as the catalytic product of ace, ang ii, regulates ace2. however, the role ang ii plays in regulating ace2 is not yet fully elucidated ; despite decreasing ace2 expression in response to ang ii in most models there is evidence of ang ii - mediated increases in ace2 in hepatic stellate cells. although angiotensin peptides, as well as other peptide and steroid hormones, appear to modulate its expression, few studies have been done on other factors that may control its regulation, such as hormones and oxygen levels. for example, although hypoxia decreases the transcription of ace2, further investigation has revealed that hypoxia - induced hif-1 increases ace expression which, in turn, leads to an increased concentration of ang ii. ang-(1 - 7) has also been shown to affect ace2 expression : cardiac and renal ace2 were decreased in both hypertensive and normotensive rat models in response to ang-(1 - 7) infusion although no effects on blood pressure were demonstrated and no mechanism of action was proposed. administration of aldosterone or endothelin-1 to rat myocytes has also been shown to downregulate ace2 mrna levels. micromolar concentrations of aldosterone were shown to decrease ace2 mrna expression significantly in the myocytes of hormone - infused rats although, in contrast to other models, no change in ace2 mrna levels were seen when these rats were infused with ang ii. when treated with endothelin-1, myocytes isolated from neonatal rats decrease ace2 expression via erk1/erk2 signalling, a decrease that was blocked by cotreatment with ang-(1 - 7). the effect of oestrogen on ace2 expression has recently also been explored in light of clinical evidence which has established that hormone replacement therapy is protective against cardiovascular disease. treating rats with oestrogen previous in vitro studies had shown that oestradiol (e2) treatment was protective against ang ii - induced fibroblast proliferation. the beneficial effects of oestrogen were coupled with a dose - dependent increase in ace2 but no significant change in blood pressure was seen and no protective mechanism proposed. a high - fat diet has been shown to increase ace2 mrna levels in mouse adipocytes both in vivo and in vitro although these changes were not evident in the adipose tissues of mice with heart failure. tissue culture models of adipose differentiation have shown that the increase in ace2 mrna over time was accompanied by an increase in adam 17 and no increase in ace2 activity. thus the activity levels of ace2 remain constant via coregulation with adam 17, which cleaves ace2 at the cell membrane. an increase in ace2 expression has been reported in adipose tissue when rats were fed a high - sucrose diet, although in preliminary form only. the effects of all - trans - retinoic acid have been investigated on ace2 expression revealing an increase in ace2 mrna levels and reportedly in protein. a decrease in blood pressure in the treated rats was also seen, which was attributed to the increased ace2 levels. ace2 expression is not only subject to posttranslational modifications, such as glycosylation and phosphorylation, but also subject to posttranslational regulation, when released from the cell membrane by shedding through the action of adam 17 as described above [21, 96, 97 ]. short peptide mimics around the likely cleavage site region are hydrolysed by recombinant adam 17 at an arg - ser bond (corresponding to arg and ser in ace2), in a sequence - dependent manner [93, 94 ]. however, mutation of these critical cleavage residues in a cell - based system failed to inhibit shedding suggesting that the specificity of adam 17 is topographically determined, rather than sequence dependent [97, 98 ]. the function (if any) of the catalytically active soluble, shed form is unknown, although for some other proteins, for example, the amyloid precursor protein and acetylcholinesterase, the released protein acts as a ligand for stimulating cell - cell interactions. the catalytic activity of any shed ace2 may be masked by the presence of an endogenous inhibitor of ace2 in the plasma, which currently remains uncharacterised. binding of the sars spike protein induced adam-17-dependent shedding of ace2 n - terminal domain (figure 3). this shedding has been reported by different groups to be both essential for viral replication and unnecessary. the sars virus undergoes clathrin - dependent endocytosis upon receptor binding ; this process internalises both the sars virus and its receptor further clearing ace2 from the cell membrane (figure 3) and hence allowing enhanced (and damaging) reactivity towards circulating ang ii. in contrast to ectodomain shedding, the cytoplasmic domain of ace2 appears to play no role in the regulation of internalisation. currently the most pertinent of all questions about ace2 is whether it is going to be a useful therapeutic target, and so far all data suggest that increased expression would be beneficial in a number of diseases. until now an increase in the level of ace2 has been achieved by viral delivery, application of allosteric activators of ace2 catalysis, or administration of human recombinant ace2. aside from its effects on hypertension [2225 ], viral overexpression of ace2 has shown reduced collagen production in cultured fibroblasts as well as inhibition of ang - ii - induced fibrosis and hypertrophy in vivo, stabilisation of atherosclerotic plaques, and renoprotection. viral delivery of ace2 after induction of myocardial infarction is protective, reducing the adverse cardiac remodelling and fibrosis [111, 112 ]. similar antifibrotic effects have been seen with an ace2 activator [113, 114 ] along with attenuation of ang - ii - induced thrombus in hypertensive rats and a modest reduction in the blood pressure of spontaneously hypertensive rats. over the past 18 months a number of studies have been carried out examining the effect of recombinant human ace2 on a range of disease conditions. to date, administration of ace2 to mouse models of ang - ii - induced diseases has been shown to reverse the pathological effects of ang ii in diabetic nephropathy, heart disease, renal oxidative stress as well as reversal of ang - ii - induced hypertension. interestingly, infusion of ace2 does not appear to have any effect on nondisease states or on the basal level of ang ii in wild - type mice or ace2-knockout mice, making it a potentially valuable therapeutic. more fundamental questions about the cellular biology of ace2 remain. as is evident from this paper, questions need answering about underlying mechanisms of ace2 regulation, which could help clarify our understanding of the ras as a whole, for example, are there antagonistic transcription factors regulating ace and ace2 ? are components of the ras co - ordinately regulated and by which signalling pathways ? ace is known to signal through phosphorylation of its cytoplasmic tail modulating its own retention on the cell membrane [119, 120 ] and also to mediate transmembrane signalling, increasing its own transcription in response to ace inhibitors [121, 122 ]. exogenous ace has been shown to have transcriptional effects independent of its catalytic activity when vsmc and endothelial cells are treated with ace [123, 124 ]. does the cytoplasmic tail of ace2, despite sharing no homology with ace, have similar signalling properties ? the fate of these ectodomains is unknown and it may be a mechanism of rapid clearance, or perhaps these ectodomains are endocytosed and trafficked to the nucleus, where they elicit transcriptional changes, as has been shown with exogenous ace. for that matter the destiny of the retained cytoplasmic sections of these proteins is not known. the intracellular domains of other shed proteins, such as app and notch, are released by -secretase cleavage, after initial adam ectodomain cleavage, a process referred to as intramembrane proteolysis. the intracellular domains of both app and notch travel to the nucleus, stabilised by chaperones, where they elicit transcriptional changes. novel roles for ace2 may yet remain to be discovered and a new twist to the ace2 story has emerged with the discovery of autoantibodies targeting ace2 in the sera of patients with connective tissue diseases. this, coupled with results showing that administration of an ace2 inhibitor improved the pathology of inflammatory bowel disease, suggests that inhibition of ace2 may be beneficial in inflammatory diseases such as arthritis. the diversity of the roles of ace2 continues to surprise those in the field. given the apparent success of recombinant human ace2 in animal models, ace2 could be a valuable therapeutic. however, here it is worth noting that so far the only disease model recombinant ace2 has shown success in is type 1 diabetes. all other studies have, as yet, only shown that ace2 reverses the effects of exogenous ang ii infusion. until work is done on hypertensive - prone models, as with viral delivery and ace2 activator - mediated induction, it is hard to judge the clinical relevance of this treatment. although no immune response to recombinant ace2 or the viral delivery systems has been reported upon infusion into rodents, this is always a concern with such strategies. given these caveats perhaps a priority focus for future research should be upregulation of endogenous ace2 gene expression or catalytic activity. as highlighted in this paper, more work is required to determine how the diverse roles of ace2 interlink in order to allow chronic modulation of ace2 levels to proceed with confidence. | the renin - angiotensin system (ras) is a critical regulator of hypertension, primarily through the actions of the vasoactive peptide ang ii, which is generated by the action of angiotensin - converting enzyme (ace) mediating an increase in blood pressure. the discovery of ace2, which primarily metabolises ang ii into the vasodilatory ang-(1 - 7), has added a new dimension to the traditional ras. as a result there has been huge interest in ace2 over the past decade as a potential therapeutic for lowering blood pressure, especially elevation resulting from excess ang ii. studies focusing on ace2 have helped to reveal other actions of ang-(1 - 7), outside vasodilation, such as antifibrotic and antiproliferative effects. moreover, investigations focusing on ace2 have revealed a variety of roles not just catalytic but also as a viral receptor and amino acid transporter. this paper focuses on what is known about ace2 and its biological roles, paying particular attention to the regulation of ace2 expression. in light of the entrance of human recombinant ace2 into clinical trials, we discuss the potential use of ace2 as a therapeutic and highlight some pertinent questions that still remain unanswered about ace2. |
with attention to the role of diet or medical nutritional therapy on chronic disease, the dietary pattern approach, which involves diet quality indices, has been suggested as a powerful tool because of the complexity of diets [1 - 3 ]. in recent decades, several indices have been developed and validated, including the diet quality index (dqi), healthy eating index (hei), mediterranean diet score, healthy diet indicator (hdi ; based on the world health organization dietary guidelines), alternate hei (ahei ; including american dietary guidelines), and dqi - international (dqi - i ; developed for worldwide national dietary guidelines). the diet quality indices may be used as a quick assessment to evaluate compliance to healthy dietary guidelines and dietary changes after diet education. there have been several studies that have shown an association between diet quality indices and chronic diseases, especially cancer and cardiovascular diseases. some research has also demonstrated an association of diet quality indices with obesity and diabetes [12 - 15 ]. however, there have been few studies assessing the relationship between diet quality indices and glycemic control, and on which indices might serve as an appropriate tool to assess diet quality in patients with type 2 diabetes following diet education. in this study, we tried to determine the relationship between the diet quality indices including hdi, ahei, and dqi - i, and glycemic indices in korean patients with type 2 diabetes, independent of adiposity and energy intake. then, we also tried to determine which indices were appropriate for assessment of diet quality after dietary education in patients with type 2 diabetes. subjects were 110 consecutive korean outpatients with type 2 diabetes who visited 2 university hospitals from april 2004 to november 2006 and who were enrolled in a study to evaluate the effectiveness of the medical nutrition therapy (mnt). briefly, subjects were patients with newly diagnosed type 2 diabetes or patients who had been diagnosed with type 2 diabetes for fewer than 5 years but did not have acute and/or chronic complications. at the time of enrollment, height was measured to the nearest centimeter while the subjects were barefoot, and body weight was measured to the nearest 0.1 kg using a digital scale while the subjects wore light clothing. then, body mass index (bmi) was calculated by determining the ratio between weight and height squared (kg / m). the waist circumference was taken at the narrowest point between the lower costal margin and the superior iliac crest, and the hip circumference was measured at the horizontal level between the symphysis pubis and the greater gluteal protuberance that yielded the maximum measurement. blood pressure was recorded using a mercury sphygmomanometer twice in a sitting position after subjects had been in a relaxed state for at least 10 minutes. hypertension was defined as 2 consecutive systolic / diastolic blood pressure measurements > 140/90 mmhg or current treatment with antihypertensive medication. fasting plasma glucose (fpg), 2-hour postprandial glucose (pp2) concentration, and lipid profile levels were analyzed enzymatically, using an autoanalyzer (hitachi 760 - 210, hitachi ltd., (hba1c) was determined using high - performance liquid chromatography (hlc-723 g8, tosoh, tokyo, japan). the dqi - i focused on 4 major aspects : variety (0 - 20 points), adequacy (0 - 40 points), moderation (0 - 30 points), and overall balance (0 - 10 points). the scores for all 4 categories were totaled, from 0 to 100 (0 being the worst and 100 the best possible scores). for the variety and adequacy categories, we used the us exchange list serving size definition for cross - national comparative work. the adequacy of intakes for iron, calcium, and vitamin c were examined on the basis of the percentage attainment of the dietary reference intakes in korea. the hdi was calculated as the sum of dichotomous variables on the basis of 9 items : saturated fatty acids ; polyunsaturated fatty acids ; protein ; complex carbohydrates ; dietary fiber ; fruits and vegetables ; legumes, nuts, and seeds ; mono- and di - saccharides ; and cholesterol (range 0 - 9, 0 being the worst and 9 the best possible scores). scoring for the ahei was based on intake levels of 9 components : fruits, vegetables, ratio of white to red meat, trans fats, ratio of polyunsaturated to saturated fat, cereal fiber, nuts and soy, moderate amounts of alcohol, and multivitamins over a long period. each component contributed 0 to 10 points, with 10 indicating that recommendations were met and 0 indicating the poorest dietary behavior. intermediate intakes were scored proportionally between 0 and 10, except for the multivitamin component, which was assigned a score of either 2.5 or 7.5 to avoid overweighting of this binary variable. component scores for ahei were totaled, ranging from 2.5 (worst) to 87.5 (best). in our study, the duration of multivitamin use was not measured, because this information was not collected. therefore, the maximum possible ahei score was 82.5. we used spss version 10.0 (spss, chicago, il, usa) for statistical analysis. partial correlation coefficients were used to assess the association of the 3 indices of dietary pattern analysis with glycemic indices. subjects were 110 consecutive korean outpatients with type 2 diabetes who visited 2 university hospitals from april 2004 to november 2006 and who were enrolled in a study to evaluate the effectiveness of the medical nutrition therapy (mnt). briefly, subjects were patients with newly diagnosed type 2 diabetes or patients who had been diagnosed with type 2 diabetes for fewer than 5 years but did not have acute and/or chronic complications. at the time of enrollment, height was measured to the nearest centimeter while the subjects were barefoot, and body weight was measured to the nearest 0.1 kg using a digital scale while the subjects wore light clothing. then, body mass index (bmi) was calculated by determining the ratio between weight and height squared (kg / m). the waist circumference was taken at the narrowest point between the lower costal margin and the superior iliac crest, and the hip circumference was measured at the horizontal level between the symphysis pubis and the greater gluteal protuberance that yielded the maximum measurement. blood pressure was recorded using a mercury sphygmomanometer twice in a sitting position after subjects had been in a relaxed state for at least 10 minutes. hypertension was defined as 2 consecutive systolic / diastolic blood pressure measurements > 140/90 mmhg or current treatment with antihypertensive medication. fasting plasma glucose (fpg), 2-hour postprandial glucose (pp2) concentration, and lipid profile levels were analyzed enzymatically, using an autoanalyzer (hitachi 760 - 210, hitachi ltd., (hba1c) was determined using high - performance liquid chromatography (hlc-723 g8, tosoh, tokyo, japan). the dqi - i focused on 4 major aspects : variety (0 - 20 points), adequacy (0 - 40 points), moderation (0 - 30 points), and overall balance (0 - 10 points). the scores for all 4 categories were totaled, from 0 to 100 (0 being the worst and 100 the best possible scores). for the variety and adequacy categories, we used the us exchange list serving size definition for cross - national comparative work. the adequacy of intakes for iron, calcium, and vitamin c were examined on the basis of the percentage attainment of the dietary reference intakes in korea. the hdi was calculated as the sum of dichotomous variables on the basis of 9 items : saturated fatty acids ; polyunsaturated fatty acids ; protein ; complex carbohydrates ; dietary fiber ; fruits and vegetables ; legumes, nuts, and seeds ; mono- and di - saccharides ; and cholesterol (range 0 - 9, 0 being the worst and 9 the best possible scores). scoring for the ahei was based on intake levels of 9 components : fruits, vegetables, ratio of white to red meat, trans fats, ratio of polyunsaturated to saturated fat, cereal fiber, nuts and soy, moderate amounts of alcohol, and multivitamins over a long period. each component contributed 0 to 10 points, with 10 indicating that recommendations were met and 0 indicating the poorest dietary behavior. intermediate intakes were scored proportionally between 0 and 10, except for the multivitamin component, which was assigned a score of either 2.5 or 7.5 to avoid overweighting of this binary variable. component scores for ahei were totaled, ranging from 2.5 (worst) to 87.5 (best). in our study, the duration of multivitamin use was not measured, because this information was not collected. therefore, the maximum possible ahei score was 82.5. we used spss version 10.0 (spss, chicago, il, usa) for statistical analysis. partial correlation coefficients were used to assess the association of the 3 indices of dietary pattern analysis with glycemic indices. the mean age, bmi, and diabetes duration of subjects were 54.6 8.9 years, 25.4 2.8 kg / m, and 1.6 1.5 years, respectively (table 1). the percentages of subjects with hypertension, who regularly exercised, who currently smoked, and who consumed alcohol were 17.3%, 64.6%, 15.5%, and 28.2%, respectively. forty subjects (36.4%) were treated with oral hypoglycemic agents, but none was on insulin therapy. the mean fpg, pp2, and hba1c of subjects were 142.6 33.6 mg / dl, 218.7 78.5 mg / dl, and 7.3 1.2%, respectively. the average intakes of energy, carbohydrate, and fat in these subjects were 1935 492 kcal / day, 277 78 g / day, and 51 22 g / day, respectively (table 2). the average scores for dqi - i, hdi, and ahei in this study were 68.9 8.2 (table 3), 5.0 1.3 (table 4), and 39.4 8.9 (table 5). in the dqi - i, the score of overall - balance aspect was the lowest of the 4 aspects (2.4 2.6 ; 24% of the total score of aspect). however, the variety aspect demonstrated the highest score (16.5 2.6 ; 82% of the total score of aspect), closely followed by the adequacy aspect (32.5 4.7 ; 81% of the total score of aspect). in the hdi, the score for saturated fatty acid (1.0 0.20) was the highest, followed by oligosaccharides (0.9 0.3), polyunsaturated fatty acids (0.8 0.4), and polysaccharides (0.7 0.5). in the ahei, the score for trans - fat was the highest (10.0 0.2), followed by polyunsaturated fatty acids : saturated fatty acids (10.0 0.2), vegetables (7.2 2.5), and nuts and soy proteins (5.5 4.7). after adjustment for age, the dqi - i and ahei scores did not correlate with hba1c, while the hdi score showed a significant negative correlation with hba1c (table 6). however, the dqi - i, ahei, and hdi scores had a significant negative correlation with fpg and pp2. when age, bmi, and energy intake were adjusted for, the dqi - i and hdi scores had a significant negative correlation with hba1c. this study focused on assessing the association between dietary quality indices and glycemic indices in korean patients with type 2 diabetes. we found that the dqi - i and hdi had a significant negative correlation with hba1c, fpg, and pp2, but the ahei did not have a significant negative correlation with hba1c. a few studies have shown an association between glycemic status and the dqi - i and hdi in patients with diabetes. however, similar to our study, the dietary guideline index, which assessed compliance with established dietary guidelines, was inversely associated with glycemic indices in type 2 diabetes. interestingly, our results showed that the total scores of the dqi - i and hdi had an association with glycemic indices, but each component of the dqi - i and hdi did not. consistent with our findings, the multiple food groups characterized as the 4-principle component analysis dietary pattern score and a priori -defined low - diabetes - risk food pattern score were associated with lower risk of type 2 diabetes, but individual food groups were not independently associated. this observation suggests that dietary pattern indices are a better approach to assess diet quality for diabetes than traditional approach of using single - nutrient or food group exposures. several studies have shown an association between eating patterns according to the ahei and risk of type 2 diabetes. contrary to our findings, higher scores on the ahei were reported to be associated with a lower risk of type 2 diabetes (comparing the top to the bottom quintile : 0.64 ([95% confidence interval, 0.58 - 0.71 ], ptrend <.0001), and the ahei score was negatively correlated with change in hba1c level (r = -0.024, p < 0.05). a limitation of this study was that the 3 dietary pattern indices were derived from one - day dietary intake data obtained by the 24-hour recall method, which may not reflect the ordinary diet of the subjects. however, 24-hour recall data maybe a stronger predictor of more recent diet than distant diet for type 2 diabetes, and the timing of food frequency questionnaires may not capture short - term improvement. this might explain why a correlation was observed between the 3 dietary quality indices and fpg and pp2, but not between ahei and hba1c. additionally, our data were cross - sectional, thereby precluding the determination of causality. taken together, these findings suggest the need for longitudinal research to examine in more detail the relationship between diet quality and glycemic changes in type 2 diabetes. in addition, for the variety and adequacy aspects for some of the components of the dqi - i, we used the us food guide pyramid serving size definitions to assess food group intake on the basis of how the number of servings consumed compared with the recommendations. this study examined whether dietary quality indices are appropriate to assess diet quality and predict adherence to dietary recommendations in korean patients with type 2 diabetes. further research is needed to examine the long - term dietary and glycemic changes in patients with diabetes following nutrition education. the dqi - i and hdi may be useful tools in assessing diet quality and adherence to dietary recommendations in patients with type 2 diabetes. future research is required to determine whether dietary quality indices have predictive validity for dietary and glycemic changes after diet education in a clinical care setting. | the present study was performed to evaluate the relationship between dietary quality indices including the diet quality index - international (dqi - i), alternate healthy eating index (ahei), and healthy diet indicator (hdi) and glycemic status in korean patients with type 2 diabetes. a total of 110 consecutive outpatients with type 2 diabetes who visited 2 university hospitals in seoul and seongnam from april 2004 to november 2006 were enrolled as subjects. at the time of enrollment, anthropometric parameters, dietary habits, experience of exercise, and metabolic parameters were obtained. experienced registered dietitians collected one - day dietary intake using the 24-hour recall method. the mean scores for dqi - i, ahei, and hdi were 68.9 8.2, 39.4 8.9, and 5.0 1.3, respectively. after adjustment for age, body mass index, and energy intake, dqi - i and hdi were found to have a significant correlation with hemoglobin a1c (hba1c) (r = -0.21, p < 0.05 ; r = -0.28, p < 0.05), fasting plasma glucose (r = -0.21, p < 0.05 ; r = -0.23, p < 0.05), and postprandial 2-h glucose (r = -0.30, p < 0.05 ; r = -0.26, p < 0.05, respectively). however, ahei did not have a significant correlation with hba1c. in conclusion, the dqi - i and hdi may be useful tools in assessing diet quality and adherence to dietary recommendations in korean patients with type 2 diabetes. future research is required to determine whether the dietary quality indices have predictive validity for dietary and glycemic changes following diet education in a clinical setting. |
the cath database is a hierarchical classification of domains into sequence- and structure - based families and fold groups. table 1 shows the population of the latest release of cath (version 2.5.1, released january 2004). in the lowest level of the hierarchy, sequences are clustered according to significant sequence similarity (35% identity and above, the s - level). at higher levels, domains are grouped according to whether they share significant sequence, structural and/or functional similarity (homologous superfamilies, h - level) or just structural similarity (fold or topology group, the t - level). fold groups sharing similar architectures, i.e. similarities in the arrangements of their secondary structures regardless of connectivity are then merged into the common architectures (the a - level). at the top of the hierarchy, domains are clustered depending on their class, i.e. the percentage of helices or -strands (the c - level). below we describe some new cath associated resources and protocols that increase the speed and reliability of classifying newly determined protein structures in the cath database. the cath associated dictionary of homologous superfamilies (dhs) (http://www.biochem.ucl.ac.uk/bsm/dhs/) was established in 1997 (1) and contains a variety of sequence, structural and functional information for each superfamily in cath. it was updated recently for cath version 2.5.1, which contains 1467 homologous superfamilies, 334 of which are populated with three or more remote homologues (85 against all relatives in the subgroup. these are generated using the cora algorithm (3) and displayed using coraplot (3). highly conserved sequence positions, which may be associated with functionally important sites, are highlighted. two new methods have been devised to illustrate the degree of structural divergence across the superfamily. both exploit a multiple structure alignment to identify equivalent secondary structures across the superfamily and inserted secondary structures. plots give information on highly conserved secondary structures that are diagnostic for the particular superfamily and on the degree of structural embellishment occurring in diverse relatives. putative homologues to a particular cath superfamily can be aligned against structural relatives in order to determine whether their structural characteristics fall within the range of structural diversity observed across the superfamily. information on the population of the superfamily is also provided so that users can gauge how well the superfamily has been sampled to date. functional annotations are also provided for each superfamily in the dhs by recruiting relevant functional data from the protein data bank (pdb) (4), genbank (5), enzyme (6), kegg (7) and gene ontology (8) databases. the more than 10-fold expansion in the extended cath database (from 43 299 cath structural domain sequences to 616 470 by including related genbank sequences and genome sequences) has significantly increased the amount of functional data available for a particular superfamily. expansion in the functional information together with more informative descriptions of structural variability in each cath superfamily considerably assists in validating new homologues classified in cath. furthermore, links to the dhs are provided for structural matches identified using the cath server. profile based methods for sequence comparison were developed in the early 1980s and allowed recognition of more distant homologues than pairwise based approaches (9). benchmarking of several publicly available methods, including those using position - specific scoring matrices and hidden markov models (hmms) have been undertaken by several groups (10,11). these approaches used datasets of distant homologues selected from the structural classifications, such as scop and cath, to determine the sensitivity of various profile based methods, e.g. hmms (12) and psi - blast (13). we recently used a dataset of remote structural homologues from the cath database (85 against all relatives in the subgroup. these are generated using the cora algorithm (3) and displayed using coraplot (3). highly conserved sequence positions, which may be associated with functionally important sites, are highlighted. two new methods have been devised to illustrate the degree of structural divergence across the superfamily. both exploit a multiple structure alignment to identify equivalent secondary structures across the superfamily and inserted secondary structures. plots give information on highly conserved secondary structures that are diagnostic for the particular superfamily and on the degree of structural embellishment occurring in diverse relatives. putative homologues to a particular cath superfamily can be aligned against structural relatives in order to determine whether their structural characteristics fall within the range of structural diversity observed across the superfamily. information on the population of the superfamily is also provided so that users can gauge how well the superfamily has been sampled to date. functional annotations are also provided for each superfamily in the dhs by recruiting relevant functional data from the protein data bank (pdb) (4), genbank (5), enzyme (6), kegg (7) and gene ontology (8) databases. the more than 10-fold expansion in the extended cath database (from 43 299 cath structural domain sequences to 616 470 by including related genbank sequences and genome sequences) has significantly increased the amount of functional data available for a particular superfamily. expansion in the functional information together with more informative descriptions of structural variability in each cath superfamily considerably assists in validating new homologues classified in cath. furthermore, links to the dhs are provided for structural matches identified using the cath server. profile based methods for sequence comparison were developed in the early 1980s and allowed recognition of more distant homologues than pairwise based approaches (9). benchmarking of several publicly available methods, including those using position - specific scoring matrices and hidden markov models (hmms) have been undertaken by several groups (10,11). these approaches used datasets of distant homologues selected from the structural classifications, such as scop and cath, to determine the sensitivity of various profile based methods, e.g. hmms (12) and psi - blast (13). we recently used a dataset of remote structural homologues from the cath database (< 35% sequence identity), which had been validated by structure comparison and manual inspection to assess the performance of several hmm based strategies (strategies for improved fold and superfamily recognition in genome annotation ; i. sillitoe, personal communication). a total of 23 876 hmm models were built for representative sequences from each sequence family in the extended cath database (containing 616 470 domain sequences). the extended model library gives a 10% increase in coverage for remote homologue detection compared to the standard cath hmm model library, with a low error rate (0.1%) (i. sillitoe, personal communication). it can be seen from figure 1 that on average, nearly 87% of homologues classified in cath over the last two years could be recognized using sequence comparison methods, both pairwise sequence alignment and scans against the more sensitive extended cath - hmm model library. we have recently devised protocols for identifying sequence relatives to cath superfamilies in completed genomes (15). to date, nearly one million sequences from 150 completed genomes have been scanned against the cath - hmm model library (15). between 40 and 60% of sequences or partial sequences from each genome could be assigned to a cath superfamily. genome sequences were also scanned against libraries of hmm models from the pfam database (release 10) (16) in order to extend the domain annotation of each genome sequence and provide more comprehensive information on domain partnerships. sequence relatives to cath superfamilies, identified in this way are displayed in the cath related dhs and gene3d resources. cath family data in the gene3d resource has revealed some intriguing insights into the expansion of superfamilies involved in metabolism and regulation in bacterial genomes (17). figure 2 shows that the power - law like trends first detected in the structural classifications are mirrored when sequence relatives from the genomes are also included. considering the structural data alone, it can be seen from figure 2a that fewer than 10 of the most highly populated folds in the cath database account for nearly 25% of all superfamilies in the pdb. these folds were previously described as superfolds as they are adopted by many diverse homologous superfamilies (18). when genome sequences are included it can be seen from figure 2b that the same fold groups dominate the genomes, as they are adopted by nearly 45% of all close sequence families (relatives have 35% or more sequence identity), of known structure, in the genomes. a new protocol has been developed for searching cath with a newly determined protein structure. structures submitted to the server (http://www.biochem.ucl.ac.uk/cgi-bin/cath/cathserver.pl) are first processed by the ddmake suite of programs that generate derived data from the pdb coordinate files (e.g. secondary structure data, residue accessibilities and data, sequence data in the fasta format, etc.). the query sequence is scanned against the cath - hmm model library to identify more remote homologues. threshold e - values used to recognize homologues are predetermined by benchmarking with validated structural homologues from cath (i. sillitoe, personal communication). if the sequence returns a significant match to any relative in one or more cath superfamilies, representatives from all close sequence families within those superfamilies are structurally compared with the query structure using the ssap structure alignment program (2). the top 10 structural matches, sorted in the order of ssap score are then displayed together with information on the degree of sequence and structural similarity and with links to the cath page and the dhs page for each cath superfamily identified. rasmol images are also provided for the top 10 matches. any query structure unmatched by the cath - hmm library is scanned against a library of representative structures from each close sequence family in cath using the rapid structure comparison algorithm, cathedral (19). cathedral uses a robust statistical framework based on the extreme value distributions observed for random similarities to assess significance. if the query structure significantly matches one or more cath superfamilies, ssap comparisons are performed for all sequence representatives in those superfamilies and the top 10 matches are displayed, as before. | the cath database of protein domain structures (http://www.biochem.ucl.ac.uk/bsm/cath/) currently contains 43 229 domains classified into 1467 superfamilies and 5107 sequence families. each structural family is expanded with sequence relatives from genbank and completed genomes, using a variety of efficient sequence search protocols and reliable thresholds. this extended cath protein family database contains 616 470 domain sequences classified into 23 876 sequence families. this results in the significant expansion of the cath hmm model library to include models built from the cath sequence relatives, giving a 10% increase in coverage for detecting remote homologues. an improved dictionary of homologous superfamilies (dhs) (http://www.biochem.ucl.ac.uk/bsm/dhs/) containing specific sequence, structural and functional information for each superfamily in cath considerably assists manual validation of homologues. information on sequence relatives in cath superfamilies, genbank and completed genomes is presented in the cath associated dhs and gene3d resources. domain partnership information can be obtained from gene3d (http://www.biochem.ucl.ac.uk/bsm/cath/gene3d/). a new cath server has been implemented (http://www.biochem.ucl.ac.uk/cgi-bin/cath/cathserver.pl) providing automatic classification of newly determined sequences and structures using a suite of rapid sequence and structure comparison methods. the statistical significance of matches is assessed and links are provided to the putative superfamily or fold group to which the query sequence or structure is assigned. |
a literature search of the pubmed online database was carried out to identify studies that reported the causes of death after tavr on august 10, 2014 (figure1). the search terms were as follows : (percutaneous or transcatheter or transfemoral or transapical or transsubclavian or transaortic or transaxillary) and (aortic valve) and (replacement or implantation). only articles in english the inclusion criteria were : (1) studies that reported the specific causes of death after tavr, (2) sufficient data (the number of patients who died because of 1 certain reason and the time interval after tavr when they died) available, and (3) studies that at least covered both short - term (30 days) and long - term (> 30 days) death events. studies were excluded if 1 of the following existed : (1) causes of death were listed vaguely, (2) death events related to valve - in - valve procedures, (3) studies concerning death predictors instead of the actual causes of death, (4) published before 2002, or (5) studies were case reports, reviews, abstracts, guidelines, comments and conference presentations. if more than 1 study was published by the same authors using the same case series or overlapping case series, studies with the largest sample size two main reviewers (t.y.x and y.b.l) independently extracted the data and reached a consensus on all items. the following items were extracted from each study if available : first author s name, publication year, study period, region, study design, single or multicenter study, tavr case number, patients age, male proportion, new york heart association classification, ejection fraction, comorbidities, logistic european system for cardiac operative risk evaluation (euroscore), the society of thoracic surgeons (sts) score, chosen valve and access, and follow - up length. when conducting reclassification, cardiac death was defined as any death directly involving cardiac integrity and function (heart failure, acute myocardial infarction, sudden death / arrhythmia, and tamponade). vascular complications and bleedings are regarded as procedure - related causes of death, and together with stroke, are grouped into cardiovascular causes of death in our study in accordance with valve academic research consortium (varc) consensus document.7 before reclassification of causes of death in included studies into different categories, all involved causes were discussed and double - checked by authors to reach the consensus on whether one certain cause of death belongs to a cardiovascular or noncardiovascular group. as for those already grouped into other cardiovascular or other noncardiovascular in original reports, we can only keep this classification even if certain causes may be separately listed in other studies. those causes with only few case numbers were also grouped into other categories for the sake of clear illustrations. study quality was assessed using the cross - sectional / prevalence study quality assessment form or newcastle - ottawa quality assessment scale (nos).8,9 results are expressed as counts and percentages for categorical variables. dersimonian and laird s random - effects model was used to pool the estimates of proportions of cardiovascular deaths in each duration and subgroup by comprehensive meta analysis software version 3. differences in the proportions of cardiovascular deaths were compared with the test by using spss software 19.0. a literature search of the pubmed online database was carried out to identify studies that reported the causes of death after tavr on august 10, 2014 (figure1). the search terms were as follows : (percutaneous or transcatheter or transfemoral or transapical or transsubclavian or transaortic or transaxillary) and (aortic valve) and (replacement or implantation). only articles in english the inclusion criteria were : (1) studies that reported the specific causes of death after tavr, (2) sufficient data (the number of patients who died because of 1 certain reason and the time interval after tavr when they died) available, and (3) studies that at least covered both short - term (30 days) and long - term (> 30 days) death events. studies were excluded if 1 of the following existed : (1) causes of death were listed vaguely, (2) death events related to valve - in - valve procedures, (3) studies concerning death predictors instead of the actual causes of death, (4) published before 2002, or (5) studies were case reports, reviews, abstracts, guidelines, comments and conference presentations. if more than 1 study was published by the same authors using the same case series or overlapping case series, studies with the largest sample size two main reviewers (t.y.x and y.b.l) independently extracted the data and reached a consensus on all items. the following items were extracted from each study if available : first author s name, publication year, study period, region, study design, single or multicenter study, tavr case number, patients age, male proportion, new york heart association classification, ejection fraction, comorbidities, logistic european system for cardiac operative risk evaluation (euroscore), the society of thoracic surgeons (sts) score, chosen valve and access, and follow - up length. when conducting reclassification, cardiac death was defined as any death directly involving cardiac integrity and function (heart failure, acute myocardial infarction, sudden death / arrhythmia, and tamponade). vascular complications and bleedings are regarded as procedure - related causes of death, and together with stroke, are grouped into cardiovascular causes of death in our study in accordance with valve academic research consortium (varc) consensus document.7 before reclassification of causes of death in included studies into different categories, all involved causes were discussed and double - checked by authors to reach the consensus on whether one certain cause of death belongs to a cardiovascular or noncardiovascular group. as for those already grouped into other cardiovascular or other noncardiovascular in original reports, we can only keep this classification even if certain causes may be separately listed in other studies. those causes with only few case numbers were also grouped into other categories for the sake of clear illustrations. study quality was assessed using the cross - sectional / prevalence study quality assessment form or newcastle - ottawa quality assessment scale (nos).8,9 dersimonian and laird s random - effects model was used to pool the estimates of proportions of cardiovascular deaths in each duration and subgroup by comprehensive meta analysis software version 3. differences in the proportions of cardiovascular deaths were compared with the test by using spss software 19.0. a total of 3934 results were identified after an initial search from the pubmed database. after careful review of abstracts, 36 were deemed suitable for data extraction ; 8 studies were excluded due to overlapping case series, leaving 28 results for further study1037 (figure1). follow - up time ranges from 6 months to (3.82) years with acceptable follow - up rate. summary of included studies i - ta indicates italian registry of transapical aortic valve implantation ; mcv indicates medtronic corevalve ; sts, society of thoracic surgeons ; ta - tavr, transapical tavr ; tavr, transcatheter aortic valve replacement ; tf - tavr, transfemoral tavr. randomized by single or double antiplatelet therapy after the procedure. the study by attias, d was used for tf - tavr subgroup analysis. the study by guinot, p was used for ta - tavr subgroup analysis. inoperable patients were randomly assigned to standard therapy (including balloon aortic valvuloplasty) or tf - tavr. the quality of eligible cohort studies was assessed using the newcastle - ottawa quality assessment scale scale, while quality of single - arm studies was evaluated by the cross - sectional / prevalence study quality. overall quality of these included studies was good (evaluation forms for each study were not shown). within 30 days, 8.4% (265/3155) of patients died, 46.4% (123/265) of which were deemed noncardiovascular. noncardiovascular causes accounted for 51.6% (282/546) of 546 deaths after 30 days. as for individual causes, within 30 days, infection / sepsis accounted for 18.5% of deaths, followed by heart failure and multiorgan failure, accounting for 14.7% and 13.2% of deaths, respectively. beyond 30 days, infection / sepsis each accounted for 14.3% of deaths, followed by heart failure and sudden death as the reported cause of 14.1% and 10.8% of deaths, respectively (figures3 through 4). because of the broad - spectrum character of noncardiovascular causes, detailed modes of infection / sepsis and other noncardiovascular causes are provided in table2 separately from figures for the sake of clear illustrations and better understanding. detailed modes of noncardiovascular deaths in overall analysis overall analysis of causes of death within the first 30 days following transcatheter aortic valve replacement. overall analysis of causes of death after the first 30 days following transcatheter aortic valve replacement (tavr). figure 2 and 3 show causes of death post - tavr per time interval, with total patients included (n), deaths (n), and the blue parts standing for noncardiovascular causes and the green parts for cardiac / procedure - related causes. overall analysis in both durations, within 30 days and after 30 days of the valve procedure. causes of death by transfemoral tavr (tf - tavr)12,15,2629,31,37 and transapical tavr (ta - tavr)14,17,19,21,24,27,28,30,32,33,36 were individually delineated (figure5 with absolute numbers). for the tf - tavr group, 6.6% (39/589) and 17.3% (95/550) of patients died within and beyond 30 days, which were 10.1% (123/1215) and 13.8% (342/2479) for ta - tavr. in general, multiorgan failure, heart failure, and vascular complications were the 3 leading causes of death for tf - tavr within 30 days (17.9%, 15.4%, and 15.4%, respectively) ; beyond 30 days, infection / sepsis, heart failure, and sudden death accounted for 15.8%, 14.7%, and 8.4% of deaths, respectively. within 30 days of ta - tavr, infection / sepsis, multiorgan failure, and heart failure accounted for 22.8%, 18.7%, and 13.8% of deaths, while beyond 30 days, heart failure, infection / sepsis, and sudden death accounted for 14.0%, 12.0%, and 9.9% deaths. the pooled estimate of proportions of cardiovascular deaths in the tf - tavr subgroup were 61.5% (95% ci : 44.4% to 76.2%) within 30 days post - tavr and 35.7% (95% ci : 23.4% to 50.3%) after 30 days. in the ta - tavr subgroup, the pooled estimate of proportions of cardiovascular deaths were 44.7% (95% ci : 31.2% to 59.0%) and 39.6% (95% ci : 28.1% to 52.4%), respectively. no significant differences were found between the 2 subgroups in terms of the proportion of cardiovascular deaths (p=0.067 within 30 days ; p=0.514 after 30 days) (figure s1). ta indicates transapical ; tf, transfemoral. two most - used valve types, balloon expandable edwards valve (edwards lifesciences, irvine, ca) and the self - expanding corevalve system (medtronic, inc, minneapolis, mn), were included in this subgroup analysis (figure6 with absolute numbers). since the corevalve system can not be delivered transapically, only tf - tavr studies were included in this subgroup analysis.12,23,26,29,31,34,37 for the edwards sapien valve group, 6.8% (25/370) and 22.3% (77/345) of patients died within and beyond 30 days, which were 10.7% (35/326) and 10.3% (30/291) for medtronic corevalve. overall, multiorgan failure, vascular complications, and stroke with 20.0%, 16.0%, and 12.0% of deaths were the 3 individual and specific leading causes of death for tavr using only edwards sapien valve within 30 days, while infection / sepsis, heart failure, and renal disease took the lead after 30 days (16.9%, 14.3%, and 6.5% of deaths, respectively). for medtronic corevalve, heart failure, vascular complications, infection / sepsis, and aortic regurgitation accounted for 25.7%, 14.3%, 8.6%, and 8.6% of deaths within 30 days, while heart failure was the most common cause after 30 days (10.0% of deaths). the pooled estimates of proportions of cardiovascular deaths in the edwards sapien valve subgroup were 66.8% (95% ci : 45.7% to 82.7%) within 30 days post - tavr and 40.2% (95% ci : 18.7% to 66.2%) after 30 days. in the medtronic corevalve subgroup, the pooled estimates of proportions of cardiovascular deaths were 57.5% (95% ci : 36.6% to 76.0%) and 27.6% (95% ci : 14.4% to 46.2%), respectively. no significant differences were found between the 2 subgroups in terms of the proportion of cardiovascular deaths (p=0.394 within 30 days ; p=0.189 after 30 days) (figure s2). subgroup analysis by types of prosthesis. according to mean sts score, studies conducted in patients with a mean sts score between 4 and 8 (regarded as moderate risk) and with mean score larger than 8 (regarded as high risk) were divided into 2 subgroups (figure7 with absolute numbers). for moderate - risk patients, 6.8% (31/457) and 10.1% (43/426) died within and beyond 30 days, which were 9.2% (157/1706) and 15.6% (242/1549) for high - risk patients. multiorgan failure and heart failure accounted for 22.6% and 19.4% of deaths in the moderate - risk group within the first 30 days, while infection / sepsis, heart failure, and multiorgan failure took the lead in the high - risk group (19.7%, 15.3%, and 15.3%, respectively). after 30 days, sudden death, infection / sepsis, and heart failure (23.3%, 18.6%, and 11.6% of deaths) were the top 3 killers for moderate - risk patients ; on the other hand, infection / sepsis, heart failure, and cancer (19.8%, 15.7%, and 7.9% of deaths) were the 3 leading causes of death for high - risk patients. comparison for the proportion of cardiovascular deaths saw a significant difference in 2 subgroups after 30 days post - tavr (moderate risk versus high risk : 55.5% [95% ci : 37.3% to 72.3% ] versus 45.6% [95% ci : 32.6% to 59.1% ], p=0.360, within 30 days ; 56.0% [95% ci : 40.6% to 70.4% ] versus 33.5% [95% ci : 22.1% to 47.3% ], p=0.005, after 30 days) (figure s3). studies that reported causes of death both in patients who underwent tavr and those who underwent other treatment choices were used for further comparison. in 3 single - center studies that compared outcomes after tavr and savr in a nonrandomized fashion,15,35,38 tavr saw a trend of increased proportion of noncardiovascular causes of death while savr had a decreased trend within and after 30 days following the procedure, although the difference is not significant. in cohort b of the placement of aortic transcatheter valves (partner) trial29 inoperable patients were randomly assigned to either tavr or standard therapy (including balloon aortic valvuloplasty). again, a decreased trend was seen for noncardiovascular causes of death in the traditional treatment group but not in the tavr group (figure s4). a total of 3934 results were identified after an initial search from the pubmed database. after careful review of abstracts, 36 were deemed suitable for data extraction ; 8 studies were excluded due to overlapping case series, leaving 28 results for further study1037 (figure1). follow - up time ranges from 6 months to (3.82) years with acceptable follow - up rate. summary of included studies i - ta indicates italian registry of transapical aortic valve implantation ; mcv indicates medtronic corevalve ; sts, society of thoracic surgeons ; ta - tavr, transapical tavr ; tavr, transcatheter aortic valve replacement ; tf - tavr, transfemoral tavr. randomized by single or double antiplatelet therapy after the procedure. the study by attias, d was used for tf - tavr subgroup analysis. the study by guinot, p was used for ta - tavr subgroup analysis. inoperable patients were randomly assigned to standard therapy (including balloon aortic valvuloplasty) or tf - tavr. the quality of eligible cohort studies was assessed using the newcastle - ottawa quality assessment scale scale, while quality of single - arm studies was evaluated by the cross - sectional / prevalence study quality. overall quality of these included studies was good (evaluation forms for each study were not shown). within 30 days, 8.4% (265/3155) of patients died, 46.4% (123/265) of which were deemed noncardiovascular. noncardiovascular causes accounted for 51.6% (282/546) of 546 deaths after 30 days. as for individual causes, within 30 days, infection / sepsis accounted for 18.5% of deaths, followed by heart failure and multiorgan failure, accounting for 14.7% and 13.2% of deaths, respectively. beyond 30 days, infection / sepsis each accounted for 14.3% of deaths, followed by heart failure and sudden death as the reported cause of 14.1% and 10.8% of deaths, respectively (figures3 through 4). because of the broad - spectrum character of noncardiovascular causes, detailed modes of infection / sepsis and other noncardiovascular causes are provided in table2 separately from figures for the sake of clear illustrations and better understanding. detailed modes of noncardiovascular deaths in overall analysis overall analysis of causes of death within the first 30 days following transcatheter aortic valve replacement. overall analysis of causes of death after the first 30 days following transcatheter aortic valve replacement (tavr). figure 2 and 3 show causes of death post - tavr per time interval, with total patients included (n), deaths (n), and the blue parts standing for noncardiovascular causes and the green parts for cardiac / procedure - related causes. overall analysis in both durations, within 30 days and after 30 days of the valve procedure. causes of death by transfemoral tavr (tf - tavr)12,15,2629,31,37 and transapical tavr (ta - tavr)14,17,19,21,24,27,28,30,32,33,36 were individually delineated (figure5 with absolute numbers). for the tf - tavr group, 6.6% (39/589) and 17.3% (95/550) of patients died within and beyond 30 days, which were 10.1% (123/1215) and 13.8% (342/2479) for ta - tavr. in general, multiorgan failure, heart failure, and vascular complications were the 3 leading causes of death for tf - tavr within 30 days (17.9%, 15.4%, and 15.4%, respectively) ; beyond 30 days, infection / sepsis, heart failure, and sudden death accounted for 15.8%, 14.7%, and 8.4% of deaths, respectively. within 30 days of ta - tavr, infection / sepsis, multiorgan failure, and heart failure accounted for 22.8%, 18.7%, and 13.8% of deaths, while beyond 30 days, heart failure, infection / sepsis, and sudden death accounted for 14.0%, 12.0%, and 9.9% deaths. the pooled estimate of proportions of cardiovascular deaths in the tf - tavr subgroup were 61.5% (95% ci : 44.4% to 76.2%) within 30 days post - tavr and 35.7% (95% ci : 23.4% to 50.3%) after 30 days. in the ta - tavr subgroup, the pooled estimate of proportions of cardiovascular deaths were 44.7% (95% ci : 31.2% to 59.0%) and 39.6% (95% ci : 28.1% to 52.4%), respectively. no significant differences were found between the 2 subgroups in terms of the proportion of cardiovascular deaths (p=0.067 within 30 days ; p=0.514 after 30 days) (figure s1). two most - used valve types, balloon expandable edwards valve (edwards lifesciences, irvine, ca) and the self - expanding corevalve system (medtronic, inc, minneapolis, mn), were included in this subgroup analysis (figure6 with absolute numbers). since the corevalve system can not be delivered transapically, only tf - tavr studies were included in this subgroup analysis.12,23,26,29,31,34,37 for the edwards sapien valve group, 6.8% (25/370) and 22.3% (77/345) of patients died within and beyond 30 days, which were 10.7% (35/326) and 10.3% (30/291) for medtronic corevalve. overall, multiorgan failure, vascular complications, and stroke with 20.0%, 16.0%, and 12.0% of deaths were the 3 individual and specific leading causes of death for tavr using only edwards sapien valve within 30 days, while infection / sepsis, heart failure, and renal disease took the lead after 30 days (16.9%, 14.3%, and 6.5% of deaths, respectively). for medtronic corevalve, heart failure, vascular complications, infection / sepsis, and aortic regurgitation accounted for 25.7%, 14.3%, 8.6%, and 8.6% of deaths within 30 days, while heart failure was the most common cause after 30 days (10.0% of deaths). the pooled estimates of proportions of cardiovascular deaths in the edwards sapien valve subgroup were 66.8% (95% ci : 45.7% to 82.7%) within 30 days post - tavr and 40.2% (95% ci : 18.7% to 66.2%) after 30 days. in the medtronic corevalve subgroup, the pooled estimates of proportions of cardiovascular deaths were 57.5% (95% ci : 36.6% to 76.0%) and 27.6% (95% ci : 14.4% to 46.2%), respectively. no significant differences were found between the 2 subgroups in terms of the proportion of cardiovascular deaths (p=0.394 within 30 days ; p=0.189 after 30 days) (figure s2). according to mean sts score, studies conducted in patients with a mean sts score between 4 and 8 (regarded as moderate risk) and with mean score larger than 8 (regarded as high risk) were divided into 2 subgroups (figure7 with absolute numbers). for moderate - risk patients, 6.8% (31/457) and 10.1% (43/426) died within and beyond 30 days, which were 9.2% (157/1706) and 15.6% (242/1549) for high - risk patients. multiorgan failure and heart failure accounted for 22.6% and 19.4% of deaths in the moderate - risk group within the first 30 days, while infection / sepsis, heart failure, and multiorgan failure took the lead in the high - risk group (19.7%, 15.3%, and 15.3%, respectively). after 30 days, sudden death, infection / sepsis, and heart failure (23.3%, 18.6%, and 11.6% of deaths) were the top 3 killers for moderate - risk patients ; on the other hand, infection / sepsis, heart failure, and cancer (19.8%, 15.7%, and 7.9% of deaths) were the 3 leading causes of death for high - risk patients. comparison for the proportion of cardiovascular deaths saw a significant difference in 2 subgroups after 30 days post - tavr (moderate risk versus high risk : 55.5% [95% ci : 37.3% to 72.3% ] versus 45.6% [95% ci : 32.6% to 59.1% ], p=0.360, within 30 days ; 56.0% [95% ci : 40.6% to 70.4% ] versus 33.5% [95% ci : 22.1% to 47.3% ], p=0.005, after 30 days) (figure s3). studies that reported causes of death both in patients who underwent tavr and those who underwent other treatment choices were used for further comparison. in 3 single - center studies that compared outcomes after tavr and savr in a nonrandomized fashion,15,35,38 tavr saw a trend of increased proportion of noncardiovascular causes of death while savr had a decreased trend within and after 30 days following the procedure, although the difference is not significant. in cohort b of the placement of aortic transcatheter valves (partner) trial29 inoperable patients were randomly assigned to either tavr or standard therapy (including balloon aortic valvuloplasty). again, a decreased trend was seen for noncardiovascular causes of death in the traditional treatment group but not in the tavr group (figure s4). aortic stenosis is the most common valvular disease in the world, with a 3-year survival rate less than 30% after symptom onset with medical therapy.39 although savr is the established therapy for aortic stenosis, over 30% patients are considered at high or prohibitive risk for this procedure40 and tavr has evolved as a promising alternative.41 certainly, there have been an increasing number of large multicenter studies and numerous single - center small studies trying to identify mortality after tavr. however, when managing a postprocedural patient, predicted mortality alone is far from enough. a delineation of the causes of death after tavr is fundamental to the optimization of acute and late outcomes. we saw noncardiovascular causes of death as important modes of death, particularly beyond 30 days : infection / sepsis, heart failure, and multiorgan failure were the top 3 individual and specific causes of death within the first 30 days and infection / sepsis, heart failure, and sudden death beyond 30 days, leaving infection / sepsis and heart failure being the biggest killers in noncardiovascular and cardiovascular causes of death, respectively. this is in line with prior works in this area.17,20 although tavr is minimally invasive, patients usually have predisposing factors for infection including age ; poor pulmonary, renal, and immune function ; diabetes ; and need for ventilation and central venous access and monitoring.42 also, as high - efficiency particulate air - filtered laminar airflow is absent in most catheterization labs, this may increase the risk for procedure - related infection.43 thus, the use of broad - spectrum antibiotics for prophylaxis beforehand merits further investigation. it is accepted that aortic stenosis leads to an adaptive hypertrophic response of the myocardium, which allows for maintenance of cardiac output but may lead to progressive myocardial failure. monrad identified the remodeling of left ventricular volume, mass, and shape as a process that may proceed up to almost a decade after correction of the valvular abnormality. thus, the long - standing left ventricular hypertrophy, increased myocardial oxygen demand, and increased left ventricular end - diastolic pressure may play an important role in cardiac deaths seen. previous studies demonstrated that after an early phase of high risk of cardiovascular deaths, risk of cardiovascular death was substantially reduced in patients receiving savr.45 non - valve - related cardiac failure and malignancy were reported as the most common certified cardiac and noncardiac causes of death in octogenarians involved in the uk heart valve registry.46 in our following comparison between tavr and savr or standard therapy, decreased trends were seen for noncardiovascular causes of death within and after 30 days following savr and standard therapy, the proportion of which increased following tavr. yet, because of current selection criteria, patients undergoing tavr tend to have more comorbidities and may end up dying from those noncardiovascular comorbidities. nonetheless, tavr still proves itself to be a reliable alternative with similar mortality and mode of death even when the referred patients are deemed to be at high risk. in the following subgroup analyses, no significant differences were seen for proportions of cardiovascular deaths except the comparison between moderate and high - risk patients after 30 days post - tavr. bearing different performing characteristics and risk potentials, tf and ta as access choices at least showed evenly distributed modes of death, which may be helpful when stratifying patients to certain treatment options, as was the case for edwards sapien valve and medtronic corevalve as valve choices. on the other hand, in current practice, patient selection is often based on the evaluation of surgical risk. sts score or the euroscore i / ii are now frequently used for risk stratification. hemmann have identified sts score as the strongest predictor of long - term survival following tavr. thus, we used the mean value of each study s sts score as a risk stratification indicator. the finding of a higher proportion of cardiovascular deaths in moderate - risk patients after 30 days may be explained by the increasing deaths from more comorbidities in high - risk patients. first, because of the huge variation of the number of causes of death, we have made some grouping such as putting accidental death and bone fracture into the also, although the varc-2 criteria were used, further distinguishing of some possible procedure - related deaths was difficult because the lack of original information could cause overestimation of the proportion of noncardiovascular deaths, especially within 30 days after tavr. second, heterogeneity should not be forgotten since our study did include large multicenter registries and also single - center studies with relatively small samples. third, our results were related to time ; however, for those results in the period after 30 days following tavr, the death toll will be altered if follow - up time changes. thus, there is a limitation for our included studies without a uniform time to end follow - up. the presented data provide important insights into the causes of early and late deaths after tavr. many of these causes are potentially preventable and merit specific attention to areas including infection prophylaxis and heart failure optimization. we believe that bearing all the most possible adverse events in mind and with the assistance of future technological advances, cardiologists will further improve patients outcomes after tavr. the work was funded by a grant from the national natural science foundation of china (grant numbers : 81370219 and 81400267, beijing, china). | backgroundtranscatheter aortic valve replacement (tavr) is an effective alternative to surgical aortic valve replacement in patients at high surgical risk. however, there is little published literature on the exact causes of death.methods and resultsthe pubmed database was systematically searched for studies reporting causes of death within and after 30 days following tavr. twenty - eight studies out of 3934 results retrieved were identified. in the overall analysis, 46.4% and 51.6% of deaths were related to noncardiovascular causes within and after the first 30 days, respectively. within 30 days of tavr, infection / sepsis (18.5%), heart failure (14.7%), and multiorgan failure (13.2%) were the top 3 causes of death. beyond 30 days, infection / sepsis (14.3%), heart failure (14.1%), and sudden death (10.8%) were the most common causes. all possible subgroup analyses were made. no significant differences were seen for proportions of cardiovascular deaths except the comparison between moderate (mean sts score 4 to 8) and high (mean sts score > 8) -risk patients after 30 days post - tavr (56.0% versus 33.5%, p=0.005).conclusionscardiovascular and noncardiovascular causes of death are evenly balanced both in the perioperative period and at long - term follow - up after tavr. infection / sepsis and heart failure were the most frequent noncardiovascular and cardiovascular causes of death. this study highlights important areas of clinical focus that could further improve outcomes after tavr. |
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