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latin america and the caribbean have the second highest urbanization level in the world. for every 13 persons there are in the region, 10 of them live in cities (78.3% in 2007) ; and 20 of the region 's largest cities are home to nearly 20% of its population. jamaica is a predominantly afro - caribbean society, 75% black and 13% mixed, with a class structure based on land and wealth rather than race. while there is much industrialization and modernization, customs, cultural and social habits of several centuries are common - place. jamaica is the third largest english speaking caribbean island (total area of 10,991 km) with an estimated population of 2.7 million (2007). the country is classified into three geographical planes (cornwall, middlesex and surrey) and has 14 parishes. cornwall covers the western belt which includes parishes such as westmoreland, hanover, st. middlesex constitutes the middle proportions of the island with parishes such as clarendon and st. another classification is cities (urban areas) which constitute 27.3% of the population, peri - urban 30.2% and rural areas, 42.5% in 2007. in 2007, jamaica 's poverty rate was 9.9%, and this was 15.3% in rural areas, 4.0% in peri - urban areas and 6.2% in urban areas. furthermore, the mean annual per capita consumption for country was us $ 2,059.91 while it was us $ 2,736.60 for urban dwellers, us $ 2,231.04 and us $ 1,513.17 for rural jamaicans. statistics for the same period showed that the sex ratio of the population was 97 per 100 and 84 per 100 for older ages (60 years and over). this indicates that there are marginally less men than women in the population, and an even greater feminization at older ages. it was estimated that 10.9% of the population was 60 years and over which indicates an ageing population that began in the 1960s[24 ], 28.3% under 15 years, and 53.5% in the reproductive years of 15 to 49 years. women comprised 50.7% of the population and elderly women accounted for 13.0% compared with 11.4% for elderly men. it was also found that 46.6% of household heads were women ; life expectancy at birth for women was 77.1 years (2002 to 2004). the unemployment rate in 2007 was 65.4% for women[35 ] with women participation rate being 55.4% compared to 72.9% for men. fifty - three percent of women in the poorest quintile were heads of households compared to 46.9% of men. statistics revealed that the mean annual consumption for male headed - household was us $ 2,188.03 compared to us $ 1,892.92 for female headed - household. it is well established that health status is determined by socio - physiological factors (age, income, education, culturalization, crime and negative psychology) and that lifestyle practices also account for good (or poor) health status[68 ]. women 's health therefore is intricately a mix of socio - physiological response or outlay and is expressed through behavior relating to culture, religion, and legal norms. although recent attention has been directed towards exploring the ramifications of women 's health in the western hemisphere including the caribbean, an extensive review of the literature revealed that only a few studies have examined health determinants of women in the caribbean, including jamaica. using secondary data from a stratified probability survey on political culture of 1,338 respondents, bourne extracted a sample of 722 women in investigating the determinants of quality of life of women in jamaica. the study showed that the mean quality of life of jamaican women was moderately high (6.8 out of 10 ; sd = 1.7). six variables (social class, employment, income, religiosity, governance of the nation and interpersonal trust) accounted for 18.5% of the variability of quality of life. eldermire investigated the general life situation of elderly jamaican women and found that their life situations were on an average good. many economic indicators showed that women are disadvantaged in jamaica and the wider caribbean when compared to men[911 ]. in 2007, statistics for jamaica showed that the mean consumption per capita on food was us $ 2,378.39 for male head - household compared to us $ 1,898.56 for women. studies have showed that women seek more health care then men[1214 ], and that this commences in earlier childhood. therefore, the similarities and dissimilarities based on area of residence of women was examined (via econometric models) in order to determine the composition of women 's health status. econometric models such as bourne 's health determinant model denotes that an individual 's health is a function of cost of medical care and other factors such as : educational level, age, the environment, gender, marital status, area of residence, psychological status which include positive and negative affective status, occupancy per room, home tenure, property, and crime and victimization. bourne 's work modeled health determinants of jamaicans, and with the aforementioned issues surrounding women as were outlined above, a study on jamaicans is not necessarily providing an understanding of women 's health and significant of particular factors determining their health status or the disparity in health status of women based on the 3 geographic sub - regions in the island. this study sought to examine 1) the consumption expenditure of women in the different income quintiles (or social classes) ; 2) health insurance coverage, and visits to health care facilities by area of residence ; 3) health status by age cohorts (i.e. young, other adults and elderly women) ; 4) diagnosed illness by age cohorts ; diagnosed illness by area of residence ; 5) the health status of women in jamaica using a modification of bourne 's health determinant model ; 6) the health status of women in and sub - regions namely urban, peri - urban and rural residence ; and 7) the strength of those factors which affect health status of women in the nation and the sub - regions. the sub - sample for the current study was all 8,541 women (ages of 15 to 100 years) extracted from a nationally representative cross - sectional survey of 25,018 jamaicans, the jamaica survey of living status (jslc, 2002). the design was two - stage stratified random sampling, where there was a primary sampling unit (psu) and a selection of dwelling from the primary units. the psu is an enumeration district (ed), which constitutes a minimum of 100 dwellings in rural areas and 150 in urban areas. this means that the country was grouped into strata of equal size based on dwellings (eds). based on the psus, a listing of all the dwellings was made and this became the sampling frame from which a master sample of dwelling was compiled which provided the sampling frame for the labor force. this study used jslc 2002 which was conducted by the statistical institute of jamaica (statin) and planning institute of jamaica (pioj) between june and october 2002. the researchers selected this survey because it was the second largest sample size for the survey in its history (since 1988 to 1998), and in that year, the survey had questions on crime and victimization, and the physical environment unlike previous years. a self - administered questionnaire was used to collect the data, which was stored and analyzed using spss for windows 16.0. the questionnaire was modeled from the world bank 's living standards measurement study (lsms) household survey. there were some modifications to the lsms as jslc was more focused on policy impacts. the questionnaire covered questions such as : socio - demographic, economic and wealth, crime and victimization, social welfare, health status and services, nutrition, housing, immunization of infants and physical environment. descriptive statistics such as mean, standard deviation (sd), frequency and percentage were used to analyze the socio - demographic characteristics of the sample. chi - square was used to examine association between non - metric variables ; an analysis of variance (anova) was used to evaluate the relationships between metric and non - dichotomous categorical variables. logistic regression examined the relationship between the dependent variable and some predisposed independent (explanatory) variables because the dependent variable was a binary one (self - reported health status, with 1 if good health status was reported and 0 if poor health). results were presented using unstandardized b - coefficients, wald statistics, odds ratio and confidence interval (95% ci). the predictive power of the model was tested using omnibus test of model and hosmer and lemeshow was used to examine goodness of fit of the model. the correlation matrix was examined in order to ascertain whether autocorrelation (or multi - collinearity) existed between variables. based on cohen and holliday, correlation can be low (weak), from 0 to 0.39 ; moderate, 0.4 to 0.69, and strong, 0.7 to 1.0. this was used to exclude (or allow) a variable in the model as any variable that had at least moderate correlation was excluded from the final model. wald statistics was used to determine the magnitude (or contribution) of each statistically significant variables in comparison with the others, and the odds ratio (or) for interpreting each significant variables. multivariate regression framework was used to assess the relative importance of various demographic, socio - economic characteristics, physical environment and psychological characteristics in determining the health status of women in jamaica. secondly, the dependent variable is a binary dichotomous one which has enabled the use of this statistic technique to be utilized in the past to do similar studies. having identified determinants of health status from previous studies, using logistic regression techniques, final models were build for women in general as well as for each geographical sub - regions (rural, peri - urban and urban areas) using only those predictors that independently predict the outcome. equation 1 is a modification of bourne health determinant model which was previously used to determine the health status of the elderly in jamaica. hi = (wi, hhi, pmci, ci, mri, ari, edi, ssi, cri, (nai, pai), mi, fi, chi, at, ai, hii, lli, eni, yi, vi,i) (1) the health status of person i, hi, is a function of wi, the two wealthiest quintiles of person i with 1 if yes or 0 for the two poorest quintiles ; hhi, household head of person i, with 1 if yes or 0 if otherwise ; pmci, cost of medical care of person i, in united states (us) dollars ; ci, average consumption per person in household, in us dollars ; mri, is marital status of person i ; ari, area of residence of person i ; edi, educational level of person i ; ssi, having social support of person i with 1 if yes or 0 if no ; cri, crowding of person i, in numbers ; (nai, pai), psychological status which is the summation of negative affective status of person i, na i where values are in continuous number ; pai, positive affective psychological status of person i, where values are in continuous numbers ; mi, number of men in household of person i ; fi, number of women in household of person i ; chi, number of children below the age of 14 years of person i ; at, asset owned of person i, in continuous numbers ; ai, age of person i, in continuous numbers ; hii, of private health insurance (proxy) ; lli, living arrangement where 1 is living with family members or relative, 0 if otherwise ; eni, physical environment of person i, with 1 if affected by flood, landslides, soil erosion or 0 if not affected ; yi, average income per person in household (this variable is proxied by total expenditure) ; vi, crime of person i, where values are continuous numbers, and i is the residual error. self - reported health status is self - assessed illness (cold, diarrhea, asthma attack, hypertension, diabetes mellitus or any other illnesses) reported by respondents in the last 4-weeks of the survey period. good health status is a dummy variable ; where 1 is good health (not reporting an ailment, injury or dysfunction) and 0 is poor health (self - reported illness, injury or ailment). household crowding is the average number of persons living in a room excluding kitchen, bathroom and verandah. physical environment is the summation of responses reported by respondents on suffering landsides ; property damage due to rains, flooding ; or soil erosion in the last 4-weeks. psychological conditions are the psychological state of an individual, and this is sub - divided into positive and negative affective psychological status. positive affective psychological status refers to the number of responses that are hopeful and optimistic about the future and life generally. negative affective psychological status refers to the number of adverse events occurred to the respondents over the last 4-week period. age is the number of years lived, which is also referred to age at last birthday. young are ages 15 to 30 years, other adults 31 to 59 years, and elderly 60 years and over. crime and victimization index (crime index) measures the number of cases and severity of crimes committed against a person or his / her family members but not against property. social support (or network) denote different social networks with which the individual has or is involved (1= membership of and/or visits to civic organizations or having friends that visit ones home or with whom one is able to network, 0 = otherwise). living arrangement denotes whether the individual is living alone or with family, friends or associates ; where 1 = living with family members or relatives, and 0 = otherwise. self - reported health status is self - assessed illness (cold, diarrhea, asthma attack, hypertension, diabetes mellitus or any other illnesses) reported by respondents in the last 4-weeks of the survey period. good health status is a dummy variable ; where 1 is good health (not reporting an ailment, injury or dysfunction) and 0 is poor health (self - reported illness, injury or ailment). household crowding is the average number of persons living in a room excluding kitchen, bathroom and verandah. physical environment is the summation of responses reported by respondents on suffering landsides ; property damage due to rains, flooding ; or soil erosion in the last 4-weeks. psychological conditions are the psychological state of an individual, and this is sub - divided into positive and negative affective psychological status. positive affective psychological status refers to the number of responses that are hopeful and optimistic about the future and life generally. negative affective psychological status refers to the number of adverse events occurred to the respondents over the last 4-week period. age is the number of years lived, which is also referred to age at last birthday. young are ages 15 to 30 years, other adults 31 to 59 years, and elderly 60 years and over. crime and victimization index (crime index) measures the number of cases and severity of crimes committed against a person or his / her family members but not against property. social support (or network) denote different social networks with which the individual has or is involved (1= membership of and/or visits to civic organizations or having friends that visit ones home or with whom one is able to network, 0 = otherwise). living arrangement denotes whether the individual is living alone or with family, friends or associates ; where 1 = living with family members or relatives, and 0 = otherwise. self - reported health status is self - assessed illness (cold, diarrhea, asthma attack, hypertension, diabetes mellitus or any other illnesses) reported by respondents in the last 4-weeks of the survey period. good health status is a dummy variable ; where 1 is good health (not reporting an ailment, injury or dysfunction) and 0 is poor health (self - reported illness, injury or ailment). household crowding is the average number of persons living in a room excluding kitchen, bathroom and verandah. physical environment is the summation of responses reported by respondents on suffering landsides ; property damage due to rains, flooding ; or soil erosion in the last 4-weeks. psychological conditions are the psychological state of an individual, and this is sub - divided into positive and negative affective psychological status. positive affective psychological status refers to the number of responses that are hopeful and optimistic about the future and life generally. negative affective psychological status refers to the number of adverse events occurred to the respondents over the last 4-week period. age is the number of years lived, which is also referred to age at last birthday. young are ages 15 to 30 years, other adults 31 to 59 years, and elderly 60 years and over. crime and victimization index (crime index) measures the number of cases and severity of crimes committed against a person or his / her family members but not against property. social support (or network) denote different social networks with which the individual has or is involved (1= membership of and/or visits to civic organizations or having friends that visit ones home or with whom one is able to network, 0 = otherwise). living arrangement denotes whether the individual is living alone or with family, friends or associates ; where 1 = living with family members or relatives, and 0 = otherwise. the sub - sample consisted of 8,541 respondents (ages 15 to 100 years), with a mean age of 40.1 years (sd 19.29 years). of the sub - sample of respondents, 65.2% were never married, 24.7% married and 10.1% divorced, separated or widowed. the mean annual consumption per person per household was us$762.35 (sd us$917.81) (rate in 2002 : 1us$ = ja$50.97) with the maximum consumption being us$136,822.08. moreover, 36.6% of the sample was in poverty with 17.5% being below the poverty line (i.e. poorest poor) compared to 44% who were in the two wealthiest quintiles, of which 23% were in the wealthiest quintile (table 1)., it was found that 21% of rural women were 60 + years compared to 15.2% of peri - urban women and 15.1% of urban women (table 1) 17.2% reported poor health status (suffering from an illness, ailment, or injuries) in a 4-week period of the survey, with 82.8% indicated good health status. of the 17.2% of women who reported poor health status, 6.5% visited public - private health care facilities for treatment. of this 6.5%, 6.3% visited public health care institutions compared to 0.2% who visited private health care facilities, 66.1% of those who had reported an illness in the 4-week survey period bought the prescribed medication, with 40.9% of them took the medication in full. some 5.6% of the sample reported that they resided alone (living arrangement), and 57.8% indicated no social support. based on table 1, there was a significant statistical correlation between good health status and area of residences rural women recorded the lowest health status among all women of the three geographic areas (table 1) : rural women recorded the least good health status (75.5%) compared to 77.0% of urban women and 81.8% of semi - urban women. more crowding was in the rural sample (1.9 1.3 persons per room) compared to 1.81.3 persons per room in peri - urban and urban areas a statistical difference was found between area of residences and mean number of visits made to health care facilities p = 0.023 : 1.6 1.1 days for rural women ; 1.7 1.3 days for peri - urban women and 2.0 2.7 days for urban women. p 0.05). however, a statistical difference existed between health insurance coverage andarea of residents in jamaica (= 24.4, p 0.05). however, a statistical difference existed between health insurance coverage andarea of residents in jamaica (= 24.4, p 0.05). however, a statistical difference existed between health insurance coverage andarea of residents in jamaica (= 24.4, p < 0.001), with there being a weak correlation (contingency coefficient = 0.167). of those who responded the least number of rural women had health insurance coverage (7%) compared to 16.5% of peri - urban women and 18.7% of urban women. health insurance, self - reported good health status by area of residence (in %) table 4 revealed that there was a negative statistical correlation between self - reported good health status and age group (= 820.397, p < 0.001), with the association being a moderate one (cc = 0.301). the findings indicated that 7.5% of young respondents reported a poor health status compared to 15.6% other adults and 40.9% of elderly respondents indicating the substantial erosion of good health status of women as they age. self - reported good health status by age group. of the 1,417 respondents who reported an illness a statistical correlation was found between illness being recurring and age group of respondents (= 413.247, p < 0.001), with relationship between the two aforementioned variables being a moderate one (cc = 0.473). based on table 5, diabetes mellitus, hypertension and arthritis were found to be more an elderly chronic illness than the other age sub - samples. simply put, as women age, chronic illness such as diabetes mellitus, hypertension and arthritis increased, with arthritis having the greatest increase in elderly compared to middle age. diagnosed with recurring illness by age group when a correlation was performed between the duration of illness (how long did the last episode of illness last ?) and area of residence, a relationship was found between the two variables (f = 7.513, p < 0.001). on an average, the mean duration for the illness was 11.09 days (sd 10.742 days), 95% ci : 10.51 - 11.67 days. rural residents reported suffering from illness for a mean of 11.74 days (sd 10.691 days), 95% ci : 11.04 - 12.44 days which was not statistically different from mean number of days reported by peri - urban residents, 10.50 days (sd 10.573 days), 95% ci : 9.21 - 11.79 days, p < 0.001. the mean number of reported days in which rural and urban resident were ill [8.20 days (sd 10.879 days), 95% ci : 6.44 - 9.96 days, p = 0.233 ]. there was no statistical difference between the mean duration of illness for peri - urban and urban residents, p = 0.091. the findings revealed that health status by area of residence had no statistical correlation, p= 0.051 (table 6). despite the no statistical difference, in excess of 30.0% in each area of residence suffered from hypertension, 16% from diabetes mellitus, 13% other and 5% arthritis. of the 1,434 respondents who indicated poor health status, 93.0% said that these were diagnosed as recurring and acute., one was excluded because the correlation coefficient between it (consumption) and income was 0.68. nine variables were found to be determinants of good health status of women in jamaica (table 7). the model had a statistical significant predictive power (= 1,249.19 p < 0.001 ; hosmer and lemeshow goodness of fit = 5.606, p = 0.691). in addition, it was revealed that overall 84.9% (n = 7,251) of the data were correctly classified : 97.7% of those who indicated good health status and 37.8% of those who indicated poor health status. logistic regression of the general good health status of jamaican women by some explanatory variables, n = 8,541 on examination of data (table 7), it was revealed that private health insurance was the most significant factor predicting good health status of women in jamaica (or = 27.5, 95% ci = 21.1 - 35.8) followed by assets owned (or = 1.1 ; 95% ci = 1.0 - 1.0) ; age of the respondents (or = 0.9, 95% ci = 0.9 - 0.9) ; positive affective psychological status (or = 1.1, 95%ci = 1.0 - 1.1) ; number of men in the household (or = 0.9, 95% ci = 0.8 - 1.0) ; income (or = 1.000, 95% ci = 1.000 - 1.000) ; marital status married (or = 1.2, 95% ci = = 1.1 - 1.6) ; crowding (or = 0.9, 95%ci = 0.8 - 1.0) ; area of residence and negative affective psychological status (or = 1.0, 95% ci = 0.9 - 1.0). all the factors explain 36.0% of the variability in health status of women in jamaica. income positively influences good health status of women (or = 1.0, 95%ci 1.0 - 1.0) (table 7). the current work has found that women who have health insurance were 27.5 times likely to report good health than those who do not have health insurance coverage. rural women were less likely to report good health status compared to urban women ; and that peri - urban women were 1.1 times more likely to report good health status compared to urban women. married women were 1.2 times more likely to report good health status with reference to those who were never married. women who were experiencing greater positive affective psychological conditions were 1.1 times more likely to report good health status ; and women who experienced greater negative affective psychological conditions were 0.03 times less likely to report good health status. the older women get, they are 0.19 times less likely to report good health status (table 7). using a sub - sample of 4,962 rural residents, 20 initial predisposed explanatory variables were tested to ascertain factors and degree of significance of each factor (p < 0.05), one was omitted (consumption, because the correlation coefficient between it and income was 0.68). of the 19 predisposed variables that were examined in the initial model, nine of them explained 38.6% of the variability in health status of rural women in jamaica (table 8). logistic regression of the good health status of rural - jamaican women by some explanatory variables, (n : 3,498). the model had a statistical significant predictive power (= 884.476 p < 0.001 ; hosmer and lemeshow goodness of fit = 8.498, p = 0.386). overall, 84% (n = 2,940) of the data were correctly classified : 96.8% (n = 2,593) of those who had indicated good health status and 42.3% (n = 347) of those with poor health status. continuing, table 8 revealed that health insurance was the most influential factor determining the good health status of rural women in jamaica (or = 25.0, 95% ci = 18.0 - 34.9) followed by assets owned (or = 1.0, 95% ci = 1.0 - 1.1) ; age (or = 0.9, 95% ci = 0.8 - 0.9) ; number of men in household (or = 0.8, 95% ci = 0.7 - 0.9) ; positive affective psychological status (or = 1.1, 95% ci = 1.0 - 1.1) ; educational attainment secondary and post - secondary level education (or = 1.4, 95% ci = 1.1 - 1.8) ; social support (or = 1.3, 95% ci = 1.1 - 1.6) ; marital status married (or = 1.4, 95% ci = 0.8 - 2.3), and lastly income (or = 1.0, 95% ci = 1.0 - 1.0). the current findings revealed that income plays the least role in determining good health status of rural women ; women with health insurance are 25.0 times more likely to have good health status than those without health insurance coverage ; married rural women are 1.4 times more likely to report good health status with reference with those who were never married ; those rural women with social support were 1.3 times more likely to report good health status compared to those who did not have social support, and as rural women become older, they are 0.102 times less likely to report good health status. more males in the household will reduced the good health status of rural women (or = 0.83, 95% ci = 0.75 - 0.93) : indicating that more males in a household will decrease rural women 's good health status by 0.17 times compared to less males in the household. with regard to peri - urban areas in jamaica, a sub - sample of 2,283 respondents were used to establish the good health status model. this model had a statistical significant predictive power (= 285.807 p < 0.001 ; hosmer and lemeshow goodness of fit = 7.226, p= 0.512). upon reviewing the classification table, overall, 88.2% of the data were correctly classified : 98.8% of those classified as having had good health status and 35.5% of those who had indicated poor health status (table 9). logistic regression of the good health status of peri - urban - jamaican women by some explanatory variables, n=2,283 of the 19 predisposed variables that were tested in the initial model, six factors accounted for 36.6% of the variability in good health status of women in peri - urban area in jamaica (table 9). the factors that predict good health status of peri - urban jamaican women in descending order were health insurance (or=57.7 ; 95%ci : 29.8 - 111.7) ; asset ownership (or=1.0 ; 95%ci : 1.0 - 1.0) ; age of respondents (or=0.9 ; 95%ci : 0.9 - 1.0) ; number of men in household (or=0.8 ; 95%ci : 0.6 - 1.0) ; negative affective psychological status (or=0.9 ; 95%ci : 0.9 - 1.0) ; positive affective psychological status (or=1.1 ; 95%ci : 1.0 - 1.1) and consumption (or=1.0 ; 95%ci : 1.0 - 1.0). the findings revealed that income contributed the least to good health status of peri - urban residents. another interesting finding of the current study is peri - urban women who had health insurance coverage is 57.7 times more likely to report good health status compared another who do not have this coverage. the older peri - urban women get, they are 0.1 times less likely to record good health ; more men contributes 0.2 times less to their good health ; the more asset they own this increased their good health by 1.0 times more another with less assets and that the more they are positive, this direct increase their good health status and the converse is the case for those with greater scores in negative affective psychological conditions. a sub - sample of 1,296 women of urban jamaica was used to build the good health status model. the model had a statistical significant predictive power (model chi - square = 263.08 p < 0.001 ; hosmer and lemeshow goodness of fit = 8.481, p = 0.388). upon observation of classification, overall, 83.4% of the data were correctly classified : 97.1% of those who had indicated good health status and 31.8% of those who reported poor health status. of the 19 predisposed variables that were examine in the initial model, six of them accounted for 30.7% of the variability in good health status of urban women in jamaica (table 10). health insurance had the most impact on good health status of urban women (or = 22.2 ; 95%ci : 11.3 - 43.7) followed by in descending order are age of respondents (or = 0.94 ; 95% ci= 0.9 - 1.0) ; two wealthiest quintiles (or = 1.8 ; 95%ci : 1.1 - 2.9) ; asset ownership (or=1.0 ; 95%ci : 1.0 - 1.0) ; positive affective psychological status (or = 1.1 ; 95%ci : 1.0 - 1.1) and number of men in the household (or = 1.2 ; 95%ci : 1.0 - 1.5). logistic regression of the good health status of urban - jamaican women by some explanatory variables, n = 1,296 embedded in the current findings are that urban women with health insurance coverage were 22.2 times more likely to record good health status compared to those who do not have health insurance coverage ; the older urban women get, they are 0.1 times less likely to record good health status and that more men in urban household contributed 1.2 times more likely to good health status. concomitantly, urban women in the two wealthiest quintiles were 1.8 times more likely to report good health status with reference to women in the poor - to - poorest 20%. the findings of the current study showed that poverty for rural women was 2.4 times more than that for urban women and 1.9 times more than that for peri - urban women. an interesting finding is that on average urban women received income which was 1.6 times more than rural women and 1.2 times that of peri - urban women. rural women 's consumption expenditure was 45% less than that for urban women and 31% less than for peri - urban women. another fundamental disparity was in education as 161 rural women for every 100 urban women had at most primary education and the ratio was 127 to 100 rural women for every peri - urban woman respectively. those socioeconomic disparities between sub - regions in jamaica, accounted for rural women having the lowest good health status. those with poor health status were more likely to report having hypertension followed by diabetes mellitus, and the rates of these two chronic diseases were similar in the three geographical locations. hypertension (43.2%) and diabetes mellitus (23.6%) was more prevalent in the elderly than in the other adult and young respondents. only 7.5% elderly had private health insurance coverage and the mean consumption expenditure for the poorest was 13% of that for those in the wealthiest income group, supporting the that poverty was a rural phenomenon and that this significantly retards consumption pattern of rural women in jamaica. a critical finding of this study was that health insurance coverage accounted for the most influence on good health status of women in the 3 sub - regions ; but that it had the most impact on good health for peri - urban women and the least for urban women. another important finding was that income played a secondary role to factors such as health insurance, age of respondents and other psychosocial factors. education did not explain good health status for peri - urban or urban women ; and that more males contributed positively to the health status of urban women and negatively for women in the two other sub - regions. when health status of jamaican women was deconstructed into area of residence, some major similarities were observed among them. the study revealed that the most significant factor predicting health status of women in jamaica across the three sub - regions was health insurance coverage. embedded in this study is the fact that health insurance aids in the health care - seeking behavior of women ; but that it is more so for peri - urban women. for peri - urban women, those with health insurance coverage were approximately 60 times more likely to report good health status than those without this coverage, suggesting that lifestyle practices of these women account for their health status. this finding can be supported by the fact that women in peri - urban zones visited health care practitioners more than that of rural but less than urban women. the resources regarding health care decision - making could be health insurance or monetary resources. health insurance is important for access to health care and being uninsured significantly reduces access to health services and substantially increases health problems. uninsured persons with poor health status are much more likely than their insured counterparts to report that they or a family member did not receive doctor 's care or prescription medicines. shi reported that income was the most significant predictor of lack of health insurance coverage, which explains why rural women in this study had the least health insurance coverage, the lowest income and consumption and the lowest good health status. low - income adult women tended to have lower health status and uninsured women tended to have problems accessing health care services, which are concurred by this study. mead noted that low - income women were less likely to have health insurance, while they were more likely to have health care access problems, chronic illness and lower overall health status than their richer counterparts. in jamaica, life of jamaica and blue cross jamaica limited are the only total health insurance companies catering to the widest cross- section of jamaica 's population. these companies offer a wide range of health insurance products to best suit the needs of clients from individuals, students, executives, associations and companies. this study revealed that an overall 11 out of every 100 of sample had health insurance coverage. in terms of geographical areas, 7 out of every 100 rural women, 17 out of every 100 peri - urban women and 19 out of every 100 urban women possessed health insurance coverage, which reinforced the aforementioned findings that income plays a critical role in health insurance coverage and health status. these results are not in agreement with findings from a study by wong and diaz, who found that almost three - quarters of the urban population (73%) have coverage compared to 38% of those in rural areas ; women showed a slightly higher and similar coverage (56%) than men (52%). postulated that rural residents were more likely to be uninsured than urban residents (17.8% versus 15.3%), and that rural respondents were more likely than urban counterparts to report having deferred health care because of cost (15.1% versus 13.1%). in studies done in rural areas, the probability of a worker being covered by an employer - sponsored insurance plan is lower than for urban workers ; and therefore account for the health insurance disparity between the 3 sub - regions. the authors found that small firm size and low wages in rural areas are the main reasons for this difference. in this study 7.5% of women residents in all three regions reported having health insurance coverage, which is similar to 7.6% reported in a previous study. hence this justifies why rural women recorded the least number of visits to health care practitioners ; because health care cost will be substantially an out of pocket expense that they would be unable to afford. unemployed women were reported to have poorer mental and physical health status than employed women. however, low - income women often experience conflicts between their poor health status and lack of resources. wagstaff and doorslaer reported that an individual 's absolute income affected his / her mortality. these authors supported rodgers argument that the relationship between an individual 's health and income is concave. this means that each additional dollar of income raises an individual 's health status, but the increase gets smaller as income increases and justifies why income plays a secondary role to health insurance coverage. another fact that this study highlights is the increased indirect role that income plays, which is weaker than it direct role. an individual or household 's position can decline or improve over short periods according to changing circumstances such as illness, unemployment, eviction or other events. the causes of urban poverty are interlinked, stemming from such factors as employment insecurity, sub - standard housing, poor health, low levels of income generation, vulnerability to market shocks, and limited education[3639 ]. according to hinrichson, most urban poverty does not result from a lack of jobs, but from a lack of well - paying, steady jobs. unemployment rates are generally below 15% in most developing country cities, but wage rates are depressed in the formal sector, and many are self - employed in the informal sector. average incomes in rural areas are often lower than in urban areas. in rural areas, poverty leads to health - related problems not only for single mothers but also for mothers with partners, while in urban areas this problem is usually observed in single mother - headed households. rural americans are more likely to be poorer and less healthy than their urban counterparts, which is also the case in jamaica. this study goes farther as it found that urban women in the two wealthy quintiles were 1.8 times more likely to report good health, and this was not the case for rural or peri - urban women. although social class (i.e. wealthy class) is a predictor of good health status of urban women, once again peri - urban women had the greatest good health status. this indicates that after certain sum of wealth, income adds increasing less to good health status. income therefore will add substantially more to the good health status of poor women than it is likely to increase good health for middle and wealthy women 's health status. non - communicable diseases such as cardiovascular diseases, cancer, chronic respiratory diseases and diabetes mellitus are rapidly increasing problems for the socially disadvantaged. in this study, findings of diagnosed chronic health conditions show patterns of worse health status among elderly women living in rural areas. the prevalence of hypertension and diabetes mellitus among respondents in the three regions were similar. however, reports of cancer, influenza, asthma and arthritis are low compared with hypertension and diabetes mellitus. the self - report of disability and chronic status is higher for older than younger residents. rural women tend to have higher rates of chronic status of hypertension, arthritis, spinal disorders, bursitis, hearing, and visual impairments than their urban counterparts. furthermore, when seeking medical services, they are more likely to be ill, hospitalized than women in urban areas. in this study, the duration of sickness in women residents in rural areas was longer than their counterparts in urban and peri - urban areas. in addition, health care facilities in rural areas are unfavorable compared to non - rural areas due to limited medical resources and shortage of physicians. we can deduce from current study that with rural women having less economic resources and lowered visits to health care facilities, they would be using more home remedy or non - traditional healers to treat their ill - health. hence this would account for an aspect of pre - mature mortality of these women. in this study a higher percentages of the elderly in the rural areas reported poor health status. found that residents in rural area were more likely to report fair to poor health status than were residents of urban counties (19.5% versus 15.6%). rural adults were more likely to report having diabetes mellitus than were urban adults (9.6% versus 8.4%). the authors also found that urban residents are more likely to use preventative care than their rural counterparts, but there seemed to be no differential use of doctor visits or hospitalizations. according to brenzel. chronic diseases such as diabetes mellitus and hypertension are either undetected or medically untreated, or in the case of those who do receive treatment, the clinical management of the status is poor. in jamaica, available hospital records show that between 1990 and 98 showed that twice as many women than men were admitted for hypertension and diabetes mellitus. the predominance of women with chronic disease visiting health care facilities (82%) is in keeping with the experience of other public health areas for chronic diseases. in addition, women are more likely to report an illness ; with 15% women compared to 12% men reported suffering from an illness or injury in the previous four weeks in 1991. the gender gap is widest for hypertension with twice as many women as men (12% vs. 6%) reporting having the disease. in the current study, the researchers found that diabetes mellitus for elderly women was 11.2 times more than that for young women and 1.6 times more than for middle aged adult women. continuing, hypertension in elderly women was 6.0 times more than that in young women and 1.2 times more than in middle aged adults. arthritis was 10.8 times more in elderly women compared to young women and 4.0 times more in elderly than in middle aged adult women. on the other hand, acute dysfunctions such as cold, diarrhea and asthma decreases as women become older and the same was recorded for unspecified illness. the more highly educated are likely to better understand the importance of proper health care. ross and miroswky reported that education significantly improved self - reported health and physical functioning. in addition, knowledge of and experiences with health care were found to affect an individual 's health care behavior more so than age. education is strongly associated with the level of health service utilization, the type of provider, the choice of private versus public provider, dietary and child - feeding, and sanitary practices. however, studies have found that it is not just general education, but also health - specific knowledge that is important. barrera and caldwell argued that educated mothers are more likely than the uneducated ones to take advantage of modern medicine and comply with recommended treatments because education changes the mother 's knowledge and perception of the importance of modern medicine in the care of her children. in contrast, rosenzweig and schultz viewed women schooling and health care services as partial substitutes for information regarding knowledge of diseases, treatment of illness and child - care practices, and hypothesized that the effect of education on child health becomes less important as access to public health care services improves. presumably, in areas where such services are readily accessible, they are used by both educated and uneducated women, and thus the advantage conferred by schooling on health outcomes is narrowed. it is unlikely that the observed effects of maternal education on child - health outcomes simply reflects health knowledge and habits acquired in school, although they may play some role. education could thus influence a woman 's beliefs about disease causation and cure and the value she places on modern medicine. they found that rural women adopted more health practices overall than their urban counterparts, and younger women in both groups exhibited more awareness of health promotion. in addition, they found that there is higher utilization of doctors visits and preventative care among persons with the highest level of education and in the highest income groups. however, higher education or income seems to have no association with differential use of hospitalizations. education was not found to be significantly correlated with good health status of jamaican women. however, this was not the case for rural women. an irony that lies in this study is the fact that there is a health disparity between women who have had a most primary education compared to those with secondary education, but there was none between tertiary and at most primary education for rural women. human emotions are a mix of not only positive status but also negative factors. hence, depression, anxiety, neuroticism and pessimism are seen as measures of the negative psychological status that affect subjective wellbeing. negative psychological status (loss of family members, friends etc) affect subjective wellbeing in a negative manner (guilt, fear, anger, disgust and that the positive factors influence self - reported wellbeing in a direct way. this was concurred in a study conducted by fromson and other researchers. in this study, negative affective psychological status was inversely affect good health status of jamaican women, and the opposite was true for positive affective psychological conditions. on disaggregating the good health status by the 3 sub - regions, only positive affective conditions influence good health status of urban women while positive and negative affective psychological conditions determined good health status for rural and peri - urban women. rural residents are more likely than their urban counterparts to experience negative circumstances such as unemployment, lower rate of health insurance coverage, poor health status, and lowered consumption and earnings and this retards their health care seeking behaviors and further becomes challenges for their health. found that an individual 's psychological state influences his / her health status, which this study concurs with. people in rural areas are more likely to have characteristics that are strongly associated with depression, poor health status, chronic diseases and poverty. found the prevalence of depression were slightly higher in residents in rural than in urban areas. depression is subsumed in negative affective psychological condition, and so this work agrees with the literature. the current study however found that there is no significant statistical difference between the negative psychological state of peri - urban and urban women in jamaica as well as between positive affective psychological conditions and urban and urban women. embedded in these findings are the higher over affective conditions of peri - urban women, and this fact accounts for peri - urban women having the greatest health status. some limitations must be considered in interpreting these results as this study was completely based on data reported by interviewed residents, and of course, persons do not always answer factually in interview surveys. interviewers and supervisory staff were aware of this problem, and interviewer instructions included directions for probing participants on these issues. however, the strength of the study 's sample design and data collection procedures compensated for these limitations. the findings revealed that rural women had the least good health, while peri - urban women recorded the greatest self - reported good health. concurrently, rural women were older ; poorer ; received the lowest income per person ; had the greatest percentage of primary level education ; recorded the highest negative affective psychological conditions ; were the least likely to have health insurance coverage and they recorded the lowest consumption expenditure. this study therefore provides a comprehensive understanding of health of women in jamaica and the 3 sub - regions as well as the disparity in socio - demographic correlates of health based on the different geographical regions. concomitantly, poverty continues to reduce the self - rated health status of women and while they are living 6 years longer than men, this does not mean that we neglect the reality that poverty is eroding their health status. | background : a comprehensive review of the literature revealed that less information is available in literature on health status of women, and health status of women in 3 geographical zones in jamaica.aims:this study examined data on the health status of women in jamaica in order to provide some scientific explanation of those factors that account for their health status ; and differences based on area of residence.materials and methods : the sub - sample for the current study was 8,541 women ages of 15 and 100 years extracted from a national survey of 25,018 respondents. stratified random sampling technique was used to draw the sample. data were stored, retrieved and analyzed using spss 16.0. descriptive statistics were used to provide background information on the subsample, and logistic regressions were utilized to model health statuses.results:rural women had the lowest health status (or = 0.819, 95% ci = 0.679 - 0.989) among all women (peri - urban or = 1.054, 95% ci = 0.842 - 1.320 ; urban or = 1.00) and that they were the least likely to have health insurance coverage. health insurance was the critical predictor of good health status of women in jamaica, and this was equally the same across the 3 geographic areas ; and that married women were 1.3 times more likely (or 1.3, 95 ci = 1.036 - 1.501) to report good health compared to those who were never married.conclusion:this study provides an understanding of women 's health status in jamaica as well as the disparity which correlates based on the different geographical regions. |
the treatment of septic patients emphasizes the optimization of oxygen utilization by tissues through maintenance of an adequate oxygen supply, minimizing the cellular dysfunction progression. blood cell transfusion is frequently used with the intention of augmenting arterial oxygen content and its utilization by the tissues. blood cell transfusion efficacy in septic patients is still not convincingly demonstrated and previous studies report conflicting results. when oxygen consumption is calculated by fick 's method, it is demonstrated to have increased following red blood cell (rbc) transfusion. discrepancies of results may be explained by the mathematical coupling of data used to calculate both oxygen transport and consumption. blood cell transfusion has been implicated with a deterioration in gastric oxygenation when it was measured by gastric intramucosal ph (phi), determined by tonometry. this deterioration has been attributed to prolonged storage time of transfused red blood cells which may lead to cellular stiffening, decreasing their deformation capacity and exacerbating the rheological abnormalities observed in sepsis. we conducted an interventional, prospective, randomized, controlled study to evaluate the immediate consequences of rbc transfusion on hemodynamic parameters and on systemic and gastric oxygen consumption, using indirect calorimetry and gastric tonometry. this study was performed in a general intensive care unit, in a private tertiary hospital, from january to december 1996 after approval by the internal ethics committee. as a result of the clinical condition of patients septic patients with hemoglobin concentration values less than 10 g%, who were being mechanically ventilated, with an oxygen inspired fraction (fio2) less than 60% and with pulmonary artery catheter monitoring (swan - ganz ccombo cco / svo2 : baxter, edwards critical care division, irvine, usa) were included. exclusion criteria included : age 80 years, pregnant women, patients on dialysis, and patients who had recently undergone a gastrointestinal surgical procedure and established septic shock. a tonometry probe device (tonometrics : datex, helsinki, finland) was inserted and its position was confirmed by x - ray. all patients received gastric protection with antacids and enteral diet was interrupted during the study period. a calorimeter (deltatrac ii metabolic monitor : datex, helsinki, finland) was installed in the mechanical ventilator circuit to measure carbon dioxide and oxygen concentrations in the inspired and expired air and to calculate systemic oxygen consumption and carbon dioxide production. a radial artery catheter (radial artery catheter : arrow, reading, usa) was introduced to measure mean arterial pressure. mechanical ventilation (servo 900 c : siemens elema, lnd, sweden) parameters were adjusted in order to maintain carbon dioxide arterial tension within the normal range and an oxygen arterial saturation of more than 92%. after initial measurements, patients were randomized 2:1 in order to receive one packed rbc unit or 5% albumin, 500 ml (5% albumin : baxter ag, vienna, austria), respectively. patients were not aspirated, decubitus changes were not allowed nor any other physiotherapeutic procedures. arterial lactate measurements, hemodynamic, tonometric and calorimetric parameters were calculated immediately before initiation of the rbc infusion and repeated immediately after the infusion was completed. hemodynamic parameters included measurement of systemic arterial pressure, pulmonary arterial pressure, central venous pressure and pulmonary capillary pressure. cardiac output was assessed by continuous thermodilution (vigilance monitor : baxter, edwards critical - care division, santa ana, usa). blood specimens were collected from the radial artery catheter and from the distal extremity of the pulmonary artery catheter in order to determine blood gas values (abl-725 : radiometer, copenhagen, denmark), oxygen saturation (abl-725), lactate (vitrus 500, johnson & johnson clinical diagnosis) and hemoglobin (stks : coulter electronics, hialeah, florida, usa) concentrations. oxygen consumption, oxygen transport, left ventricular systolic work index, systemic vascular resistance index and pulmonary vascular resistance index were calculated by the standard methods. a tonometry catheter is a nasogastric tube with a silicon balloon in its tip, which is extremely sensitive to carbon dioxide. this balloon was filled with 2.5 ml of saline solution until an equilibrium with the gastric mucosa was achieved (60 min before each measurement). once equilibrium was reached, the balloon was aspirated and the carbon dioxide level was determined. the phi was calculated from the carbon dioxide value by applying the henderson - hasselbach equation. the calorimeter measures carbon dioxide and oxygen concentrations in the inspired and expired air in each minute and calculates carbon dioxide production as well as the oxygen consumption in each minute. thirty minutes after stabilization of the patient, oxygen consumption was measured for a period of 15 min. the apache ii index was used to evaluate the severity of the patient 's condition. comparisons taken before versus after intervention were evaluated using the wilcoxon signed rank test in each group. in order to study the relationship between the age of the rbcs and the effect of this on phi we used pearson 's correlation coefficient. we resubmitted the data for variance analysis with repeated measures and related designs with the intention to compare hemodynamic response to the interventions between study groups. for statistical significance we considered p 80 years, pregnant women, patients on dialysis, and patients who had recently undergone a gastrointestinal surgical procedure and established septic shock. a tonometry probe device (tonometrics : datex, helsinki, finland) was inserted and its position was confirmed by x - ray. all patients received gastric protection with antacids and enteral diet was interrupted during the study period. a calorimeter (deltatrac ii metabolic monitor : datex, helsinki, finland) was installed in the mechanical ventilator circuit to measure carbon dioxide and oxygen concentrations in the inspired and expired air and to calculate systemic oxygen consumption and carbon dioxide production. a radial artery catheter (radial artery catheter : arrow, reading, usa) was introduced to measure mean arterial pressure. mechanical ventilation (servo 900 c : siemens elema, lnd, sweden) parameters were adjusted in order to maintain carbon dioxide arterial tension within the normal range and an oxygen arterial saturation of more than 92%. after initial measurements, patients were randomized 2:1 in order to receive one packed rbc unit or 5% albumin, 500 ml (5% albumin : baxter ag, vienna, austria), respectively. patients were not aspirated, decubitus changes were not allowed nor any other physiotherapeutic procedures. arterial lactate measurements, hemodynamic, tonometric and calorimetric parameters were calculated immediately before initiation of the rbc infusion and repeated immediately after the infusion was completed. hemodynamic parameters included measurement of systemic arterial pressure, pulmonary arterial pressure, central venous pressure and pulmonary capillary pressure. cardiac output was assessed by continuous thermodilution (vigilance monitor : baxter, edwards critical - care division, santa ana, usa). blood specimens were collected from the radial artery catheter and from the distal extremity of the pulmonary artery catheter in order to determine blood gas values (abl-725 : radiometer, copenhagen, denmark), oxygen saturation (abl-725), lactate (vitrus 500, johnson & johnson clinical diagnosis) and hemoglobin (stks : coulter electronics, hialeah, florida, usa) concentrations. oxygen consumption, oxygen transport, left ventricular systolic work index, systemic vascular resistance index and pulmonary vascular resistance index were calculated by the standard methods. a tonometry catheter is a nasogastric tube with a silicon balloon in its tip, which is extremely sensitive to carbon dioxide. this balloon was filled with 2.5 ml of saline solution until an equilibrium with the gastric mucosa was achieved (60 min before each measurement). once equilibrium was reached, the balloon was aspirated and the carbon dioxide level was determined. the phi was calculated from the carbon dioxide value by applying the henderson - hasselbach equation. the calorimeter measures carbon dioxide and oxygen concentrations in the inspired and expired air in each minute and calculates carbon dioxide production as well as the oxygen consumption in each minute. thirty minutes after stabilization of the patient, oxygen consumption was measured for a period of 15 min. the apache ii index was used to evaluate the severity of the patient 's condition. comparisons taken before versus after intervention were evaluated using the wilcoxon signed rank test in each group. in order to study the relationship between the age of the rbcs and the effect of this on phi we used pearson 's correlation coefficient. we resubmitted the data for variance analysis with repeated measures and related designs with the intention to compare hemodynamic response to the interventions between study groups. for statistical significance we considered p < 0.05. table 1 shows baseline patient characteristics and table 2 shows hemodynamic data, oxygen utilization parameters and hemoglobin concentration in the group that received rbc transfusion. patient baseline characteristics results from the group receiving red blood cell transfusion mean sd. ci, cardiac index ; do2, systemic oxygen delivery index ; hb, hemoglobin ; hr, heart rate ; ht, hematocrit ; lvswi, left ventricular stroke work index ; map, mean arterial pressure ; mpap, mean pulmonary artery pressure ; phi, gastric intramucosal ph ; pvri, pulmonary vascular resistance index ; rap, right atrial pressure ; svri, systemic vascular resistance index ; vo2, systemic oxygen consumption index ; wp, wedge pressure. blood transfusion increased hemoglobin concentration from 9.4 0.5 to 10.1 0.8 g% (p < 0.001). this was associated with an increase in the left ventricular systolic work index (38.6 12.6 to 41.1 13.0 g / min / m ; p < 0.05). although systemic vascular resistance was unchanged, pulmonary vascular resistance was increased (203.7 58.0 to 238.0 49.8 dyne / s / cm / m ; p < 0.05). filling pressures, and systemic and regional oxygenation indices were not significantly changed. table 3 depicts hemodynamic data and oxygen utilization parameters for the colloid (control) group. although global oxygen consumption did not change, regional gastric mucosa oxygenation was improved, with an increase in phi from 7.14 0.2 to 7.24 0.1 but did not reach statistical significance (p = 0.1). ci, cardiac index ; do2, systemic oxygen delivery index ; hb, hemoglobin ; hr, heart rate ; ht, hematocrit ; lvswi, left ventricular stroke work index ; map, mean arterial pressure ; mpap, mean pulmonary artery pressure ; phi, gastric intramucosal ph ; pvri, pulmonary vascular resistance index ; rap, right atrial pressure ; svri, systemic vascular resistance index ; vo2, systemic oxygen consumption index ; wp, wedge pressure. we were unable to find a correlation between the age of the rbcs and the effects on phi (r = 0.257, p = 0.473). figure 1 shows the relationship between the age of rbcs and the change in phi. figure 2 shows that the systemic oxygen delivery index (do2) response to the interventions was effective in both groups. analysis of variance with repeated measurements showed that do2 increased (group 1 : from 607.3 123.5 to 647.5 167.7 ml / min / m and group 2 from 699.6 183.0 to 732.0 194.8 ml / min / m, p < 0.05). change in intramucosal ph (phi) versus red blood cell (rbc) age. systemic oxygen delivery index (do2) response after intervention in both groups. repeated measures anova (p < 0.05). in this group of septic patients, red blood cell transfusion did not improve systemic oxygen consumption, whether indirectly calculated by the fick method or actually measured by calorimetry, despite increased arterial oxygen content. this decreases 2,3-diphosphoglycerate levels and renders red cells less capable of offering oxygen to tissues. the fact that we were unable to detect an acute increase in the oxygen consumption could be because we did not allow sufficient time for the above - mentioned cellular changes to revert. when oxygen consumption is calculated by the fick method, the formula couples oxygen consumption (vo2) to oxygen delivery (do2) : vo2 = ci (cao2 - cvo2) where ci = cardiac index (l / min / m), cao2 = oxygen content of arterial blood (ml / l) and cvo2 = oxygen content of venous blood (ml / l). thus, an increase in oxygen supply could result in a false increase in the oxygen consumption. on the other hand, indirect calorimetry offers the possibility of directly measuring oxygen consumption and is probably a more reliable method. another disadvantage with the fick method is that it could potentially underestimate systemic oxygen consumption because of its inability to measure data from bronchial circulation and thebesian 's circulations. in the study reported here, we did not observe the effect of mathematical coupling of supply to consumption ; rbc transfusion did not result in increased o2 consumption, whether calculated by the fick method or measured by indirect calorimetry methods. in this study another possible explanation for our findings is the fact that our patients would not be in a delivery - dependent state as we might speculate observing the baseline lactate values. gastric tonometry with phi calculation has been utilized as an indicator of regional gastric perfusion. regional gastric oxygenation behavior in this study was similar to that of systemic oxygenation and did not change in response to rbc transfusion. marik and sibbald reported worsening of gastric oxygenation when transfused blood cells were stored for more than 15 days and they postulated that prolonged storage time could make the cells rigid and this could, in turn, lead to capillary obstruction and consequent decrease in local blood flow. worsening of gastric mucosal and splanchnic oxygenation could be associated with increased mucosal permeability and bacteria translocation. blood viscosity is increased by rbc transfusion and this may decrease oxygen transport to tissues. in the study reported here, pulmonary vascular resistance increased significantly after rbc transfusion and there was a nonsignificant trend towards an increase in systemic vascular resistance. these changes can be deleterious in septic patients, who often present with associated myocardial dysfunction. an infusion of 500 ml of 5% albumin was used in this study with the intention of promoting a plasma expansion varying from 250 to 500 ml to serve as a control group for the 10 patients that received rbc transfusion. the cochrane injuries group has reviewed the use of albumin in critically ill patients and has concluded that it could increase mortality, but this review has been severely criticized and albumin is widely used as a colloid expander. colloid expansion did not change filling pressures, cardiac index or systemic oxygen consumption but caused a nonsignificant increasing in gastric phi, denoting improved oxygenation of the gastric mucosa. the trend towards an increase in gastric oxygenation could be related to some degree of hemodilution and amelioration of gastric mucosal rheology, although neither of these factors was demonstrated in our study, perhaps because of the small number of patients involved. boldt 's group randomized septic patients to receive hydroxyethyl starch or albumin (both groups also received saline infusion) for a period of 5 days. the phi was low in both groups and was increased by hydroxyethyl starch but not by albumin, but this effect was only evident after 48 h. forrest reported that 10% pentastarch infusion did not change phi in hypovolemic septic patients, but measurements were only taken for 2 h after infusion and it is known that the effects of colloidal infusion are not immediately evident. a recent study failed to demonstrate reduced mortality in critically ill patients with hemoglobin levels between 7 and 10 g% who received rbc transfusion. anemia does not aggravate gastric mucosal acidosis, whereas rbc transfusion can worsen it. in this study we found that albumin (and probably other colloid expansions) may improve gastric phi. it seems clear that restoration of circulating blood to normal levels is capable of restoring gastric perfusion and it should precede blood cell transfusion in septic patients with hemoglobin levels above 8 g%, who are not suffering from associated coronary heart disease. blood transfusion does not lead to acute improvement in systemic or regional oxygen utilization and can hamper right ventricular ejection by increasing pulmonary vascular resistance, whereas restoration of circulating blood with colloidal solutions may improve gastric perfusion in septic patients. we are indebted to frederico rafael moreira, gianni yanaguibashi and professor clovis de araujo peres for their statistical analysis. | backgroundred blood cell (rbc) transfusion is commonly used to increase oxygen transport in patients with sepsis. however it does not consistently increase oxygen uptake at either the whole - body level, as calculated by the fick method, or within individual organs, as assessed by gastric intra - mucosal ph.aimthis study evaluates the hemodynamic and oxygen utilization effects of hemoglobin infusion on critically ill septic patients.methodsfifteen septic patients undergoing mechanical ventilation whose hemoglobin was < 10 g% were eligible. ten patients (apache ii : 25.5 7.6) received an infusion of 1 unit of packed rbc over 1 h while sedated and paralyzed. the remaining five control patients (apache ii : 24.3 6.0) received a 5% albumin solution (500 ml) over 1 h. hemodynamic data, gastric tonometry and calorimetry were obtained prior to and immediately after rbc transfusion or 5% albumin infusion.resultstransfusion of rbc was associated with an improvement in left ventricular systolic work index (38.6 12.6 to 41.1 13.0 g / min / m2 ; p = 0.04). in the control group there was no significant change in the left ventricular systolic work index (37.2 14.3 to 42.2 18.9 g / min / m2). an increase in pulmonary vascular resistance index (203 58 to 238 49 dyne / cm5/m2 ; p = 0.04) was also observed, while no change was produced by colloid infusion (237 87.8 to 226.4 57.8 dyne / cm5/m2). oxygen utilization did not increase either by fick equation or by indirect calorimetry in either group. gastric intramucosal ph increased only in the control group but did not reach statistical significance.conclusionhemoglobin increase does not improve either global or regional oxygen utilization in anemic septic patients. furthermore, rbc transfusion may hamper right ventricular ejection by increasing the pulmonary vascular resistance index. |
since the initial development of inactivated influenza vaccines in the 1940s, vaccine manufacturers have continually sought to improve their products in response to three main drivers : increased safety and comfort of vaccinated individuals, improved manufacturing security and productivity, and greater vaccine efficacy. the most notable example of such improvements was the development in the 1970s of subvirion vaccines to replace whole virion inactivated vaccines to reduce vaccine reactogenicity, although these latter vaccines are still commercially available in some countries. the vast majority of influenza vaccines, both licensed and in development, are produced from virus propagated in embryonated hens eggs. in recent years, considerable efforts have been made to develop alternative production systems, such as cellculture vaccines, as a complementary source of influenza vaccine to further increase the global production capacity. most vaccine manufacturers have cellculture influenza vaccine development programmes and in 2007, a first cellculturebased seasonal influenza vaccine received european licensure. other recent developments include the reformulation of vaccine with novel adjuvants including virosomes, influenza viruslike particles and the production of recombinant influenza vaccines., the intense political and public interest surrounding avian and pandemic influenza in recent years has stimulated interest in influenza research by governments, nongovernmental organizations, vaccine manufacturers and other researchers. the benefits of this renewed interest extend beyond our preparedness for the next influenza pandemic and into seasonal vaccination. there is increased awareness of the immediate need to increase seasonal influenza vaccination coverage and to optimize the immunogenicity of current influenza vaccines. here, we review sanofi pasteur s epidemic and pandemic influenza research and development programme with emphasis on two key projects : intradermal influenza vaccine for seasonal vaccination and pandemic influenza vaccine development. the elderly are at high risk of serious disease from respiratory infections, particularly influenza, and this population accounts for the majority of the disease burden (for a review, see ref. influenza vaccination is thus recommended for all individuals older than 65, 60 or even 50 years, depending on the country. while many studies attest to the efficacy of influenza vaccination, the exact level of vaccine efficacy in the elderly remains a matter of debate.,,,,,,,, in healthy adults aged < 65 years, one dose of trivalent, inactivated vaccine is considered to be highly immunogenic. with increasing age, however, changes in the immune system result in a lower immunogenicity of influenza vaccines compared with that in younger adults. consequently, the efficacy of conventional intramuscular influenza vaccines is also lower among the elderly. one approach to increase influenza vaccine immunogenicity is the use of a vaccine adjuvant, such as mf59 in the licensed vaccine fluad., a second approach is to formulate the vaccine with virosomes that mimic the structure of viral particles with the aim of improving antigen presentation, invivac). a recent study comparing both of these vaccines, together with a licensed nonadjuvanted, subunit vaccine, found all three vaccines to have similar immunogenicity profiles. influenza vaccines change each year, so by definition, vaccination of individuals in atrisk groups is recommended before each influenza season, and thus individuals can be vaccinated every year for many years. although such data do not exist at an individual level, current formulations of conventional inactivated splitvirion vaccines are well known and do have a good longterm safety record.,, during its 40 years of existence, approximately 1 billion doses of the vaccine vaxigrip (sanofi pasteur, lyon, france) have been distributed. to further improve seasonal influenza vaccines, the approach adopted at sanofi pasteur has been to identify alternative methods to increase immunogenicity without using adjuvants or other additives. the immunological potential of the intradermal route for immunization has been known for a long time. since the 1930s, studies with a variety of antigens have shown that vaccination via the dermis can induce comparable immune responses to intramuscular vaccination, but with a fraction of antigen dose (for a review, see ref. intradermal vaccination against rabies is routinely used in some countries as a way of sparing antigen, and against hepatitis b, the intradermal route has also been shown to induce immune responses in individuals who were previously unresponsive to intramuscular vaccination. although the exact mechanism remains unclear, the efficiency of intradermal immunization is thought to be due to the capture and presentation of antigen by dendritic cells, predominantly dermal dendritic cells, which then drain through the extensive lymph network to the lymph nodes, as well as the direct migration to the nodes of freeantigen, resulting in the stimulation of resident lymph node dendritic cells. together, these processes result in the activation of lymph node t cells and an efficient initiation of cellular arm immune responses. although the potential of intradermal vaccination has been known for some time, it is only recently that advances in vaccine delivery systems have allowed this route to be considered for the largescale production of vaccines such as influenza. despite the successes attributable to the historical intradermal injection methods (such as the standard mantoux intradermal injection technique that involves inserting a 27 g, 3/8 inch, short bevel needle attached to a plastic, 1ml disposable syringe into the skin at a very slight angle, the bifurcated needle for smallpox vaccination, multipuncture devices for bcg and needlefree jet injectors), all have important drawbacks. the development of an easytouse, disposable microdelivery system for intradermal vaccination (soluvia ; becton dickinson, franklin lakes, nj, usa) led to the reconsideration of the intradermal route for influenza vaccination. briefly, it consists of a prefilled, readytouse syringe with an integral, narrow (30 gauge), shortbevel microneedle that protrudes only 15 mm from the proximal end of a glass syringe. it was designed and engineered based on skin anatomical requirements to ensure a consistent and reliable injection of 01 ml of fluid into the papillary and reticular dermis., sanofi pasteur s intradermal influenza vaccine programme for the elderly is based on the hypothesis that the natural potential of the intradermal route can be exploited to increase immune responses to a vaccine produced using essentially the same process as that used to produce a licensed vaccine (vaxigrip). the physical properties of the skin limit the volume of fluid that can be injected into the dermal layer. the volume of a typical intradermal vaccination is 01 ml, five times less than most intramuscular vaccinations. consequently, the challenges of developing an intradermal influenza vaccine have included not only the identification of the appropriate antigen dosage, but also the production of vaccine containing the appropriate dosage in onefifth of the volume. in a phase 2 study, the immunogenicity and safety of two different intradermal vaccine formulations containing 15 or 21 g of haemagglutinin per strain were compared with that of vaxigrip, which contains 15 g of haemagglutinin per strain. the study was designed to test whether one or both of the investigational intradermal vaccines induced statistically superior immune responses compared with the control vaccine. superiority was demonstrated in terms of the primary endpoint (geometric mean titres 21 days after vaccination) for all three strains, and higher responses were observed in all but one of the nine secondary analyses based on immunogenicity criteria defined by the european committee for medicinal products for human use (chmp) (day 21 seroprotection rates, day 21 seroconversion rates and day 021 mean titre increases for each of the three strains). differences between responses in the two intradermal vaccine groups (15 or 21 g haemagglutinin per strain) were not significant. study subjects were aged 6085 with a median age of 70 years. as could be expected, this was the case with both intradermal and intramuscular vaccines for each of the three strains and importantly, seroprotection rates remained higher after intradermal vaccination than after intramuscular vaccination in the older age group (table 1). this study led to the continued development of an intradermal influenza vaccine for adults aged 60 years, containing 15 g of haemagglutinin per strain, which is equivalent to the quantity of antigen contained in conventional intramuscular vaccines. the results of a subsequent phase 3 trial with this vaccine confirm the superiority observed in the phase 2 trial. affect of age class on the difference in postvaccination seroprotection rate after intradermal and intramuscular vaccination in a phase 2 study results are expressed as the absolute difference in seroprotection rate and 95% confidence interval of the difference for each strain after with intradermal vaccine containing 15 g haemagglutinin per strain or intramuscular vaccine containing 15 g haemagglutinin per strain. while the greatest challenge surrounding seasonal influenza vaccination for elderly adults is increasing vaccine immunogenicity and thus the level of protection, for younger adults the challenge is to increase the vaccination coverage. in five western european countries in 20062007, coverage rates among adults in their twenties and thirties were around 10%. in these countries, coverage rates increase with age to 15% of adults in their forties and 21% of adults in their fifties. comparable coverage rates are reported in the usa and in countries in asia, latin america and eastern europe., these numbers are considerably lower than the target coverage rates of between 50% and 90% set by national and international health organizations.,, even among healthcare professionals a population assumed to be better informed of the risks of influenza for themselves and for their patients influenza vaccination coverage remains low in many cases. the morbidity and mortality associated with influenza are lower among adults younger than 60 years than among the elderly. complications are less frequent in young adults than in the elderly, but do occur. for example, in a study of over 20 years of us national hospital discharge survey data (1980s and 1990s), the average annual rate of primary pneumonia and influenza hospitalizations attributable to influenza was 68 per 1 00 000 personyears among persons aged 549 years and 379 among persons aged 5064 years. typically, however, influenza infection of a healthy young adult results in uncomplicated illness with 37 days of high fever with other symptoms including cough, headache and myalgia. the benefits of influenza vaccination in this age group are therefore often expressed in terms of cost benefits because of prevented work absenteeism. studies among the general adult population or among working adults have shown that influenza vaccination can be costeffective, and even cost saving.,, in addition to cost benefits, the benefits of vaccinating adults against influenza include reduced family and social disruption, and reduced transmission to others who may be at increased medical risk themselves. indeed, while an individual s decision to get vaccinated is motivated predominantly to protect oneself, it is the prevention of transmission to others who are at risk that motivates health authorities to specifically recommend influenza vaccination for healthcare workers and anyone with a household member who is in a high risk group. two recent surveys in europe and the usa found that around 1416% of individuals cited a dislike of needles and injections as one of the reasons for not getting vaccinated., other barriers included the common misperception that being healthy is sufficient protection against influenza, that the risk of contracting the disease is low (most frequent responses in these two studies), or a lack of recommendation by an individual s family practitioner, or a lack of reimbursement. these findings suggest that, together with improved education about influenza disease and vaccination, and reimbursement programmes, alternative vaccination methods that avoid the need for a classic syringe and needle have the potential to contribute to increase vaccination uptake among such populations. this is the rationale for the development of an intradermal influenza vaccine for young adults using the same microinjection system as described above. in contrast to the vaccine for elderly adults, the intradermal vaccine for young adults was developed with the aim of providing equivalent immunogenicity to current intramuscular vaccines. phase 2 trials have demonstrated that a 9 g intradermal dose of haemagglutinin per strain is sufficient to elicit an equivalent (statistically noninferior) immune response to vaxigrip (j. beran., vaccination and travel medicine centre, hradec kralove, unpublished results). clinical trial results obtained to date reveal no safety issues with either the 9 or the 15g formulation of the intradermal vaccine., the rates of solicited systemic reactions and unsolicited adverse events observed in each trial performed to date have been comparable between intradermal and intramuscular groups. furthermore, a study conducted over 3 consecutive years, with (re)randomization to intramuscular or intradermal vaccination each year revealed that the rates of reaction did not increase from year to year in any of the randomized subgroups, suggesting that intradermal vaccination could be safely administered repetitively or in alternation with intramuscular vaccine from year to year (j. beran. intradermal vaccination with the microinjection system is inherently safer than vaccination via the intramuscular route, since the 15 mm long microneedle limits the possibility of mechanical damage to nerves or blood vessels. also inherent to intradermal vaccination is the higher frequency of minor visible injection site reactions, such as redness, swelling or induration, around the point of intradermal vaccination. higher rates of these injection site reactions after intradermal vaccination have been observed in all the clinical trials performed to date, but importantly the incidence of injection site pain has been comparable between groups, and as with intramuscular vaccination, these reactions are shortlived and disappear spontaneously. a marketing authorization dossier on both the 9g formulation for adults < 60 years and the 15g formulation for adults 60 years was submitted to the european medicines agency and is currently (september 2008) under review. the elderly are at high risk of serious disease from respiratory infections, particularly influenza, and this population accounts for the majority of the disease burden (for a review, see ref. influenza vaccination is thus recommended for all individuals older than 65, 60 or even 50 years, depending on the country. while many studies attest to the efficacy of influenza vaccination, the exact level of vaccine efficacy in the elderly remains a matter of debate.,,,,,,,, in healthy adults aged < 65 years, one dose of trivalent, inactivated vaccine is considered to be highly immunogenic. with increasing age, however, changes in the immune system result in a lower immunogenicity of influenza vaccines compared with that in younger adults. consequently, the efficacy of conventional intramuscular influenza vaccines is also lower among the elderly. one approach to increase influenza vaccine immunogenicity is the use of a vaccine adjuvant, such as mf59 in the licensed vaccine fluad., a second approach is to formulate the vaccine with virosomes that mimic the structure of viral particles with the aim of improving antigen presentation, invivac). a recent study comparing both of these vaccines, together with a licensed nonadjuvanted, subunit vaccine, found all three vaccines to have similar immunogenicity profiles. influenza vaccines change each year, so by definition, vaccination of individuals in atrisk groups is recommended before each influenza season, and thus individuals can be vaccinated every year for many years. although such data do not exist at an individual level, current formulations of conventional inactivated splitvirion vaccines are well known and do have a good longterm safety record.,, during its 40 years of existence, approximately 1 billion doses of the vaccine vaxigrip (sanofi pasteur, lyon, france) have been distributed. to further improve seasonal influenza vaccines, the approach adopted at sanofi pasteur has been to identify alternative methods to increase immunogenicity without using adjuvants or other additives. the immunological potential of the intradermal route for immunization has been known for a long time. since the 1930s, studies with a variety of antigens have shown that vaccination via the dermis can induce comparable immune responses to intramuscular vaccination, but with a fraction of antigen dose (for a review, see ref. intradermal vaccination against rabies is routinely used in some countries as a way of sparing antigen, and against hepatitis b, the intradermal route has also been shown to induce immune responses in individuals who were previously unresponsive to intramuscular vaccination. although the exact mechanism remains unclear, the efficiency of intradermal immunization is thought to be due to the capture and presentation of antigen by dendritic cells, predominantly dermal dendritic cells, which then drain through the extensive lymph network to the lymph nodes, as well as the direct migration to the nodes of freeantigen, resulting in the stimulation of resident lymph node dendritic cells. together, these processes result in the activation of lymph node t cells and an efficient initiation of cellular arm immune responses. although the potential of intradermal vaccination has been known for some time, it is only recently that advances in vaccine delivery systems have allowed this route to be considered for the largescale production of vaccines such as influenza. despite the successes attributable to the historical intradermal injection methods (such as the standard mantoux intradermal injection technique that involves inserting a 27 g, 3/8 inch, short bevel needle attached to a plastic, 1ml disposable syringe into the skin at a very slight angle, the bifurcated needle for smallpox vaccination, multipuncture devices for bcg and needlefree jet injectors), all have important drawbacks. the development of an easytouse, disposable microdelivery system for intradermal vaccination (soluvia ; becton dickinson, franklin lakes, nj, usa) led to the reconsideration of the intradermal route for influenza vaccination. briefly, it consists of a prefilled, readytouse syringe with an integral, narrow (30 gauge), shortbevel microneedle that protrudes only 15 mm from the proximal end of a glass syringe. it was designed and engineered based on skin anatomical requirements to ensure a consistent and reliable injection of 01 ml of fluid into the papillary and reticular dermis., sanofi pasteur s intradermal influenza vaccine programme for the elderly is based on the hypothesis that the natural potential of the intradermal route can be exploited to increase immune responses to a vaccine produced using essentially the same process as that used to produce a licensed vaccine (vaxigrip). the physical properties of the skin limit the volume of fluid that can be injected into the dermal layer. the volume of a typical intradermal vaccination is 01 ml, five times less than most intramuscular vaccinations. consequently, the challenges of developing an intradermal influenza vaccine have included not only the identification of the appropriate antigen dosage, but also the production of vaccine containing the appropriate dosage in onefifth of the volume. in a phase 2 study, the immunogenicity and safety of two different intradermal vaccine formulations containing 15 or 21 g of haemagglutinin per strain were compared with that of vaxigrip, which contains 15 g of haemagglutinin per strain. the study was designed to test whether one or both of the investigational intradermal vaccines induced statistically superior immune responses compared with the control vaccine. superiority was demonstrated in terms of the primary endpoint (geometric mean titres 21 days after vaccination) for all three strains, and higher responses were observed in all but one of the nine secondary analyses based on immunogenicity criteria defined by the european committee for medicinal products for human use (chmp) (day 21 seroprotection rates, day 21 seroconversion rates and day 021 mean titre increases for each of the three strains). differences between responses in the two intradermal vaccine groups (15 or 21 g haemagglutinin per strain) were not significant. study subjects were aged 6085 with a median age of 70 years. as could be expected, this was the case with both intradermal and intramuscular vaccines for each of the three strains and importantly, seroprotection rates remained higher after intradermal vaccination than after intramuscular vaccination in the older age group (table 1). this study led to the continued development of an intradermal influenza vaccine for adults aged 60 years, containing 15 g of haemagglutinin per strain, which is equivalent to the quantity of antigen contained in conventional intramuscular vaccines. the results of a subsequent phase 3 trial with this vaccine confirm the superiority observed in the phase 2 trial. affect of age class on the difference in postvaccination seroprotection rate after intradermal and intramuscular vaccination in a phase 2 study results are expressed as the absolute difference in seroprotection rate and 95% confidence interval of the difference for each strain after with intradermal vaccine containing 15 g haemagglutinin per strain or intramuscular vaccine containing 15 g haemagglutinin per strain. while the greatest challenge surrounding seasonal influenza vaccination for elderly adults is increasing vaccine immunogenicity and thus the level of protection, for younger adults the challenge is to increase the vaccination coverage. surveys of vaccination uptake repeatedly show that coverage rates among young adults are low. in five western european countries in 20062007, coverage rates among adults in their twenties and thirties were around 10%. in these countries, coverage rates increase with age to 15% of adults in their forties and 21% of adults in their fifties. comparable coverage rates are reported in the usa and in countries in asia, latin america and eastern europe., these numbers are considerably lower than the target coverage rates of between 50% and 90% set by national and international health organizations.,, even among healthcare professionals a population assumed to be better informed of the risks of influenza for themselves and for their patients influenza vaccination coverage remains low in many cases. the morbidity and mortality associated with influenza are lower among adults younger than 60 years than among the elderly. complications are less frequent in young adults than in the elderly, but do occur. for example, in a study of over 20 years of us national hospital discharge survey data (1980s and 1990s), the average annual rate of primary pneumonia and influenza hospitalizations attributable to influenza was 68 per 1 00 000 personyears among persons aged 549 years and 379 among persons aged 5064 years. typically, however, influenza infection of a healthy young adult results in uncomplicated illness with 37 days of high fever with other symptoms including cough, headache and myalgia. the benefits of influenza vaccination in this age group are therefore often expressed in terms of cost benefits because of prevented work absenteeism. studies among the general adult population or among working adults have shown that influenza vaccination can be costeffective, and even cost saving.,, in addition to cost benefits, the benefits of vaccinating adults against influenza include reduced family and social disruption, and reduced transmission to others who may be at increased medical risk themselves. indeed, while an individual s decision to get vaccinated is motivated predominantly to protect oneself, it is the prevention of transmission to others who are at risk that motivates health authorities to specifically recommend influenza vaccination for healthcare workers and anyone with a household member who is in a high risk group. two recent surveys in europe and the usa found that around 1416% of individuals cited a dislike of needles and injections as one of the reasons for not getting vaccinated., other barriers included the common misperception that being healthy is sufficient protection against influenza, that the risk of contracting the disease is low (most frequent responses in these two studies), or a lack of recommendation by an individual s family practitioner, or a lack of reimbursement. these findings suggest that, together with improved education about influenza disease and vaccination, and reimbursement programmes, alternative vaccination methods that avoid the need for a classic syringe and needle have the potential to contribute to increase vaccination uptake among such populations. this is the rationale for the development of an intradermal influenza vaccine for young adults using the same microinjection system as described above. in contrast to the vaccine for elderly adults, the intradermal vaccine for young adults was developed with the aim of providing equivalent immunogenicity to current intramuscular vaccines. phase 2 trials have demonstrated that a 9 g intradermal dose of haemagglutinin per strain is sufficient to elicit an equivalent (statistically noninferior) immune response to vaxigrip (j. beran., vaccination and travel medicine centre, hradec kralove, unpublished results). clinical trial results obtained to date reveal no safety issues with either the 9 or the 15g formulation of the intradermal vaccine., the rates of solicited systemic reactions and unsolicited adverse events observed in each trial performed to date have been comparable between intradermal and intramuscular groups. furthermore, a study conducted over 3 consecutive years, with (re)randomization to intramuscular or intradermal vaccination each year revealed that the rates of reaction did not increase from year to year in any of the randomized subgroups, suggesting that intradermal vaccination could be safely administered repetitively or in alternation with intramuscular vaccine from year to year (j. beran., vaccination and travel medicine centre, hradec kralove, unpublished results). intradermal vaccination with the microinjection system is inherently safer than vaccination via the intramuscular route, since the 15 mm long microneedle limits the possibility of mechanical damage to nerves or blood vessels. also inherent to intradermal vaccination is the higher frequency of minor visible injection site reactions, such as redness, swelling or induration, around the point of intradermal vaccination. higher rates of these injection site reactions after intradermal vaccination have been observed in all the clinical trials performed to date, but importantly the incidence of injection site pain has been comparable between groups, and as with intramuscular vaccination, these reactions are shortlived and disappear spontaneously. a marketing authorization dossier on both the 9g formulation for adults < 60 years and the 15g formulation for adults 60 years was submitted to the european medicines agency and is currently (september 2008) under review. the challenges of developing a pandemic influenza vaccine candidate differ from those of developing improved vaccines against seasonal influenza in almost every respect. although an influenza pandemic is expected, it is not known which strain of virus, or even which subtype, will cause it. the challenge is therefore to develop a vaccine against a disease that does not yet exist. currently, the most widely accepted scenario is that the next pandemic strain will evolve out of one of the highly pathogenic avian influenza a (h5n1) strains that have been in circulation among both wild bird and poultry populations, and have caused almost 400 human cases over the past 5 years. seed strains used to produce most of the current pandemic vaccine candidates are derived by reverse genetics from one of these h5n1 avian strains., other possibilities include the emergence of an h7 or h9 strain, or the resurgence of a human h2n2 strain, which caused 1957 pandemic. when a pandemic occurs, the priority will be to vaccinate as many people as possible, as quickly as possible. among the many industrial, logistical and political challenges that this implies, the identification of a minimum dose of antigen needed to confer an acceptable level of protection against severe disease to optimize the manufacturing capacity is critical. sanofi pasteur s strategy for pandemic influenza vaccine development has been to provide a firstgeneration vaccine as quickly as possible for stockpiling, whilst developing secondgeneration vaccines with improved immunogenicity profiles. first, using a manufacturing process adapted from seasonal vaccine production (fluzone sanofi pasteur, swiftwater, pa, usa), a nonadjuvanted h5n1 vaccine was produced in the usa under governmental contract and provided to the us national institute of allergy and infectious diseases (niaid) for clinical evaluation. a dose ranging study, which tested doses of 7590 g of haemagglutinin of this nonadjuvanted vaccine, revealed that a twodose regimen of 90 g of haemagglutinin was needed to elicit a response expected to reduce the risk of getting influenza in 45% of subjects. this led to the fda s approval of this vaccine as a first measure to ensure the nation s readiness, pending the development of the next generation of vaccines, and confirmed the need for formulations with improved immunogenicity. this vaccine has been further evaluated in a second study of the response to a third, booster vaccination 6 months later, which found that titres were higher after the booster injection than after the second injection. a second project with an adjuvanted vaccine has therefore been conducted with the dual objective of improving the immune response to the vaccine whilst reducing the dose of antigen needed. aluminiumbased adjuvants are readily available, are widely used in licensed vaccines and have documented safety. the first clinical trial with an aluminium hydroxide adjuvant this trial was also designed as a dose ranging and formulation finding trial and evaluated three antigen dosages (75, 15 and 30 g of haemagglutinin), each with or without adjuvant. although the adjuvanted 30 g formulation was the most immunogenic and 67% of subjects seroconverted after two vaccinations, there was no adjuvant effect at either of the two lower dosages. furthermore, the lowest dose (75 g) appeared to perform better without adjuvant than with adjuvant. these findings led us to select the adjuvanted 30 g formulation and 75 g nonadjuvanted formulation for further investigation. results from a clinical trial of these two formulations in children in thailand confirm the immunogenicity of the vaccine, with the adjuvant providing a clear immunogenic advantage. the mf59 oilinwater emulsion adjuvant used in novartis licensed seasonal influenza vaccine for the elderly, fluad, has been shown to increase immune responses to h5n1 after both homologous vaccination as well as after heterologous vaccination with a nonpathogenic h5n3 virusderived vaccine., another proprietary oilinwater emulsionbased adjuvant has been developed by glaxosmithkline biologicals (gsk) and has been tested in an h5n1 prototype vaccine formulation. these studies have shown that these adjuvanted vaccine formulations are well tolerated and significantly dosesparing compared with nonadjuvanted vaccine, and are able to induce crossneutralizing antibody responses to various h5n1 clades and subclades. our third project, undertaken in parallel with the aluminiumbased adjuvanted vaccine project, was the development of vaccine containing an oilinwater emulsion of the same class as those described above. this proprietary adjuvant is a squaleneinwater emulsion with a very fine particle size and a narrow particle size distribution. the potential of this emulsion in a clade 1 influenza a / vietnam/1194/2004 (h5n1) vaccine has been evaluated in preclinical studies in two species, as well as in a phase 1 clinical study. in a macaque viral challenge model, two injections of this emulsionadjuvanted h5n1 vaccine with 30 g of haemagglutinin were found to reduce the incidence and severity of interstitial pneumonia, and protect against infection in the lungs and upper respiratory tract after intratracheal challenge 3 months later with homologous wildtype virus. protection against disease and death was seen in a ferret model in which groups of animals received two injections of either 19, 38, 75 or 15 g of haemagglutinin with emulsion adjuvant or a saline control and were challenged 2 months later with the parental wildtype virus. all vaccinated animals survived challenge, whereas five of the six controls died. vaccinated animals also showed fewer and milder clinical signs of disease (temperature increase, body weight loss), shed less virus in nasal samples 2 and 4 days after challenge, and had fewer and milder lesions in the lungs upon histopathological examination. both species mounted robust haemagglutination inhibition responses against the vaccine strain, as well as crossreactive responses against the clade 2 indonesia/5/05 strain. strong homologous immunogenicity and crossreactive responses were also seen in the first clinical trial with this vaccine. this study in healthy 18 to 40year olds, evaluated vaccine with antigen dosages ranging from 19 to 15 g. results showed that after two injections, even the lowest dosage elicited haemagglutination inhibiting antibody titres 32 geometric mean titres were in the same range as 30 g with aluminium hydroxide adjuvant and as 90 g without adjuvant, i.e. a potential dose sparing of up to 48fold (figure 1). geometric mean titres of haemagglutination inhibiting antibodies against clade 1 h5n1 influenza strains 2128 days after two vaccinations with nonadjuvanted or adjuvanted vaccine in groups of healthy adults in three clinical trials. adapted from data in ref. none of the clinical or preclinical studies performed so far have revealed any clinically significant adverse events or safety signals after vaccination with any of the nonadjuvanted or adjuvanted formulations.,, clinical trials comparing adjuvanted and nonadjuvanted vaccine show that adjuvantation does increase the incidence of minor injection site reactions, such as redness, but that increasing the antigen dosage does not. recent advances in vaccine delivery systems have allowed the intradermal route of administration to be reconsidered for annual vaccination against seasonal influenza. this strategy avoids the unknowns associated with the repetitive annual administration of vaccine adjuvants and has led to the development of a new vaccine based on a current, wellknown, trivalent, inactivated splitvirion vaccine, with two dosage formulations designed specifically for younger and older adults. in contrast, adjuvanted vaccines will play a pivotal role in protection against the next human pandemic of influenza, helping to prime and immunize as many people as possible against a newly emerging strain of influenza with a finite vaccine production capacity. the ability of this new generation of vaccine to induce broadly crossreactive antibodies opens up the possibility of priming with stockpiled vaccine to mitigate the impact of infection during the first few months of the next pandemic, pending the availability of vaccine against the actual pandemic strain. | abstract challenges facing seasonal and pandemic influenza vaccination include : increasing the immunogenicity of seasonal vaccines for the most vulnerable, increasing vaccination coverage against seasonal influenza, and developing vaccines against pandemic strains that are immunogenic with very low quantities of antigen to maximize the number of people who can be vaccinated with a finite production capacity. we review sanofi pasteur s epidemic and pandemic influenza research and development programmes with emphasis on two key projects : intradermal influenza vaccine for seasonal vaccination of both elderly and younger adults, and pandemic influenza vaccine. |
ubiquitin is an evolutionarily highly conserved small protein of 76 amino acids (8.6 kda). ubiquitination is a post - translational protein modification, carried out by three classes of enzymes, namely the ubiquitin - activating- (e1), ubiquitin - conjugating- (e2), and ubiquitin - ligating - enzymes (e3). the consecutive activity of these enzymes leads to the attachment of ubiquitin via its c terminus to a target protein (hershko and ciechanover, 1998). ubiquitin itself can be ubiquitinated by attachment of the incoming ubiquitin to either of seven different lysine (k) residues (k6, k11, k27, k29, k33, k48, k63) or the n - terminal methionine (m1). thus, depending on the linkage type(s) target proteins can be decorated with ubiquitin chains that are diverse in their compositions and exhibit different three - dimensional conformations (kulathu and komander, 2012). whereas k48-ubiquitin linkages serve to signal for protein degradation by the proteasome (hershko and ciechanover, 1998), non - degradative ubiquitin chains have emerged as important regulators of signals emanating from diverse immune receptors including tnfr1, nod2, cd40, tlr2, tlr4, and il-1r. upon stimulation by their respective ligands, components within the primary receptor - associated signaling complexes (scs) are modified by addition of k63- and m1-linked and, in certain cases, also other types of ubiquitin chains (fiil and gyrd - hansen, 2014, iwai., 2014, shimizu., 2015, formation of k63 chains is mediated by various e3 ubiquitin ligases specific for individual scs. the linear ubiquitin chain assembly complex (lubac), consisting of hoil-1, sharpin, and the catalytically active subunit hoip, is the only currently known e3 capable of forming m1 chains de novo (gerlach., 2011, haas., 2009, ikeda., 2011,, 2006, tokunaga., 2011). in all of the above signaling pathways, lubac has been determined to be responsible for m1 chain formation (damgaard., 2012, emmerich., 2013, gerlach., 2011, rodgers., 2014). k63 chains are recognized by the ubiquitin binding domains of tab2 or tab3 (kanayama., 2004, wang., 2001), resulting in recruitment of the tak / tab complex as well as lubac (haas., 2009, lubac then enables efficient recruitment of nemo and, consequently, of the nemo / ikk/ikk (nemo / ikk) complex (haas., these two functional units then cooperatively trigger activation of the nf-b and mapk signaling pathways (walczak., absence of lubac therefore attenuates gene induction by the above receptors and causes early embryonic lethality in mice due to aberrant tnfr1-induced endothelial cell death. importantly, this cell death is due to increased formation of complex ii of tnfr1 and not caused by attenuated gene activation from the tnf - rsc (peltzer., 2014). to signal at the physiological level in response to a given stimulus, it is not only required that the corresponding sc forms, but it also has to disassemble with the appropriate kinetics. regulated assembly and disassembly of ubiquitin chains within scs are essential to achieve this. the enzymes responsible for removing ubiquitin moieties from target proteins and cleaving polyubiquitin chains are deubiquitinases (dubs). dubs implicated in the regulation of signaling by tnfr1 and other immune receptors are cyld, a20 (harhaj and dixit, 2012), and the m1-specific dub otulin, which was recently proposed to specifically antagonize lubac at scs, including in the context of the tnf - rsc and the nod2-sc (fiil., 2013, keusekotten., 2013, rivkin., 2013)., 2003, wright., 2007), it cleaves various linkages in vitro, albeit with preference for k63 and m1 linkages (komander., 2008, ritorto., 2014). a20 is induced by nf-b upon stimulation of various immune receptors and hydrolyzes k11, k63, and k48 but not m1 linkages (mevissen., 2013, ritorto., 2014, wertz., 2004). a20 binds to both k63 and m1 linkages via its zinc finger (znf) domains 4 and 7, respectively (bosanac., 2010, tokunaga., 2012, otulin deficiency is embryonically lethal due to vascular defects (rivkin., 2013). cyld deficiency causes cylindromatosis in humans, a disease characterized by formation of benign tumors in the skin of affected individuals (bignell., 2000, deficiency in a20, but interestingly not inactivation of its dub activity, causes early death in mice due to severe inflammation, implying that a20 likely exerts major functions independently from its dub activity (lee., 2000, recently, hoip was found to directly associate with both cyld and otulin in non - stimulated cells (elliott., 2014,, 2013, schaeffer., 2014, takiuchi., 2014). here, we analyzed the interplay between lubac, m1-ubiquitin, and the various before - mentioned dubs in assembly and disassembly of immune scs and the functional impact this interplay has on the regulation of their signaling output. as linear ubiquitination is crucial for various immune signaling pathways (gerlach., 2011, haas., 2009), we aimed to understand how it is regulated in scs. to do so, we created cell lines that are genetically deficient in hoip, the m1-chain - forming component of lubac, and re - expressed tandem affinity purification (tap)-tagged (2 strep - tag ii followed by a prescission cleavage site and 1 flag) hoip in these cells (figure s1a). we first analyzed the unstimulated lubac obtained from these cells by mass spectrometry following tap. in line with recent reports (elliott., 2014, fu., 2014, rivkin., 2013, schaeffer, our data showed that prior to stimulation, both cyld and otulin interacted with hoip (figures s1b s1d). we therefore deemed it likely that lubac would mediate recruitment of both of these dubs to scs. unexpectedly, however, a kinetic analysis following tumor necrosis factor (tnf) stimulation revealed that otulin was not recruited to the tnf - rsc following tnf stimulation, despite being present in lysates, whereas recruitment of cyld was evident, interestingly with kinetics reminiscent of lubac recruitment (figure 1a). it is currently unclear how cyld is recruited to scs, including the tnf - rsc, although optineurin was previously suggested to be involved (nagabhushana., 2011). as cyld interacts with lubac prior to stimulation, and because it is recruited to the tnf - rsc with similar kinetics as lubac, we wondered whether recruitment of cyld may require hoip. analyzing the native tnf - rsc in hoip - proficient versus -deficient cells revealed that while cyld was present in the tnf - rsc of hoip - proficient a549 (figure 1b) and hacat (figure s1e) cells, it was absent from it in their hoip - deficient counterparts (figures 1b and s1e). thus, hoip is required for recruitment of cyld to the tnf - rsc. to determine whether absence of m1 chains from the tnf - rsc affected cyld presence and otulin absence, we reconstituted hoip - deficient cells with either tap - tagged wild - type (wt) or enzymatically inactive hoip - c885s (smit., 2012, stieglitz., 2012). when examining the effects of absence of lubac activity on the constitutive interaction of hoip with cyld and otulin, we found that neither of these interactions required hoip activity (figure 1c). regarding the tnf - rsc, in line with our previous results, this complex is less stable in the absence of hoip or its activity (haas., 2009). consequently, all proteins were retained less efficiently in the complex (figure 1d). importantly, however, cells expressing hoip - c885s maintained the capacity to recruit cyld to the tnf - rsc, yet otulin remained absent from this complex (figure 1d). we therefore conclude that the enzymatic activity of hoip is not required for recruitment of cyld to the tnf - rsc. as mentioned above, the mechanism of cyld recruitment to scs was responsible for tnf - rsc recruitment, we wondered whether lubac might also be responsible for recruitment of cyld to other scs. to test this hypothesis, we analyzed the receptor - associated complex that forms upon stimulation of nod2 because, despite of being composed quite differently from the tnf - rsc, signaling via nod2 also involves lubac (damgaard. nod2 is an intracellular pattern - recognition receptor (prr) recognizing components of the bacterial cell wall, and it plays a critical role in gastrointestinal host defense (chen., 2009). as previous studies employed a system triggered by overexpression of nod2 that does not require ligand - induced stimulation and to avoid possible non - physiological events resulting from ligand - independent signaling by nod2 overexpression, we generated stable clones expressing tap - tagged nod2 at intermediate levels. in these cells, nod2-sc formation requires ligand - induced stimulation, which more closely resembles the physiological situation. stimulation of these cells with synthetic muramyl dipeptide (mdp) l18-mdp, a known ligand for nod2 (grimes., 2012), induced strong nf-b activation in hoip - proficient, but not in hoip - deficient cells (figures 2a and s2a). in line with reports on hoip s requirement for erk activation upon cd40 and tnfr1 stimulation (peltzer., 2014, sasaki., 2013), we found that activation of erk was also substantially inhibited in hoip - deficient nod2-stimulated cells (figure s2a). we next analyzed the nod2-sc and found that cyld also forms part of this complex (figures 2b and s2b). as with the tnf - rsc, hoip is essential for recruitment of cyld to the nod2-sc (figures 2b and s2b), while otulin also does not form part of this complex (figure 2b). cyld was previously described as an inhibitor of tnf signaling (brummelkamp., 2003,, we isolated bone - marrow derived macrophages (bmdms) from wt and cyld - deficient mice and stimulated them with l18-mdp, as these cells constitutively express nod2. this revealed that in the absence of cyld, nod2-induced gene activation via nf-b and mapks (erk, p38, and jnk) is enhanced (figure 2c). as it was unclear why otulin could be absent from lubac - containing scs despite interacting with lubac in the cytosol prior to stimulation, we next aimed to find a biochemical explanation for this unexpected phenomenon. the crystal structure of the hoip pub domain bound to the otulin pim peptide (aa 4967) shows that tyr56 in otulin and asn102 in hoip are crucial for their interaction (elliott., 2014, schaeffer., 2014). accordingly, phosphorylation of an otulin - derived peptide on the residue corresponding to tyr56 prevented its association with hoip s pub domain (elliott. therefore, one possible explanation of otulin s absence from scs could be its release from lubac as a result of tyr56 phosphorylation. this event would have to be postulated to occur at, or shortly following, recruitment. tnf stimulation, however, did not abolish the interaction between hoip and otulin (figures 1c and s3a). furthermore, induction of global tyrosine phosphorylation by pervanadate treatment, which induces irreversible inhibition of phosphatases, did not prevent this association either (figure 3a). conversely, reduction of overall tyrosine phosphorylation by phosphatase treatment did not increase it (figure 3a). thus, stimulation - associated tyrosine phosphorylation of otulin, including at tyr56, can not be responsible for otulin intriguingly, hoip s relatively small pub domain mediates the interaction with both cyld and otulin and even short deletions in this domain abolished interaction with both factors (figures s3b s3d). this suggested that steric hindrance may prevent simultaneous interaction of hoip with cyld and otulin. if that was the case, they would compete for binding to hoip so that cyld - interacting lubac would be devoid of otulin and vice versa. to test this possibility, we precipitated tap - tagged otulin and checked for binding of lubac components and cyld. while lubac components were co - precipitated, cyld was not (figure 3b). thus, cyld and tap - tagged otulin do not form part of the same individual lubac complexes, implying that they indeed can not simultaneously interact with an individual hoip protein. to address whether this also holds true for endogenous otulin, we next quantitatively immunoprecipitated otulin from wt cells, which would result in removal of the fraction of lubac bound to endogenous otulin, yet without co - precipitating cyld. in accord with this hypothesis, cyld did not form part of endogenous otulin - associated lubac complexes. subsequent cyld immunoprecipitation from otulin - depleted samples revealed that a second fraction of lubac is bound to cyld (figure 3c). hence, there are two distinct fractions of lubac in the cell, one associated with cyld, and another one bound to otulin. the hoip - otulin interaction can be disrupted by mutation of a critical residue (n102) in hoip s pub domain (elliott., 2014). to test whether the cyld - hoip interaction would also be affected by this mutation, we reconstituted hoip - deficient cells with hoip - n102a and checked for association with cyld. this revealed that hoip - n102a was unable to bind to cyld (figure 3d). together, these results show that cyld and otulin interact with hoip via the same or an overlapping site and that hoip s interactions with cyld and otulin are mutually exclusive. based on these results, in combination with our findings regarding the composition of the tnf - rsc (figure 1) and the nod2-sc (figure 2), it can be concluded that cyld - associated lubac is recruited to scs, whereas lubac associated with otulin is not. tnf stimulation induces expression of various response genes, which help counteract invading pathogens but also promote autoinflammation (walczak, 2011). we therefore analyzed the tnf - rsc in cells expressing hoip n102a to assess functional consequences of the loss of dub interaction with hoip. the recruitment of cyld was abrogated in cells expressing this hoip variant (figure 3e), confirming that the interaction with lubac is required for cyld recruitment. following tnf stimulation, these cells showed increased m1 ubiquitination at the tnf - rsc (figure 3e) and enhanced activation of nf-b (figure 3f). similar results were obtained with cells expressing hoip devoid of the pub domain (figure s3e). furthermore, the lack of hoip s pub domain resulted in increased tnf - induced expression of irf1, icam1, tnf, and ib (figure s3f). nemo was previously shown to be linearly ubiquitinated (gerlach., 2011, tokunaga., 2009), and this polyubiquitination event, albeit weak, could be observed in cells expressing hoip - wt but not in cells lacking hoip or expressing inactive hoip. in cells expressing hoip - n102a, however, the polyubiquitination of nemo was enhanced (figure s3, abolishing hoip s capacity to interact with both cyld and otulin enhances m1 ubiquitination in the tnf - rsc and, consequently, tnf - induced gene activation. as hoip - associated otulin was not recruited to scs, we next studied whether otulin deficiency may increase levels of linear ubiquitin found in the cytosol. using crispr - cas9, we generated otulin - deficient cells. already prior to stimulation, these cells contained significantly more m1-linked ubiquitin chains in their cytosol than control cells (figures 4a and s4a). these aberrant m1 chains were not free chains but conjugated to substrates (figure s4b). the accumulation of linear ubiquitin linkages was found to be due to absence of otulin s enzymatic activity (figure s4c). however, in accordance with our observation that otulin is not present at the tnf - rsc, linear ubiquitination within the tnf - rsc was not increased in otulin - ko as compared to wt cells (figures 4a and s4a). these circumstances led us to suspect that, instead of antagonizing m1-ubiquitin at scs, otulin might regulate lubac components themselves. to identify which proteins show increased linear ubiquitination in lysates of otulin - deficient cells, we devised a new strategy to enrich for m1-ubiquitinated proteins. this method, which we will refer to as m1-affinity purification (m1-ap), employs a bead - resin - immobilized, enzymatically inactive portion of otulin (aa 58352, c129a) that binds to, but does not cleave, m1 linkages with high affinity and specificity (keusekotten., 2013) prior to m1-ap, proteins were completely denatured in 1% sds and subsequently renatured so that only proteins directly modified by ubiquitin chains containing m1 linkages, and not proteins that are only non - covalently associated with such chains, could be detected in these assays. notably, m1-ap showed that m1 ubiquitination of hoil-1 and sharpin was substantially increased in otulin - deficient cells (figure 4c). in accord with the results of the tnf immunoprecipitation, analysis of the components of the tnf - rsc by m1-ap showed that m1 ubiquitination of rip1, a known lubac target in the tnf - rsc (gerlach., 2011), was not increased in otulin - deficient cells (figure 4d). hence, rather than serving as a negative regulator of lubac activity at scs, otulin keeps components of lubac free of aberrant m1 linkages prior to stimulation. since its association with lubac was the recruiting principle for cyld and because expression of hoip - n102a, unable to recruit cyld to the tnf - rsc, increased the amount of m1 linkages in this sc, we next sought to determine whether cyld could be responsible for antagonizing lubac activity in scs. we employed crispr - cas9 to create cyld - deficient a549 cells and stimulated them with tnf before subjecting them to m1-ap or k63-ap, the latter being based on isolation of k63-ubiquitin linkages with tuim (sims., 2012) when determining which proteins were modified by these linkages, we found that both m1 and k63 ubiquitination of rip1, tnfr1, and tradd were increased in the absence of cyld (figures 5a and s5b). this suggested that each one of these proteins carries m1 and k63 chains when present in the tnf - rsc, and that cyld antagonizes both of them on all of these proteins. to determine whether tnfr1 and tradd are bona fide lubac targets, subsequent to tnf stimulation and isolation by m1-ap we treated m1-ubiquitinated proteins with two different recombinant dubs (figure s5c). treatment with otulin resulted in removal of all m1-linked chains from these proteins (figure 5b). crucially, this treatment significantly reduced the high - molecular - weight species of tnfr1, tradd, and rip1 (figure 5b). thus, prior to otulin treatment m1-linked ubiquitin chains had been present on these proteins. it should be noted that complete removal of ubiquitin chains by otulin would be expected only for exclusively linearly ubiquitinated targets. however, in line with our previous results regarding rip1 (gerlach., 2011), tradd and tnfr1 also carry other chain types, and the degree of reduction in their overall ubiquitination by otulin treatment is indicative of the ratio between m1 linkages and other linkages present on these proteins. in contrast, the dub votu is capable of cleaving all ubiquitin linkages except for the m1 linkage (akutsu., 2011). treatment with votu resulted in removal of all linkages from m1-affinity purified tnfr1, tradd, and rip1 (figures 5b and s5d). as this includes the ubiquitin moieties through which m1 chains are linked to these proteins, a ladder of linear ubiquitin chains that are shed from these proteins by votu treatment becomes apparent. as they are completely hydrolyzed when otulin is added together with votu, it can be concluded that these chains are pure m1-linked chains (figure 5b). importantly, no linear ubiquitination of rip1, tnfr1, or tradd was observed in cells lacking hoip (figure s5d). these results identify tnfr1 and tradd as previously unrecognized, additional bona fide targets of lubac in the tnf - rsc. in addition, they show that the linear chains present on these targets are of considerable length. as absence of cyld enhanced m1 ubiquitination of tnf - rsc components, we assessed whether the recombinant usp domain (aa 583956) of cyld (cyld - usp), encompassing its catalytic dub activity, could be capable of removing ubiquitin chains from them. isolation of ubiquitinated proteins following tnf stimulation and subsequent treatment with recombinant cyld - usp or votu showed that cyld s dub domain is capable of removing the majority of overall ubiquitination from tnfr1, tradd, and rip1 (figure 5c). the fact that treatment with cyld, in contrast to treatment with votu, did not release m1 chains demonstrates that cyld indeed hydrolyzes m1 linkages present on these target proteins (figure 5c). thus, cyld acts as a dub that antagonizes both linear and k63 ubiquitin linkages in the tnf - rsc. as cyld was previously described to be a positive regulator of tnf - induced necroptosis (odonnell., 2011) and because we previously showed that linear ubiquitination in the tnf - rsc protects from tnf - induced cell death (peltzer., 2014), we next wanted to assess the impact of cyld s activity on m1 chains in the tnf - rsc in relation to tnf - induced cell death. in line with previous studies, we found that cyld - reconstituted but not -deficient mefs were prone to tnf- and tnf / zvad - induced cell death (figure 5d). importantly, this coincided with a decrease in both linear ubiquitination of tnf - rsc components and tnf - induced gene activation in cyld - reconstituted mefs (figures 5e and 5f). thus, cyld - mediated removal of ubiquitin chains, including of linear chains, from components of the tnf - rsc results in diminished tnf - induced gene activation and, at the same time, enhanced cell death. another major dub involved in tnf the znf4 domain of a20 selectively recognizes k63-linked ubiquitin, and mutations in this domain were reported to impair a20 recruitment to the tnf - rsc (bosanac., 2010, lu., 2013). interestingly, the znf7 domain of a20 is involved in a20 s ability to suppress nf-b and cell death (tokunaga., 2012, verhelst., in addition, this domain binds with high affinity to linear ubiquitin chains, and tnf - rsc recruitment of a20 devoid of znf7 is reduced (tokunaga., this prompted us to study the biochemical and functional interplay between lubac, linear ubiquitination, and a20 in tnfr1 and nod2 signaling. a20 is induced by various stimuli in an nf-b - dependent manner (catrysse., 2014). in all cell lines tested, we observed, however, that a20 was already present before stimulation. consequently, it formed part of the tnf - rsc already 5 min after tnf stimulation, yet interestingly only in wt but not hoip - deficient cells (figures 6a, s6a, and s6b). prolonged stimulation of up to 3 hr resulted in increased a20 expression and recruitment to the tnf - rsc, again only in control and not in hoip - deficient cells (figure s6c). a20 functions as a negative regulator of nod2 signaling (hitotsumatsu., 2008). we therefore next analyzed the nod2-sc for a20 presence and, if there, what the role of lubac would be in its recruitment. this analysis revealed that a20 forms part of the nod2-sc and that hoip is required for this (figure 6b). as lubac deficiency reduces gene activation, we next addressed the role of hoip s enzymatic activity in induction of a20. as expected, hoip - deficient cells showed substantially decreased activation of nf-b and, consequently, reduced production of ccl2 and il-8 as well as a comparably weak upregulation of a20 upon tnf stimulation (figures 6c6e). in cells expressing catalytically inactive hoip - c885s the tnf - induced stimulation of gene - activatory signaling pathways and cytokine production interestingly, these cells had increased basal expression of a20 before stimulation, but 3 hr after stimulation the a20 levels were almost identical to the ones observed in hoip - wt cells (figure 6e). to evaluate whether the enzymatic activity of hoip is required for a20 recruitment to the tnf - rsc, we therefore compared the tnf - rsc that forms after 3 hr in cells expressing hoip - wt and hoip - c885s. this showed that hoip - c885s expressing cells are almost completely defective in a20 recruitment to the tnf - rsc (figure 6f). together, this identifies lubac - generated m1 chains as required for recruitment of a20 to the tnf - rsc. even though a20 is unable to cleave m1 linkages, we hypothesized that it could affect m1 chains in scs indirectly through cleavage of other linkages. we therefore next assessed the impact of a20 absence on the presence of linear ubiquitin chains in the tnf - rsc. to do so, we again employed crispr - cas9, this time to generate a20-deficient a549 cells. unexpectedly, rather than being increased, m1 ubiquitination was markedly decreased in the tnf - rsc of a20-deficient as compared to control cells, a finding that also applied to tnfr1 (figure 7a). a20 deficiency also reduced the amount of m1-linked ubiquitin present in the nod2-sc (figure 7b) and on rip2 in this complex (figure s7b). finally, also mefs deficient in a20 showed a marked reduction in linear ubiquitination at the tnf - rsc (figure s7c). even though this result made it unlikely that a20 s dub activity was responsible for this effect, we could not formally exclude it based on the experiments performed so far. independently thereof, we reasoned that because a20 is recruited to scs via linear ubiquitin chains, this interaction could be responsible for their a20-endowed stabilization. since a20 s znf7 was known to bind m1 chains and to be required for tnf - rsc recruitment (tokunaga., 2012), we tested whether znf7 could be responsible for a20-mediated stabilization of linear ubiquitin chains at the tnf - rsc. to do so, we used crispr - cas9 to prepare cells lacking only the znf7 domain of a20 (figure s7d). in line with previous results (tokunaga., 2012), we found in both a549 and hacat cells that absence of znf7 severely compromised recruitment of a20 to the tnf - rsc and accumulation of ubiquitin chains therein (figures 7c and s7e). to assess whether the dub activity of a20 or its m1-binding function was responsible for its capacity to stabilize m1 linkages in the tnf - rsc, we reconstituted a20-deficient mefs with a20-wt, dub - inactive a20 (a20-c103s), or an a20 znf7 point mutant c779a / c782a (a20-znf7mut), which is unable to interact with m1 chains (tokunaga., 2012). both a20-wt and a20-c103s, but not a20-znf7mut, were recruited to the tnf - rsc and stabilized linear ubiquitin linkages within this complex (figures 7d and s7f). thus, the linear - ubiquitin - binding activity of a20, but not its dub activity, is required for stabilization of linear chains. in summary, these results show that direct binding of a20 to linear ubiquitin chains enables recruitment of a20 to the tnf - rsc and that this, in turn, results in stabilization of m1-ubiquitin chains in this complex. the observation that znf7 is necessary for recruitment of a20 to the tnf - rsc fits with the finding that a20-wt and a20-c103s, but not a20-znf7mut, inhibit tnf - induced gene activation (figures 7e and s7 g) and protect cells from tnf / zvad - induced necroptosis (figure 7f) (yamaguchi and yamaguchi, 2015). hence, the activity of a20 as a binder and stabilizer of linear ubiquitin linkages appears to be functionally more significant than its activity as a dub. our analysis of lubac obtained from non - stimulated cells confirmed previous reports that cyld and otulin bind to the pub domain of hoip (takiuchi., 2014). to our surprise, we found that, unlike cyld, otulin formed part of neither the native tnf - rsc nor the nod2-sc. (2013) reported that transient overexpression of flag - tagged nod2 resulted in its interaction with otulin. however, this system does not require stimulation by a nod2 ligand, and it is therefore possible that the detected interaction could be a non - physiological event related to nod2 overexpression. even though we were able to detect cyld in two different scs, including the tnf - rsc, in all cell lines studied, we never found otulin to be recruited. these results implied that sc recruited hoip was not associated with otulin, despite the fact that otulin and hoip interacted prior to stimulation. indeed, we found that otulin antagonizes lubac - mediated linear ubiquitination in the cytoplasm but not at scs. the function of otulin is therefore likely to prevent accumulation of m1-ubiquitin linkages outside of scs (figure 7 g). aiming to find a mechanistic explanation for the unexpected absence of otulin from scs, we first turned our attention to phosphorylation, as phosphorylation of otulin tyr56 was previously reported to be capable of disrupting the interaction between hoip and otulin (elliott., 2014). our data, however, show that phosphorylation is not responsible for otulin absence from scs. the explanation was provided by our discovery that hoip can not simultaneously bind otulin and cyld as both require hoip - asn102 for binding. we next demonstrated the existence of two separate pools of cytoplasmic lubac : one associated with otulin, the other one with cyld. importantly, whereas cyld - bound lubac is recruited to the tnf - rsc and the nod2-sc, otulin - associated lubac is not. it will be interesting to determine how otulin s interaction with hoip prevents its recruitment to scs, e.g., whether otulin binding could interfere with hoip s ability to bind ubiquitin chains, a requirement for lubac recruitment to scs (gerlach. we show here that cyld is recruited to scs due to its interaction with hoip, independently of lubac s enzymatic activity. furthermore, in cells expressing hoip - n102a, which can interact with neither cyld nor otulin, cyld is not recruited to the tnf - rsc, demonstrating that cyld s interaction with hoip is essential for cyld recruitment. cyld is a dub with broad specificity, yet the enzymatically active usp domain of cyld, when produced recombinantly, was previously shown to most efficiently hydrolyze m1- and k63-linked tetra - ubiquitin in vitro (komander., 2009). it has been unclear to date, however, whether endogenous cyld would be able to regulate m1-ubiquitin, in addition to its previously demonstrated role as a dub that cleaves k63 linkages on targets within the tnf - rsc (brummelkamp., 2003, kovalenko., 2003, trompouki. employing newly devised protocols for k63-ap and m1-ap, we demonstrate here that, as a consequence of cyld deficiency, both k63- and m1-ubiquitin are increased on several components of the tnf - rsc, including tnfr1, tradd, and rip1. furthermore, the recombinant usp domain of cyld completely removes m1 linkages, and indeed the majority of other linkage types, from components of the tnf - rsc. importantly, m1 linkages are fully hydrolyzed and not merely released from complex components, as no free m1 chains appear following treatment with cyld. collectively, these results identify cyld as an antagonist of linear ubiquitination in scs, in addition to its previously described role as an antagonist of k63-linked ubiquitination (figure 7h). we show that reconstitution of cyld - deficient mefs with cyld, but not with vector control, decreases m1-ubiquitin in the tnf - rsc. this is accompanied by decreased gene activation, yet interestingly, also with enhanced tnf - induced cell death. this is in line with the fact that cyld has previously been described as being both an inhibitor of gene activation and promoter of cell death, the latter by enhancing complex ii formation upon tnf stimulation (hitomi., 2008, we previously showed that lack of linear ubiquitination in the tnf - rsc enhances formation of complex ii of tnf signaling (peltzer., 2014). based on the results presented here, cyld requires hoip for tnf - rsc recruitment. hence, in cells lacking hoip, cyld does not form part of the tnf - rsc. consequently in these cells, cyld can not mediate the transition from complex i of tnfr1 signaling (i.e., the tnf - rsc) to complex ii. decisively, however, in hoip - deficient cells this event does not require cyld activity ; it readily occurs without it (peltzer., this identifies the lack of m1 chains in complex i as decisive to render this complex unstable so that complex ii can readily form. we thus conclude that the m1-chain - antagonizing activity of cyld in complex i of tnfr1 signaling is responsible for its pro - cell death role. whether this is due to direct cleavage of m1 chains and/or their indirect removal through the previously demonstrated cleavage of k63 linkages that are extended by linear chains remains to be determined. equally, whether removal of a specific m1 chain from a particular target enables complex ii formation, or multiple such events together account for it, remains to be resolved. with respect to a20, we show that in absence of hoip or m1-ubiquitin a20 recruitment to scs is almost completely prevented and that genomic deletion of a20 s znf7, in turn, drastically impairs recruitment. thus, lubac, by placing m1 chains on sc components, recruits a20 to these scs via its znf7 (figure 7h). these results are in line with the previous finding that a20 can bind to m1-ubiquitin via its znf7 domain (tokunaga. additionally, they provide the biochemical explanation for the observation that tnf - rsc recruitment of a20 lacking znf7 was significantly reduced (tokunaga., 2012). even though a20 was shown to be incapable of cleaving m1 chains (mevissen., 2013), we were surprised to find that a20 significantly stabilized m1-ubiquitin in scs. given that m1 chains are required for full gene - activatory signaling (fiil and gyrd - hansen, 2014, iwai., 2014, walczak., 2012) and that a20 is a dub previously described to inhibit nf-b signaling (bosanac., 2010, tokunaga., 2012, wertz., 2004), this finding seemed counterintuitive, at first. indeed, results obtained in reconstitution experiments showed that it is znf7 and not a20 s enzymatic dub activity or znf4 that is required for a20-mediated restriction of gene activation (skaug., 2011). combined with our observation that znf7 is required for a20 recruitment to scs due to its interaction with lubac - generated m1 chains, which are, in turn, stabilized by it, these findings now offer a possible alternative explanation for a20 s nf-b - inhibitory activity. a20 binding to m1 chains in scs could compete with the binding of other gene - activatory factors, e.g., the nemo / ikk complex, to them. rising levels of a20 protein, induced as a consequence of nf-b activation, would render m1 chains less available for nemo / ikk retention over time so that the negative feedback loop would be completed. a20 has also been implicated as a negative regulator of tnf - induced cell death (lee., 2000, yamaguchi and yamaguchi, 2015), whereas cyld was shown to promote it (hitomi. thus, while they cooperatively restrict gene activation from various scs, they act in opposing ways on tnf - induced cell death. linear ubiquitination in the tnf - rsc prevents tnf - induced cell death by restricting complex ii formation (gerlach., 2011, ikeda., 2011, we show here that a20 binding to m1 chains in the tnf - rsc stabilizes them, whereas cyld antagonizes m1 chains in this complex. together this implies that the ability of a20 to bind m1 chains protects them from cleavage by dubs that are capable of cleaving linear ubiquitin chains in signaling complexes. having identified herein cyld as a dub with precisely this activity at the tnf - rsc, we therefore propose a model according to which a20 protects m1 chains in the tnf - rsc from cyld - mediated cleavage, thereby providing the sought after explanation for the opposing roles played by a20 and cyld with regards to tnf - induced cell death. intriguingly, both roles depend on lubac - generated linear ubiquitin in the tnf - rsc. hence, an intricate interplay between lubac, linear ubiquitination, a20, and cyld is crucial for assembly and disassembly of scs to enable efficient, yet properly controlled, immune signaling. for description of cell lines, antibodies, and plasmids, see supplemental experimental procedures. tap - tnf, untagged tnf, recombinant deubiquitinases, and proteins used for m1-ap, k63-ap, and ubi - ap were produced in e. coli. hoip- and hoil-1-deficient k562 and hoip - deficient a549 and hela cell lines were prepared by transfecting mrna encoding gene - specific zinc finger nucleases (sigma). cyld-, otulin-, a20-deficient, and a20znf7-expressing cells were prepared by lentiviral transduction with lenticrispr v2 vectors (sanjana., 2014) provided by feng zhang (addgene plasmid # 52961). coding sequences of hoip - wt, hoip - c885s, hoip - n102a, deletion mutants of human hoip fused or not at the c terminus to the tap - tag and tap - tagged nod2 (aa 281040) were inserted into the retroviral mscv vector containing gfp as selection marker. upon infection, cells were sorted using moflo facs (beckman coulter). samples were subsequently diluted to 0.1% sds before m1-, k63-, or total ubiquitin - specific recombinant affinity protein coupled to halo beads was added for overnight incubation at 4c. beads were washed, and samples were subjected to treatment with 1 m recombinant deubiquitinase for 1 hr at 37c or eluted with reducing sample buffer. to analyze the native tnf - rsc, after cell lysis, samples were subjected to anti - flag immunoprecipitation using m2 beads (sigma) or incubated with protein a / g - agarose beads (santa cruz biotechnology) coupled to indicated antibodies. for pervanadate treatment, cells were incubated with 1 mm pervanadate for 20 min before lysis. phosphatase treatment was performed on lysates using 50u of fastap (thermo scientific) per mg of protein in absence of phosphatase inhibitors. performed experiments ; s.s. performed mass spectrometric analysis of tap - purified lubac ; l.t. and e.r. | summaryubiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes. lubac - generated linear (m1) ubiquitin is important for signaling via various immune receptors. we show here that the deubiquitinases cyld and a20, but not otulin, are recruited to the tnfr1- and nod2-associated signaling complexes (tnf - rsc and nod2-sc), at which they cooperate to limit gene activation. whereas cyld recruitment depends on its interaction with lubac, but not on lubac s m1-chain - forming capacity, a20 recruitment requires this activity. intriguingly, cyld and a20 exert opposing effects on m1 chain stability in the tnf - rsc and nod2-sc. while cyld cleaves m1 chains, and thereby sensitizes cells to tnf - induced death, a20 binding to them prevents their removal and, consequently, inhibits cell death. thus, cyld and a20 cooperatively restrict gene activation and regulate cell death via their respective activities on m1 chains. hence, the interplay between lubac, m1-ubiquitin, cyld, and a20 is central for physiological signaling through innate immune receptors. |
lead - free materials are of interest as new candidates to interchange the widely used lead - based ceramics owing to their pollution free environmental friendly character throughout the preparation process. materials that can absorb microwaves will eliminate electromagnetic radiation pollution. wide unfolding applications of electromagnetic absorbers have affected engineers to explore relating to optimum design without their algorithms. the dielectric, electric, acoustic, mechanical, temperature, magnetic, and optical properties of these materials are used in a wide number of electronic applications. components based on ferroelectric phase have a wide range of commercial applications like memory cells sensors, actuators, and so forth. perovskites are excellent dielectrics characterized by extraordinarily high dielectric permittivity that rely on the ferroelectric technology for microwave applications are creating their way to the industry and commercial applications, like wireless sensor networks, safety and security systems, automotive, medical, environmental food monitoring, radio tags, and so forth. good dielectrics and electric field dependent permittivity make the parametric phase ferroelectrics attractive for the development of a wide range of tunable microwave devices for applications in agile microwave systems. the materials ' property from engineer 's perspective, device, and system (circuit) applications of the ferroelectrics plays very important role. recent dramatic changes in microelectronics and in particular wireless communications technologies have made the importance of materials with the unusual combination of high dielectric constant, less dielectric loss, and low temperature dependence of dielectric constant of great interest. relaxor ferroelectrics exhibit a high - dielectric constant over a wide temperature range around the ferroelectric phase transition. ferroelectric srxba1xnb2o6 (0.25 x 0.75) with the ttb (tetragonal tungsten bronze) structure has attracted a great deal of attention and is being investigated as a potential material for pyroelectric, electro - optic, and photorefractive devices [3, 4 ]. recently, it was reported that the hexagonal phase of banb2o6 transforms above 1200c to the orthorhombic structure. these microwave dielectrics can be synthesized by several roots syntheses like chemical methods, the coprecipitation, sol - gel, and hydrothermal and colloid emulsion techniques [59 ]. the purpose of this study was to prepare banb2o6 ceramics using simple low cost solid state technique from simple inorganic materials. a detailed significant investigation of the structural, microstructural, and mechanical properties of barium niobate has been reported. electromagnetic transmittance and absorption of samples in the x band frequency range frequencies using waveguide reflectometer technique are reported. ar grade chemicals of high purity barium carbonate (baco3 (99.95%)) and niobium penta - oxide (nb2o5 (99.999%)) were used as starting materials. this powder was again mixed in stoichiometric proportion and ground for 4 hours in acetone medium to obtain the desired compound. this mixture was initially sintered at 1200c for 10 hrs and further at 1000c for 24 hours in a muffle furnace (given by (2). the flowchart of barium niobate (banb2o6) ceramic is shown in figure 1 : (1)baco3+nb2o5banb2o6+co2 the phase analysis was confirmed by x - ray diffraction using cr - k radiations (philips diffract meter pw 3710). the structural parameters such as the lattice constants, average crystallite size, and texture coefficients this powder was pressed into pellets in a hydraulic press at 10 ton / cm for 10 minutes. the surface morphology was studied using scanning electron microscope (sem jeol - jsm 6360). transmission of microwaves due to bulk sample was measured point by point using transmission / reflection method with rectangular waveguide, consisting of the x band generator, isolator, attenuator, directional coupler, and rf detector. x - ray diffraction technique is a powerful tool to analyze the crystalline nature of the materials. if the material to be investigated is crystalline, well defined peaks will be observed. x - ray diffractogram of barium niobate 2 values from 20 to 90 is shown in figure 2. the barium niobate has tetragonal structure with lattice parameters a = b = 12.34 and c = 3.89 (table 1). nanocrystalline (particle size) characteristics of the samples depend on the broadening of the xrd lines. average particle size of the calcined powder was 37.5 nm, determined using debye - scherer formula : (2)d = kcos, where k is scherrer constant (k = 0.9), is bragg 's angle, and is full - width half maxima (fwhm) in radians. dislocations are 1d crystalline defects marking the boundary between slipped and unslipped regions of material. the amount of defects in the as - deposited film is assessed by dislocation density (d). it is defined as the deformation of an object divided by its effective - length. the dislocation density (d) and microstrain () were calculated as (3)dislocation densityd=1d2,(4)=cos4. the williamson - hall equation is used to calculate the strain () and particle size of the sample graphically given by [11, 12 ] (5)cos=0.9d+4sin, where is the lattice microstrain, d is the grain size (in), is the wavelength of the radiation (in), is the bragg angle, and is full - width half maxima (fwhm) of a xrd peak in degree. using a linear extrapolation fit to williamson - hall analysis plot (shown in figure 3), the intercept gives the particle size (d) and the slope represents the strain (). microstrain (), crystallite size (nm), and dislocation density (d) are tabulated in table 2. texture is perceived in almost all engineered materials which can have a great influence on properties of materials. diffraction patterns from samples containing a random orientation of crystallites have predictable relative peak intensities. texture frequently represents a pole figure, in which a defined axis (crystallographic) from each of a representative number of crystallites is mapped in a stereographic projection. quantitative data concerning the preferential crystal orientation can be obtained from the texture coefficient (tc) : (6)tchkl = ihkl / i0hkl1/nnihkl / i0hkl. if the crystallographic orientations are fully random, then the sample has no texture. if the orientations are not random but have some preferred orientation, then the samples have a weak, moderate, or strong texture. as tc(hkl) increases, the preferential growth of the crystallites in the direction perpendicular to the hkl plane is greater (table 3). from the texture analysis preferential grain growth which is observed at (210) plane is more dominant (tc = 2.977) and that observed at (311) plane is weak. the morphology of barium niobate (banb2o6) is like typical pebble (beach stone) structure (shown in figure 4). the image shows homogenous grains distributed over the entire volume of the samples and shows good crystallization. it is clear from the micrographs that the grains are densely packed in the sintered sample. however, a certain degree of porosity is still observed. the shape and distribution of grains in the microstructure exhibit the polycrystalline nature of the sample. the grain size of the sample (obtained from sem) is larger than of the crystallite size obtained from scherrer 's equation. the advantages of solid - state reaction are simplicity and low cost, but the disadvantages are that the high calcining temperature results in very large grain sizes confirmed by sem images. the infrared spectral analysis is effectively used to understand the chemical bonding and it provides information about molecular structure of the synthesized compound. the characteristic absorption peaks in the range from 400 to 4000 cm are shown in figure 5. using perkin elmer spectrophotometer, fourier transform infrared (ftir) spectra of sample (in pellet form mixed with kbr) were recorded in the range 4004000 cm. the sharp absorption peaks at ~3470 cm indicating the presence of hydroxide group (oh) result from surface - adsorbed atmosphere (like moisture and humidity). the strong absorption bands at ~816 cm, 641 cm, and 482 cm are associated with the coupling mode between nb o stretching modes. the insertion loss (transmission) and absorption loss of barium niobate sample were measured by the rectangular waveguide reflectometer setup shown in figure 6. the microwaves were incident on the device under test (dut) in the frequency range 8 ghz to 12 ghz (x band). the waveguide reflectometer setup consists of gunn oscillator, isolator, attenuator, two 3 db directional couplers connected in reverse directions, sample holder for device under test (dut), and the diode detector. the system was calibrated by measuring the output with and without the dut [4, 17 ]. the variation of insertion loss and absorption loss of barium niobate is a function of frequency in the x band (812 ghz) which exhibits trivial wavy like nature shown in figure 7. the average absorbance is relatively more compared to transmittance. at 9.8 ghz frequency, dip absorbance (53 db) may be the motion of active ferroelectric nbnb also because the frequency of the hopping ions could not follow the applied field frequency and it lags behind. absorption is the heat loss under the action between electric dipole or magnetic dipole in material and the electromagnetic field. these barium niobates may be used to function as sensors, actuators, detectors, filters, resonators, and so forth through special layout arrangements. lead - free ferroelectric barium niobate that has been synthesized by the solid state reaction method was investigated. it absolutely was shown by x - ray diffraction that the space temperature shows tetragonal structure, lattice parameters a = b = 12.343 and c = 3.889, and average particle size was 37.5 nm with preferred (210) textured orientation. pebble (beach stone) like morphology with grain size varied within the range of 24 m, confirmed by sem. the robust absorption bands at ~816 cm, 641 cm, and 482 cm are related to the coupling mode between nb o stretching modes. microwave studies (absorbance and reflectance) depict periodical behaviour which can be used to perform like sensors, actuators, detectors, and filters. | the structural and microwave studies of lead - free barium niobates ceramics prepared by the high temperatures solid state reaction technique are reported. the structural parameters such as the lattice constants, average crystallite size (d), texture coefficients (tc), dislocation density, and microstrain have been determined using x - ray diffraction data. surface morphological studies were carried out using scanning electron microscopy (sem) technique. the strong absorption bands at ~816 cm1, 641 cm1, and 482 cm1 are associated with the coupling mode between nb o stretching modes observed in ftir studies. the electromagnetic transmittance, absorption, studies of barium niobates in the x band frequency range frequencies using waveguide reflectometer technique are reported. |
mechanical amplification is something we experience every day, in the form of gears, pulleys, and levers. while climbing a hill on a bicycle, for instance, shifting gears increases the force on the wheels while limiting the pressure required on the pedals. however, energy has to be conserved, and because mechanical work is defined as force displacement, an increase in force can only be obtained at the expense of displacement. thus, although shifting gears allows one to develop the additional force needed to go uphill, speed is reduced as each pedal stroke produces a smaller turn of the wheels. cells have similarly developed microscopic force amplification strategies during evolution. here, we discuss some amplification schemes for one of the major force generators in the cell actin polymerization. actin plays a ubiquitous role in cell motility and morphogenesis, spanning many scales of space and time. in fission yeast, for example, a miniature actin machinery only 100 nm across can induce the invagination of an endocytic vesicle in just a few seconds (picco., 2015. however, to sever the entire yeast cell, a cytokinetic ring forms with an initial perimeter of 10 m and requires 30 min to drive division (proctor., 2012). in other species, considerably larger actin assemblies exist that reach the scale of centimeters, such as in muscle cells. clearly, actin and its associated factors need to be specifically organized to achieve these different functions (fig. 1). from a functional point of view, a key problem is to understand how the global architecture of an actin network allows forces that are produced at the molecular scale to be productive for the cell. in this respect, we can distinguish two sorts of components. active components generate forces from chemical sources of energy and include molecular motors, as well as actin itself, which can push by polymerizing (kovar and pollard, 2004) and possibly pull while depolymerizing. passive components, such as actin cross - linkers, are essential but can only transmit forces generated by other elements. networks of actin filaments are essential for many biological processes at the cellular level, and the organization of the filaments in space must be adapted to the task. here, polymerization force (orange) of actin filaments (red) occurs near the plasma membrane (blue). linear filopodia bundles with fascin (black) can produce high speeds, but represent a weak configuration for force generation. lamellipodia are thin cellular extensions in which filaments are nearly parallel to the substrate on which the cell is crawling. the 2d branched network, created by arp2/3 actin - nucleating complexes (black), can produce higher forces at the expense of displacement. during endocytosis in yeast, actin forms a 3d network at the site of the invagination that appears roughly spherical, but the organization of actin filaments in space is not known. the coat structure (yellow) enables actin to pull the membrane inward and actin polymerizes near the base of the structure, where arp2/3 nucleators are shown in black (picco., 2015). the forces developed by an actin meshwork are determined by the organization of its components. ultimately, these forces must be sufficient to drive biological processes, and thus their scale depends on the physical characteristics of the cell. for example, in the case of endocytosis in yeast, the turgor pressure pushing the surface of the invagination outward reaches 1,000 pn, which the actin machinery must overcome (basu., 2014). during cytokinesis, the actomyosin ring also works against the turgor pressure, which produces high forces on the furrow (proctor., 2012). for both cases, these forces have been calculated from measured cellular parameters, particularly the turgor pressure and the dimensions over which the membrane is deformed. hence, for these processes at least, the two ends of the problem are known : the forces produced by the molecular components make up the input and the force required for the cellular process to occur represents the output. yet the force balance within the system must be considered to understand how the actin machinery harvests the input to produce this output. in this comment, we focus on the transmission of forces produced by the polymerization of actin, setting aside turnover and the contribution of molecular motors. we discuss specifically how the arrangement of the filaments in the system regulates the amount of productive force. in many ways, the actin machinery behaves analogously to a cyclist : though its power is limited, it can shift gears to favor either more displacement (high gears) or more force (low gears). indeed if an actin monomer in solution binds the barbed end of a filament, there is a change of free energy (gp) and polymerization will occur if gp 10 pn (mogilner and rubinstein, 2005) to overcome membrane tension and rigidity. in a filopod if the load is distributed over all barbed ends, then each end sustains a fraction of the total force (f / n). extension will then be possible only if the polymerization force is larger than the fraction of force experienced by each filament (f / n < fa) and thus requires sufficient barbed ends to distribute the force i.e., it is the highest gear of the actin machinery. assembling more filaments can increase the force, but because the molecular forces are always equal to the productive force, there is no mechanical amplification. if each filament can produce the same amount of force parallel to its axis, the push on the membrane can be higher as a result of the contact angle (usually = 54) at which actin filaments encounter the membrane (fig. parallel to the axis of a filament corresponds to a proportional force perpendicular to the membrane (fa / sin). the total pushing force (f) on the membrane, then, is the sum of such perpendicular forces applied by n filaments (f = n fa / sin). because sin(54) < 1, the productive force is increased. this occurs at the detriment of displacement achieved by each actin monomer, which is also proportional to the contact angle (sin). importantly, the contact angle is not solely determined by the branching angle imposed by arp2/3, the primary nucleating complex for branched actin filaments, because the branched network can adopt different orientations with respect to the leading edge (weichsel and schwarz, 2010). thus, this quasi-2d system works like a gearbox, where the coefficient (sin) can vary, allowing a lamellipod to generate nanonewton scale forces (prass., 2006) 2 e, in which two asymmetrically branched filaments engage the membrane, but only the long branch polymerizes whereas the short branch provides support by transmitting force between the membrane and the filament network. upon polymerization, the whole construction rotates around a pivot point at the base of the supporting branch, and the contact angle of the polymerizing filament becomes shallower in comparison to the symmetrically polymerizing configuration. strikingly, this configuration can develop more force than the symmetric case, as an additional amplification (x + y)/x is associated with the lever arms (compare fig. this illustrates that the network force is not solely proportional to the number of polymerizing barbed ends. the geometry of the system, particularly the angle at which the filaments contact the membrane, and the lever arms can further affect and amplify the total forces generated by the network. to interpret in vitro experiments in which actin polymerizes around beads (achard., 2010 ; dmoulin., 2014), it has been suggested that resistance from a load could cause actin to polymerize parallel to the surface. in this simple configuration, a filament is confined between a base and a load, which is pushed upward as the filament grows (fig. 2 f). the upward displacement of the load is determined by the thickness of the actin filament () and by the lever arms x and y, relative to the pivot point. 2 e, but the new device offers better performance ; whereas the long filament in fig. 2 e can bend all the more as it elongates, this configuration works well even with flexible filaments. in the geometry suggested by fig. 2 f, the load is lifted by the filament thickness once the filament has polymerized over the entire base. in a more realistic 3d network, the relationship between polymerization and displacement will not be as simple, because the arrangement of filaments in 3d networks is intricate. nevertheless, the mechanical concepts remain valid and, in particular, polymerization parallel to a surface could lead to strong orthogonal forces. in yeast endocytosis, actin polymerizes at the bottom of the network in a configuration resembling the wedge (picco., 2015). this may perhaps resolve the apparent mismatch between the number of polymerizing filaments and the force resulting from pressure (basu., the force generated by the network depends critically on the network architecture, as this determines the constraints under which filaments grow (carlsson and bayly, 2014). in general, the force that can be exerted on a load will also depend on the mechanics of the entire structure. network elasticity allows the polymerization force to be stored as stress, whereas stress relaxation by disassembly and turnover will decrease the force the network can exert (zhu and mogilner, 2012). in 1d structures, such as filopodia, force balance forbids mechanical amplification ; however, in 2d structures, the contact angle between the barbed end and the membrane provides a mechanism for tradeoff between force and displacement, and thus allows for force amplification. configurations in which filaments grow parallel to the membrane, and thus act like wedges, produce the highest forces. of course, energy conservation dictates that displacement is reduced as force is increased, such that there is a a key parameter of our considerations is the force that a polymerizing actin filament can support (fa). energetic consideration provides an upper bound of 9 pn, but so far direct measurements have yielded lower values, around 1 pn. thermal fluctuations provide a scale to which this can be compared. at a given temperature (t), the characteristic energy associated with thermal fluctuations is kbt, where kb is the boltzmann constant ; at room temperature, the associated force (kbt/) corresponds to 1.5 pn. hence, if fa is truly 1 pn, it would imply that actin polymerization is hardly more efficient than thermal fluctuations. it is to be hoped that future experimental studies, possibly closer to in vivo conditions, will reveal higher forces, as it would be truly astonishing if actin used only 10% of the available energy. in conclusion, the architecture of a network determines the productive force, often in a nonintuitive manner. hence, once a system has been well characterized experimentally, mechanical theory should be used to balance the forces within the network. when this can not be done, energetic considerations, in which the mechanical work of the forces are summed and compared, are informative. a thorough analysis of force transduction in the system makes it possible to predict the most efficient architecture for performing a given task (ward., 2015), which is of outstanding value when comparing different modus operandi across species. | the actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. we discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. |
initiation of an multimodal, customer focused service network across the service sectors for multimorbid patients : for example the hard - of - hearing. the lack in accessibility of services, flexibility of appointments and handicap - oriented communication skills often prevents the geriatric patient from partaking in the medical services offered. the geriatric hearcare service and its care for the hard - of - hearing outlines the possibilities of an integrated, multimodal setting at all levels of care for these patients. the scientific evaluation of > 2000 hard - of - hearing patients in geriatric care from 2002 to 2008. upon standardized admission patients are screened for a history in hard - of - hearing. organised and accompanied by a specially trained hearcare - nurse, a patient with a possible auditive decline is then seen by an ent and hearing - aid technician for examination and audiometry. the patient is then seen by an neuropsychologist for a cognitive evaluation. after these tests, documented by the hearcare - nurse, the patient and his next - of - kin are informed of the outcome, the treatment options and follow - up in the out - patient setting. the integration of services across sectors of care and the provided co - ordination leads to a higher degree of customer satisfaction, a lesser rate of cases of hard - of - hearing missed and better continuity in care on a long - term basis. | purposeinitiation of an multimodal, customer focused service network across the service sectors for multimorbid patients : for example the hard-of-hearing.contextthe lack in accessibility of services, flexibility of appointments and handicap - oriented communication skills often prevents the geriatric patient from partaking in the medical services offered. the geriatric hearcare service and its care for the hard - of - hearing outlines the possibilities of an integrated, multimodal setting at all levels of care for these patients.data sourcethe scientific evaluation of > 2000 hard - of - hearing patients in geriatric care from 2002 to 2008.case descriptionupon standardized admission patients are screened for a history in hard - of - hearing. organised and accompanied by a specially trained hearcare - nurse, a patient with a possible auditive decline is then seen by an ent and hearing - aid technician for examination and audiometry. the patient is then seen by an neuropsychologist for a cognitive evaluation. after these tests, documented by the hearcare - nurse, the patient and his next - of - kin are informed of the outcome, the treatment options and follow - up in the out - patient setting.conclusion and discussionthe integration of services across sectors of care and the provided co - ordination leads to a higher degree of customer satisfaction, a lesser rate of cases of hard - of - hearing missed and better continuity in care on a long - term basis. |
a 74-year - old female patient visited a pain clinic, complaining of pain in her back, and left chest wall. six years earlier, the patient had a lateral fixation at t12, l1, and l2, due to a compression fracture at l1. two years after this operation, the patient had osteoporotic compression fractures at t6 and t9. for the past three months, the patient had complained about pain at an nrs 7 on the left chest wall, which started without any serious trauma. after the patient was diagnosed in the neurosurgery department, with compression fractures at t8 and t9, she was scheduled to undergo vertebroplasty. on the physical examination, the patient stated that the left paraspinal muscles at the height of t8-t10 were tender when compressed, but did not have any percussion pain or facet joint tenderness. when the spine was bent forward, the patient complained about a sharp pain in the left mid - axillary line and radiating pain toward the abdomen. however, the patient did not have any tenderness in her ribs, and the pain was not elicited when she took a deep breath or coughed. on palpation, the 10 rib was not felt, and the rib - cage image confirmed that the left 10 rib was severed (fig. we had determined that perhaps the 10 rib was severed during the last operation 6 years previous. when applying pressure to the 10 rib from the legs to the lower margin of the 9 rib of the patient (the hooking maneuver), the pain was reproduced. therefore, we assumed that the patient had 9 rib syndrome, similar to 12 rib syndrome, and ultrasound - guided 9 and 10 intercostal nerve blocks using 2 ml of 0.25% ropivacaine and 10 mg of triamcinolone were performed around the tips of the severed 10 rib. in addition, 2 ml of 0.25% ropivacaine and 10 ml of triamcinolone were administered into the muscles beneath the 9 rib at the point of the greatest tenderness. no additional procedures were performed, but the patient was instructed to avoid postures that could cause pain. the pain has not occured again up to date, and the patient is only receiving only the treatment for osteoporosis. cyriax reported in 1919 that pain could be caused by impingement on the intercostal nerves, and in 1922 it was named the slipping rib syndrome by davies - colley. depending on the locations of the impinged intercostal nerves and the authors writing about the syndrome, various names were used to refer to the syndrome, including 12 rib syndrome, slipping rib syndrome, rib tip syndrome, clicking rib syndrome, cyriax 's rib syndrome, and painful rib syndrome. the mechanism that causes pain in the intercostal nerves is a hypermobile rib impinging on the intercostal nerve of the rib one level above due to the loosening of the costochondral cartilages of the lower rib. therefore, pain can be triggered or made more severe when patients perform lateral flexion and rotation of the trunk. slipping rib syndrome is a common disorder, and shows a higher prevalence among women than men by a ratio of 3:1. while the 12 rib syndrome occurs more frequently among women than men by a ratio of 4:1. scott. conducted a study targeting 76 patients with painful rib syndrome. the mean age of the patients at presentation was 48 years, and the mean duration of pain at presentation was 32 months. forty - three percent of the subjects had pain in the left side, 45% had pain in the right side, 4% had pain in both sides, and 7% had pain in the xiphoid area. among the entire study population, 43% said that they already underwent various tests, including barium meal, barium enema, endoscopy, ultrasound scanning, intravenous urography, and spinal radiology ; some patients had laparotomy and non - curative cholecystectomy in combination with them. if a patient tests positive using the hooking maneuver, the syndrome is suspected. if tender spots causing pain are felt upon palpation, and the pain can be relieved when intercostal nerve blocks are performed, this confirms the diagnosis of the syndrome. imaging tests such as ct, mri, or bone scan do not offer much help, and if a differential diagnosis is deemed necessary, tests for biliary tract pathology, peptic ulceration, and urological pathology can be performed. scott. stated, " with firm reassurance and explanation, the pain either disappears usually within three months, or the patient learns to live with it as a minor nuisance. if the pain does not settle within three months it will become chronic in the vast majority ". if a physician approaches patients without suspecting 12 rib syndromes, diagnosis will be delayed. so the patients ' activities will be limited by pain, the patient may lead to depression and anxiety, due to prolonged pain without knowing the cause, and the patient may suffer financial burdens due to unnecessary medical tests. the most important aspect of medical treatment is that physicians provide accurate information on diseases, give reassurance, and advise patients to refrain from physical activities, which may cause pain. if pain is not controlled by medication, an intercostal nerve block with local anesthetics and long - acting steroid infiltration to the tip of the affected rib can be performed. if the pain relief effect is not sustained, an intercostal nerve cryotherapy, costo - vertebral blocks, and a percutaneous dorsal root ganglion radiofrequency thermos - coagulation can be performed. if the pain is not controlled by the above procedures, a rib resection can be performed. in this case - study, it is suspected that the patient had a partial resection of the left 10 rib in the past, and subsequent compression fractures at t8 and t9 led to the deformation of the rib cage (fig. 3), causing the tip of the remaining 10 rib to impinge on the 9 intercostal nerves, causing pain. | the 12th rib syndrome is a disease that causes pain between the upper abdomen and the lower chest. it is assumed that the impinging on the nerves between the ribs causes pain in the lower chest, upper abdomen, and flank. a 74-year - old female patient visited a pain clinic complaining of pain in her back, and left chest wall at a 7 on the 0 - 10 numeric rating scale (nrs). she had a lateral fixation at t12-l2, 6 years earlier. after the operation, she had multiple osteoporotic compression fractures. when the spine was bent, the patient complained about a sharp pain in the left mid - axillary line and radiating pain toward the abdomen. on physical examination, the 10th rib was not felt, and an image of the rib - cage confirmed that the left 10th rib was severed. when applying pressure from the legs to the 9th rib of the patient, pain was reproduced. therefore, the patient was diagnosed with 9th rib syndrome, and ultrasound - guided 9th and 10th intercostal nerve blocks were performed around the tips of the severed 10th rib. in addition, local anesthetics with triamcinolone were administered into the muscles beneath the 9th rib at the point of the greatest tenderness. the patient 's pain was reduced to nrs 2 point. in this case, it is suspected that the patient had a partial resection of the left 10th rib in the past, and subsequent compression fractures at t8 and t9 led to the deformation of the rib cage, causing the tip of the remaining 10th rib to impinge on the 9th intercostal nerves, causing pain. |
pregnancy, delivery and puerperium can be complicated by severe maternal morbidity necessitating intensive care unit (icu) admission. management of the critically ill obstetric patient is very complex and requires cooperation of both obstetrician and intensivist / anaesthetist. one facility - based study has been performed in the netherlands, which reported an incidence of 7.6 per 1,000 deliveries. however, this study was inevitably biased by the long (12-year) inclusion period, during which technological and therapeutic changes have occurred. the primary aim of this study was to assess incidence, case fatality rate and possible risk factors of obstetric icu admission on a population - based national level. as ethnicity appeared to be a significant risk factor for severe maternal morbidity and maternal death, we were especially interested in the association of ethnicity with obstetric icu admission [13 ]. this study was part of a broader nationwide enquiry into severe maternal morbidity in the netherlands, called lemmon. in this study, which enrolled cases from 1 august 2004 until 1 august 2006, all dutch hospitals with an obstetric unit participated. this involves 10 tertiary care centres, 33 non - academic teaching hospitals and 55 general hospitals. all hospitals with an obstetric unit are equipped with an icu, subdivided into three levels. level 1 icus are equipped for monitoring and treatment of patients with single organ dysfunction, if necessary with assisted ventilation. patients with severe diseases can be monitored and treated at level 2 icus, and level 3 icus are equipped for patients with very complicated diseases with multiple organ dysfunction who need constant availability of an intensivist. according to the netherlands health care inspectorate, there are 49 level 1 units, 25 level 2 units and 24 level 3 units in the netherlands. in addition to a level 3 icu, all tertiary care centres are also equipped with an obstetric high care unit, which has one - to - one nursery care and cardiac monitoring, but no assisted ventilation. forty - one percent of all deliveries are considered low - risk pregnancies and take place under the responsibility of primary care providers, three quarters of which are home births. any complication occurring in primary care will be referred to a hospital and thus be notified. icu admission was defined as admission to an icu or coronary care unit, but not to an obstetric high care unit. short stay at an icu only because of postoperative nursery was not considered as an icu admission. requests for notification of cases of obstetric icu admission during pregnancy, delivery or puerperium were, along with other types of severe maternal morbidity, sent to all local coordinators on a monthly basis. cases were communicated to the national surveillance centre for obstetrics and gynaecology (nscog) in a web - based design. reminders were sent to non - responders every month until they had returned the monthly notification card. after notification, a completed case record form was sent to us, accompanied by anonymous photocopies of all relevant sections of the hospital case notes and correspondence. a detailed review of cases was completed by two of the authors (jz and jd), and all cases were entered into an access database. cases of maternal mortality were reported to the national maternal mortality committee of the netherlands society of obstetrics and gynaecology by the attending obstetrician as usual. we recorded maternal characteristics (age, body mass index, parity, ethnicity, smoking), and all variables concerning pregnancy and delivery. we also recorded data specifically related to the icu admission : admission and discharge date, diagnosis on admission, vital signs on admission, interventions and laboratory results. characteristics of each hospital were also recorded (university or teaching hospital, annual number of deliveries and level of icu). major obstetric haemorrhage (moh) was defined as transfusion need of four or more units of packed cells or hysterectomy or embolisation. when more than one diagnosis was provided, the case was classified according to the most serious condition. ethnicity was defined by country of origin (geographical ethnic origin) and grouped according to the most common population groups in the netherlands (western, moroccan, surinam / dutch antilles, turkish, sub - saharan african and central and eastern asian). women born in the netherlands with at least one parent born abroad were considered to be from the same origin as their non - dutch parent(s). women from other european countries, north america, japan and indonesia were considered western immigrants according to statistics netherlands because of their cultural background and socio - economic position, which is comparable with western women. denominator data for the number of births in the netherlands and national reference values for possible risk factors for obstetric icu admission were obtained from statistics netherlands and the netherlands perinatal registry (lvr-2) [6, 7 ]. the case fatality rate was calculated by dividing the number of deaths by the total number of icu admissions. relative risks and confidence intervals compared with the general pregnant population were calculated using univariable analysis. odds ratios and confidence intervals compared with women with severe maternal morbidity not admitted to the icu were calculated using multivariable logistic regression analysis. differences between groups were identified using the chi - square test ; significance was defined as p 1,500 deliveries) units 3.3 per 1,000. the incidence of icu admission was significantly increased in low - volume hospitals as compared to other non - academic hospitals (p 1,500 deliveries) units 3.3 per 1,000. the incidence of icu admission was significantly increased in low - volume hospitals as compared to other non - academic hospitals (p < 0.05) and significantly lower in intermediate - volume hospitals as compared to other hospitals (p < 0.001). in tertiary care centers, differences by icu level are shown in table 2.table 2characteristics of admission by intensive care unit levellevel 1level 2level 3p valuen%n%n% number of women admitted to icu26635.623035.134129.70.01mean duration of icu stay (days)1.92.33.2maternal mortality41.5104.3154.40.11induction of labour8331.27030.48625.20.20inotropic support103.8187.84613.5<0.001assisted ventilation3212.08737.817250.4<0.001diagnosis major obstetric haemorrhage11041.412052.215144.30.05 hypertensive disorders of pregnancy11141.74620.06719.6<0.001 cardiac disease83.0198.3288.20.02 sepsis124.5167.0277.90.23 pulmonary disease62.3125.2257.30.02 cerebral disease20.831.3144.10.01 liver / pancreatic disease41.520.982.30.39 thrombo - embolism20.841.772.10.42 anaesthetic complication31.141.751.50.85 miscellaneous83.041.792.60.65intensive care unit levels are described in the " methods"rates reflect percentage of all women with severe maternal morbiditydata missing for 22 womendata missing for 21 womendata missing for 20 women characteristics of admission by intensive care unit level intensive care unit levels are described in the " methods " rates reflect percentage of all women with severe maternal morbidity data missing for 22 women data missing for 21 women data missing for 20 women rates of icu admission for different subgroups of severe maternal morbidity were 12% for uterine rupture, 42% for eclampsia and 27% for major obstetric haemorrhage. twenty - six women (3.1%) were admitted to icu during early pregnancy, 191 (22.8%) antepartum and 620 (74.1%) postpartum. mean duration of icu stay was 2.9 days (range 1 to 71). ninety - one women (10.9%) stayed in the icu for more than 4 days.. of all women, 234 (28.0%) had at least one chronic disease (table 1). cerebral disease and thrombo - embolism had the highest case fatality rates with 26.3 and 23.1%, respectively. regarding only antepartum diagnoses, 47.6% of women were diagnosed with hypertensive disorders of pregnancy, 13.6% with moh and 9.9% with sepsis. women admitted postpartum were mainly diagnosed with moh (55.2%) and hypertensive disorders of pregnancy (21.5%). most frequent diagnoses during early pregnancy were moh (50.0%) and sepsis (26.9%), mostly caused by ectopic pregnancy or abortion. regarding differences between hospitals, moh (39.9 vs. 47.4%) and hypertensive disorders of pregnancy (16.8 vs. 30.0%) were less diagnosed in tertiary care centers as compared with general hospitals. rare life - threatening diseases like cardiac, liver / pancreatic, cerebral, septic and thrombo - embolic diseases were more frequently diagnosed in tertiary care centres (33.2 vs. 13.8%). roughly the same results were found for high - volume hospitals in comparison with low - volume hospitals.fig. 1outcome of icu admission by indication for admission outcome of icu admission by indication for admission assisted ventilation was needed in 291 women (34.8%), inotropic support in 74 (8.8%) and renal dialysis in 16 (1.9%). central venous and swan ganz catheter insertion were reported in 123 (14.7%) and 21 (2.5%) women, respectively. fresh frozen plasma and pooled platelets were administered in 365 (43.6%) and 220 (26.3%) women, respectively. in 82 (9.8%) and 92 (11.0%) cases, non - western women had a higher risk of being admitted to icus than western women. especially women from sub - sahara africa and eastern asia experienced increased risks of icu admission (table 3). other possible risk factors for icu admission as compared with the general pregnant population and with women with severe maternal morbidity not admitted to the icu are shown in table 4. a continuum of risk can be observed from lower risks in the general pregnant population to higher risks among women with severe maternal morbidity and highest risks among women with severe maternal morbidity admitted to icu.table 3unadjusted relative risks of intensive care unit admission by ethnicity n(%)rr (95% ci)western64877.41non - western18622.21.4 (1.21.7)morocco435.11.3 (0.91.7)turkey263.11.0 (0.71.4)surinam293.51.5 (1.12.2)dutch antilles141.71.7 (1.02.9)sub - saharan africa313.73.6 (2.55.1)central asia111.31.5 (0.82.7)eastern asia172.02.1 (1.33.4)unknown30.4table 4risk indicators for obstetric icu admission, as compared with non - icu admission and as compared with the general pregnant populationobstetric icu admission (n = 837)severe maternal morbidity without icu admission (n = 1,676)the netherlands, general pregnant population (n = 358,874)(%)(%)unadjusted or (95% ci)adjusted or (95% ci)(%)unadjusted rr (95% ci)patient age 35 years29.327.91.1 (0.91.3)24.71.0 (0.81.1) 40 years5.34.71.1 (0.81.7) 3.41.6 (1.12.1) body mass index (kg / m) < 18.5 (underweight)3.82.41.6 (0.92.9) 3.11.7 (1.22.5) 25 (overweight)36.636.21.0 (0.81.3) 31.72.0 (1.72.4) 30 (obese)15.612.01.4 (1.01.8)1.3 (0.91.7)9.81.7 (1.42.2)pregnancy parity 36.74.21.6 (1.12.3)1.6 (1.02.6)5.01.4 (1.01.8) prior caesarean delivery14.721.10.7 (0.50.8)0.5 (0.40.7)10.11.5 (1.31.9) artificial reproduction techniques : ivf / icsi5.64.41.3 (0.91.9) 1.93.0 (2.24.0) multiple pregnancy8.47.91.1 (0.81.4) 1.75.2 (4.16.6) initial antenatal care by obstetrician38.037.41.0 (0.91.2) 14.33.7 (3.53.9)delivery home delivery3.58.20.4 (0.30.6)0.4 (0.20.7)30.00.1 (0.050.1) induction of labour28.625.11.2 (1.01.4)1.6 (1.22.0)12.52.8 (2.43.3) caesarean delivery overall52.937.61.9 (1.62.2)1.5 (1.12.0)13.07.7 (6.78.8) prelabour caesarean delivery31.216.92.2 (1.82.7)2.0 (1.52.8)5.97.2 (6.38.4) ventouse / forceps extraction10.413.50.7 (0.61.0)0.8 (0.51.1)8.61.3 (1.11.7)or odds ratio, ci confidence intervalall significant factors in univariable analysis were included in the multivariable logistic regression model. significant values are in bold unadjusted relative risks of intensive care unit admission by ethnicity risk indicators for obstetric icu admission, as compared with non - icu admission and as compared with the general pregnant population or odds ratio, ci confidence interval all significant factors in univariable analysis were included in the multivariable logistic regression model. there were 29 maternal deaths during icu stay, giving a case fatality rate of 1 in 29 (3.5%). underlying causes of death and case fatality rates by diagnosis on admission are shown in fig. 1. the most frequent mode of death was cerebral (cerebrovascular haemorrhage, encephalopathy, brain stem compression and thrombosis). comparison of characteristics of deaths and survivors revealed no significant differences because of small numbers. compared with women with severe maternal morbidity who were not admitted to icu, women admitted to icu had a significantly higher case fatality rate (3.4 vs. 1.1%, p this report concerns by far the largest prospective cohort of obstetric icu admissions in the literature. in the only other, comparably large study inclusion was performed retrospectively, with case ascertainment relying on icd-9 codes. the incidence of 2.4 per 1,000 in the netherlands is comparable with other high income countries considering the range of incidences of 24 per 1,000 as mentioned by zeeman. however, the case fatality rate of 3.4% is well under the average of 6.8% in other studies [9, 10 ]. the average duration of icu stay was also lower than reported by others (3 vs. 5 days) [3, 9, 1122 ], and women seemed to be older (mean age 32 vs. 29 years) [3, 11, 1315, 1719, 2125 ]. with respect to the moment of admission, our findings were comparable with other studies. in this study moh was diagnosed almost twice as often as on average in other studies (45.5 against 23.6%), although incidence varied largely from 5 to 53% [3, 9, 1127 ]. on the other hand, respiratory disease and thrombo - embolism were diagnosed less than half as much in our study as compared with others (5.1 vs. 13.3% and 1.6 vs. 4.2%) [3, 1114, 1619, 2227 ]. only 20 women were admitted to icu with peripartum cardiomyopathy (1 in 20,000 pregnancies). this is few in light of the reported incidence of 1 in 100 to 1 in 15,000 pregnancies. differences could be explained by the fact that most other studies were not population based, but mainly from level 3 icus. tertiary care centres receive relatively more women with hypertensive disorders than women with moh as this concerns an acute clinical problem that is mostly treated locally. the less frequent diagnosis of hypertensive disorders of pregnancy as compared to the other studies (26.8 vs. 36.3%) was surprising in the light of the elevated incidence of eclampsia recently found in the netherlands this possibly reflects the underestimation of the risk of severe preeclamptic conditions in the netherlands. over 60 percent received packed cells, which is more than others previously reported (47.3% in canada and 32.0% in the uk) [12, 20 ]. as could be expected, we saw that tertiary care centres, high - level icus and high - volume hospitals treated more severely ill women with cardiac, liver / pancreatic, cerebral, thrombo - embolic and septic diseases as compared to general hospitals, level 1 icus and low - volume hospitals. women who had their antenatal care with an obstetrician for any preexisting medical or obstetric condition had an elevated risk of being admitted to icu, whereas women who delivered at home under supervision of the midwife had a decreased risk. these findings support the proper functioning of the system of selection between low- and high - risk pregnancies used in the netherlands. another important finding in this study is the fact that only one - third of all cases of severe maternal morbidity in the netherlands were admitted to an icu. therefore, obstetric icu admission alone is not a good surrogate for severe maternal morbidity. however, it seems appropriate to use icu admission to describe maternal characteristics and associated factors, because we found no differences between women who were and were not admitted to icu. even so, we can say that the most severe cases of severe maternal morbidity are generally included, as illustrated by the significantly higher case fatality rate and higher number of performed caesarean sections for maternal conditions of icu women as compared to non - icu women. since women with severe maternal morbidity had a baseline risk, odds ratios for icu versus non - icu women were not that high nevertheless, we found induction of labour and caesarean section to be adjusted risk factors. the protective effect of a previous caesarean section is probably caused by the fact that many of these women were included because of uterine rupture, a condition that rarely necessitates maternal icu admission. with abortion being legal in the netherlands, septic abortion one death among four women with septic abortion was found during the study period as compared to 63 in a 10-year unicentre study from argentina with a comparable case fatality rate. the main limitation of this study is that we were not able to correct population - based risk indicators for possible confounders as individual characteristics of the reference population were not available. the high relative risk among women who delivered by caesarean is probably confounded as caesarean delivery could be the consequence of the underlying disease for which the mother was admitted rather than the risk factor. icu admission is a management - based criterion and therefore by definition leads to inclusion bias. this is especially the case for tertiary care centres, where the threshold for icu admission is high due to the presence of obstetric high care units. furthermore, we saw that the threshold for icu admission was sometimes low in low - volume maternity units due to the fact that local protocols require intravenous therapy of pre - eclampsia to be monitored at an icu due to logistic reasons. this probably also explains the relatively long duration of icu stay in low - volume hospitals and the relatively high share of admissions for hypertensive disorders at level i icus. finally, results of the present study can not be merely extrapolated to other countries. reporting marked differences in medical diseases, organ failure and intensive care needs between a developed and a developing country. as shown, the management of critically ill women during pregnancy, delivery and puerperium is difficult and requires specific knowledge of the physiology and pathology of pregnancy. therefore, both obstetrician and intensivist / anaesthesist should always be involved in the management of women admitted to the icu. as obstetric icu admission is a rare event in western countries, exposure of obstetricians and intensivists / anaesthesists is low. this would plea for centralisation of obstetric care, which is a very current issue in the netherlands. although underexposure to rare but life - threatening complications might affect quality of care, this has to be balanced against the disadvantage of larger distances between obstetric services, which involves many more pregnant women. although illnesses are generally very serious, the case fatality rate is relatively low as compared to non - pregnant patients admitted to icu. proper management of obstetric icu admissions requires intensive cooperation of intensivist / anaesthesist and obstetrician. since two - thirds of all women with severe maternal morbidity in the netherlands were not admitted to the icu, icu admission is not a good parameter to assess the incidence of severe maternal morbidity in a specific population. it is, however, a good indicator of the most severe cases of maternal morbidity. | purposeas part of a larger nationwide enquiry into severe maternal morbidity, our aim was to assess the incidence and possible risk factors of obstetric intensive care unit (icu) admission in the netherlands.methodsin a 2-year nationwide prospective population - based cohort study, all icu admissions during pregnancy, delivery and puerperium (up to 42 days postpartum) were prospectively collected. incidence, case fatality rate and possible risk factors were assessed, with special attention to the ethnic background of women.resultsall 98 dutch maternity units participated in the study. there were 847 obstetric icu admissions in 358,874 deliveries, the incidence being 2.4 per 1,000 deliveries. twenty - nine maternal deaths occurred, resulting in a case fatality rate of 1 in 29 (3.5%). incidence of icu admission varied largely across the country. thirty - three percent of all cases of severe maternal morbidity were admitted to an icu. most frequent reasons for icu admission were major obstetric haemorrhage (48.6%), hypertensive disorders of pregnancy (29.3%) and sepsis (8.1%). assisted ventilation was needed in 34.8%, inotropic support in 8.8%. in univariable analysis, non - western immigrant women had a 1.4-fold (95% ci 1.21.7) increased risk of icu admission as compared to western women. initial antenatal care by an obstetrician was associated with a higher risk and home delivery with a lower risk of icu admission.conclusionspopulation-based incidence of obstetric icu admission in the netherlands was 2.4 per 1,000 deliveries. obstetric icu admission accounts for only one - third of all cases of severe maternal morbidity in the netherlands. |
nine children and their mother were exposed to vapors of metallic mercury. the source of the exposure appears to have been a 6-oz vial of mercury taken from a neighbor 's home. the neighbor reportedly operated a business preparing mercury - filled amulets for practitioners of the afro - caribbean religion santeria. at diagnosis, urinary mercury levels in the children ranged from 61 to 1,213 microg / g creatinine, with a geometric mean of 214.3 microg / m creatinine. all of the children were asymptomatic. to prevent development of neurotoxicity, we treated the children with oral meso-2,3-dimercaptosuccinic acid (dmsa). during chelation, the geometric mean urine level rose initially by 268% to 573.2 microg mercury / g creatinine (p<0.0005). at the 6-week follow - up examination after treatment, the geometric mean urine mercury level had fallen to 102.1 microg / g creatinine, which was 17.8% of the geometric mean level observed during treatment (p<0.0005) and 47.6% of the original baseline level (p<0.001). thus, oral chelation with dmsa produced a significant mercury diuresis in these children. we observed no adverse side effects of treatment. dmsa appears to be an effective and safe chelating agent for treatment of pediatric overexposure to metallic mercury.imagesfigure 1 |
|
ehrlich 's advice that treatment of infections should " hit hard and hit early, " formulated in the earliest days of antimicrobial chemotherapy, presciently summarized the principles of treatment for infections such as tuberculosis (tb) (9). these principles are embodied in modern protocols of directly observed, short - course chemotherapy, where the goal is to treat with adequate concentrations of multiple drugs and maintain treatment until the bacterial population is extinct.resistance to each of the major antituberculosis drugs is mediated by single point mutation ; therefore tuberculosis treatment is designed to prevent the ascent of subpopulations of mutant bacilli that are resistant to any one of the drugs. similar principles have been suggested for other infections in which resistance can arise by simple mutation, most notably hiv (10), although there has been some controversy on this topic (11). in these infections, the relationship between treatment, resistance in the treated person, and resistance in the community at large is relatively clear. inadequate therapy (owing to subtherapeutic drug concentrations, too few drugs, or poor adherence to therapy) results in the emergence of resistance, and possibly treatment failure, in the treated host. following the emergence of resistance in the treated host, resistant infections may be transmitted to others (figure, a ; table). four mechanisms by which antibiotic treatment can create selection for resistance in the population, showing direct effects increased resistance in treated (yellow) vs. untreated (white) hosts, and indirect effects increased resistance in others (turquoise) due to treatment of specific hosts. (a) subpopulations (usually mutants) of resistant (red) bacteria are present in a host infected with a predominantly susceptible (green) strain ; treatment fails, resulting in outgrowth of the resistant subpopulation, which can then be transmitted to other, susceptible hosts (turquoise). (b) successful treatment of an individual infected with a susceptible strain reduces the ability of that host to transmit the infection to other susceptible hosts, making those hosts more likely to be infected by resistant pathogens than they would otherwise have been, and shifting the competitive balance toward resistant infections. (c) treatment of an infection eradicates a population of susceptible bacteria carried (often commensally) by the host, making that host more susceptible to acquisition of a new strain. if the newly acquired strain has a high probability of being resistant (as in the context of an outbreak of a resistant strain), this can significantly increase the treated individual s risk of carrying a resistant strain, relative to an untreated one. (d) treatment of an infection in an individual who is already colonized (commensally) with resistant organisms may result in increased load of those organisms if competing flora (perhaps of another species) are inhibited leading to increased shedding of the resistant organism and possibly to increased individual risk of infection with the resistant organism. for example, methicillin resistance in s. aureus and vancomycin resistance in enterococcus are mediated by the acquisition of one or several new genes, rather than by point mutations in existing genes. in streptococcus pneumoniae, penicillin resistance occurs when segments of wild - type penicillin - binding protein genes are replaced with alleles whose sequences differ from the wild - type at multiple positions. these new resistance mechanisms arose and spread in large populations under conditions of antibiotic selection pressure, but they are unlikely to occur de novo in any single person because of the multiple changes involved. organisms (or plasmids) bearing these types of resistance must be acquired, generally as a consequence of cross - transmission. furthermore, most of these organisms are not obligate pathogens such as hiv or tb ; as a result, much of their exposure to antibiotics occurs during treatment directed at infections caused by other, unrelated organisms. because of these genetic and epidemiologic differences, the paradigm for tuberculosis treatment, minimizing resistance in the treated host and the community by preventing the emergence of resistant subpopulations during treatment, is often inapplicable to these organisms (12). antibiotic treatment promotes the spread of these organisms, as suggested by the rapid increases in resistance in many of the organisms after the new drug classes are introduced. however, the effects of treatment in promoting resistance occur by less direct mechanisms, which depend on competitive interactions between drug - resistant and drug - susceptible strains. for any infectious disease, the infection or colonization status of any one (index) patient affects the risk of infection or colonization of others. measures (such as vaccination or antibiotic treatment) that change the incidence or duration of infection in one person will affect that person 's contacts (13 - 14). just as vaccination programs benefit those who are not vaccinated because of the phenomenon of herd immunity, antibiotic usage by some persons may increase the risk of colonization or infection with resistant organisms in people who have not received antibiotics. members of a population experience indirect effects of antimicrobial use, defined as the enhancement of risk for acquiring a resistant organism, because of the use of antimicrobials by other persons in the group or population. for example, simply by eradicating susceptible organisms, and thereby reducing the opportunities for transmission of susceptible strains, antibiotics received by treated hosts can increase the probability that other hosts will acquire resistant variants (figure, b ; table). for many pathogens, acquisition of one strain reduces a person 's chances of acquiring other strains, either via immune responses, via direct interference (1517), or both. treatment of some patients, by eradicating susceptible strains and thereby reducing their ability to transmit to other hosts, is advantageous to resistant strains in the population. mathematical models (1822) and epidemiologic studies (23) suggest that this mechanism of shifting the competitive balance in favor of resistant strains can increase the prevalence of resistant organisms in the community, alone or in combination with other mechanisms. an important feature of this kind of indirect effect is that it need not involve an increase in a patient s own risk of carrying resistant organisms, only a reduction in the duration or probability of carrying susceptible ones. in these organisms, the increase in transmission of resistant pathogens is a consequence of successful treatment of the infected host, resulting in the eradication of drug - susceptible pathogens that colonize or infect that host. as a consequence, the more effective a treatment is at eradicating drug - susceptible populations of these organisms, the more it will promote the spread of resistant ones. this spread contrasts with tb, in which treatment failure is often associated with the emergence of resistance in treated hosts, so unsuccessful treatment is seen as a factor promoting the spread of resistance (although, over a time scale of decades, this type of indirect mechanism described here may play a role even in tuberculosis). a third mechanism by which antimicrobial use increases the number of patients colonized or infected with resistant organisms is by modifying the treated host s colonization resistance (figure, c ; table). eradication or reduction of drug - susceptible normal flora by antibiotic treatment may increase vulnerability to acquisition of new strains. this effect will increase the patient s probability of being colonized with a resistant organism if, during or shortly after treatment, he or she is exposed to others with resistant organisms. this mechanism is direct in the sense that it increases the treated patient s risk of colonization with resistant organisms but is also associated with indirect effects because of the requirement for transmission. an index host given antibiotics is placed at greater risk for colonization with resistant organisms (direct effect), but this risk is amplified by his or her exposure to other patients harboring resistant organisms, which is in turn enhanced by their use of antibiotics (indirect effect). a fourth mechanism by which antimicrobial use increases antimicrobial resistance is by increasing the density of resistant organisms within a patient who already harbors such organisms at a lower density (figure, d ; table). enhanced shedding of these organisms, resulting in an increased risk to other patients (an indirect effect), has been documented (i.e., in the case of anti - anaerobic agents that increase shedding of vancomycin - resistant enterococci (vre) (24). an increased risk of resistant infection to the treated patient (a direct effect) may occur if a higher density of resistant organisms places the patient at higher risk of infection with his or her own flora. unlike the other three ways by which antimicrobial use promotes resistance, this mechanism is mediated through antimicrobial treatment of patients already colonized with the resistant organism. there are a number of other cases in which direct and indirect effects of antibiotic treatment are combined. due to the diversity of genetic mechanisms of resistance, the risk of emergence of resistance during treatment represents a continuum, with tb at one end and vre (or mrsa) at the other. fluoroquinolone resistance in s. pneumoniae mediated by the accumulation of mutations in the dna gyrase and topoisomerase iv genes (25), or resistance to third - generation cephalosporins in enterobacteriaceae mediated by mutations in tem and shv beta - lactamases located on plasmids (26), lie between these two extremes. in these cases, multiple mutations are required to turn a fully susceptible strain into a clinically resistant one. for a patient colonized or infected with a fully susceptible strain, emergence of resistance during treatment may be highly unlikely because of the requirement for selecting multiple mutations. however, in such cases, there may be selection in consecutive hosts for small increases in levels of resistance to a particular compound, resulting eventually in the emergence of clinical resistance (27). patients may be colonized with a mixed flora of resistant and susceptible organisms, and eradication of the drug - susceptible flora may permit outgrowth of the resistant subpopulation (28). this mechanism has some formal similarity to what occurs in tb, except that for a colonizing bacterium such as the pneumococcus or the enteric colonizers, outgrowth of resistant organisms in the site of colonization need not be associated with treatment failure. in these cases, the treated patient is at increased risk of carrying resistant organisms (direct effect), but an indirect effect on the population occurs because the treated patient no longer carries susceptible organisms and is, therefore, unable to transmit them. treatment with one antimicrobial drug can select for resistance to a number of other, unrelated agents, by several means. if individual organisms are resistant to multiple drugs, then use of any one of these may promote resistance to others (29). additionally, by altering the balance of different components of the indigenous microbial flora, treatment with one agent may increase the load of a pathogen resistant to another agent, simply by killing off competing flora of different species ; this has been observed, for example, with anti - anaerobic treatments that increase the load of vre (24). these complexities increase the number of relationships that need to be studied in assessing the effects of antimicrobial use on resistance and also the number of potential confounders in any study. variation in mechanisms of resistance has implications for the choice of antimicrobial therapy and the evaluation of strategies to minimize resistance. adopting the individual and population - level perspective informs therapeutic decision - making, clinical study design, and public policy. in tb, preventing the emergence of resistance in a treated host is a sound policy for averting the emergence of resistance at the population level as well (although once resistant strains have emerged, special measures are required to contain them). with respect to antimicrobial resistance in contrast, for other types of resistance, antimicrobial treatment may exert individual - level effects that are substantially different in magnitude or even opposite in direction to that of population - level effects. treatment with a beta - lactam may produce only a small, short - lived increase in the treated patient 's odds of carrying or being infected by a resistant pneumococcus (7). in some cases, treatment may actually eradicate carriage of a resistant organism, thereby reducing the individual s risk of resistant carriage. small or unobservable effects on individual risk have been observed in other cases as well, such as vancomycin use for vre (8,31) and the use of various antibiotics for infections with resitant gram - negative rods (32). in these cases, preventing resistance in the treated patient may not be the central goal of a prudent antibiotic use policy ; instead, treatment should seek to minimize the advantage it provides to resistant organisms in the community or the hospital as a whole, subject to the constraint of providing effective treatment for the patient. the considerations of the distinctive biologic and epidemiologic mechanisms of antibiotic resistance in different pathogens lead to several broad suggestions for future studies. first, the optimal study design to estimate individual - level effects of antibiotics on colonizing organisms such as vre and beta - lactam resistant s. pneumoniae is to measure acquisition and loss rates in an observational cohort or experimental study where subjects are serially cultured before, during, and after antibiotic therapy (23,33). time - to - event statistical models (e.g., cox proportional hazards regression) are appropriate analytic methods for these kinds of studies (23,31,34). this design allows investigators to distinguish between the effects of antimicrobials on the risk for acquisition (colonization) and their effects on the risk for clinical infection once an patient has been colonized with a resistant organism. as a consequence of the mechanisms we have described, the magnitude of an antibiotic s effect on a patient s risk of resistant colonization or infection may be dependent on his or her exposure to potential transmission of resistant organisms (14). stated differently, the frequency of contact with others carrying the resistant organisms is likely an important effect - modifier of antibiotic effects for pathogens that do not follow the simple model of emergence of resistance exhibited by organisms such as m. tuberculosis. individual - level antibiotic effects mediated by alterations in colonization resistance or killing of susceptible bacteria may be greater in settings of high exposure to resistant organisms, for example, during outbreaks (7). controlling for transmission risk or measuring effects conditional on a specified level of transmission risk is advised, when possible. standard analytic approaches make the assumption that outcomes in different subject are independent, but this assumption is violated in the case of infectious diseases. use of one of these strategies to model exposure to transmission will help to account for this non - independence of outcomes in different persons in the same study (13,3537). one practical result of quantifying direct, individual - level antibiotic effects is to provide information on the short - term risk of infection with a resistant organism to a person about to initiate antibiotic treatment. this hazard needs to be taken into account when weighing the risks and benefits of use of antimicrobial agents in individual patients. however, analogous to the evaluation of vaccine programs, combined direct and indirect antibiotic effects carry increased importance from the public health and policy management perspective (38,39). the measurement of population - level effects of antimicrobials also has educational value in demonstrating to clinicians and patients the extent to which individual antibiotic use choices have negative consequences for the population as a whole. such a conflict between individual benefit and the population s harm is an example of what economists term an externality or what environmentalists have called the tragedy of the commons (40). to estimate overall antibiotic effects from data observational group - level studies may lack sufficient data to avoid confounding and other causes of ecologic bias (41). for this reason, studies that estimate the effects of individual- and group - level antimicrobial use are generally preferable to ones that contain group - level data alone. depending on the context, the appropriate group(s) may include the family, the community, the hospital, or the hospital unit or department (4244). further research is necessary to evaluate hierarchical regression methods and compare results obtained from different levels of analysis (44). for the most accurate measurement of overall antibiotic effect on resistance in communities, a cluster - randomized intervention trial is appropriate (45). in cluster - randomized trials, the unit of randomization is a group such as a community or a hospital, and multiple units (sometimes as few as six, but often more) are assigned to each of two (or more) treatment arms. we are not aware of published studies using this design to evaluate antibiotic resistance, although we know of two in progress (r. platt, pers. however, this design has been used in other areas of infectious disease epidemiology for which group level effects are important (such as vaccination programs), and it is considered the standard design for investigations of the effects of insecticide - impregnated bednets in preventing malaria (4547). in the context of antimicrobial resistance, cluster - randomized trials have two key advantages. first, unlike studies that gather individual - level data alone, they provide the opportunity to observe the indirect effects of treatment on resistance. second, they provide a clean way to avoid the statistical problems of nonindependence between patients in a study that may reduce the power or increase the false - positive rate of observational studies. in cluster - randomized studies of antimicrobial resistance, both the incidence rate of infection with resistant organisms in the population and the ratio of resistant to susceptible (or proportion of total organisms that are resistant) would be appropriate study endpoints. transmission - dynamic modeling can also play an important role in bridging the gap between individual- and group - level effects (20,21,4850). these models take information about individual - level effects as parameters and make predictions about the response of the population to changes in such parameters as transmission risk or antibiotic usage. although models can not substitute for empirical intervention studies, they can be particularly valuable in at least four ways : 1) generating hypotheses about the relationship between antibiotic use and resistance that can be used in designing and prioritizing empirical studies ; 2) defining the conditions under which a particular intervention is likely to work, thereby suggesting how empirical results can (and can not) be extrapolated to other settings ; 3) providing explanations for phenomena that have been observed but whose causes were uncertain ; and 4) identifying biological mechanisms that, while important, remain poorly understood. an example of models for generating hypotheses comes from the question of antimicrobial rotation or cycling. cycling of antimicrobial classes in hospitals has been suggested and is currently being evaluated for its ability to curtail resistance in major nosocomial pathogens (5,5154). one mathematical model of this process has suggested that using a mixture of different drug classes simultaneously (e.g., if two drug classes are available for empiric therapy of certain infections, treat half of the patients with one drug class and half with the other) will reduce resistance more effectively than cycling under a broad range of conditions (19). this suggests that such mixed regimens would be good candidates for comparison with cycling in controlled trials. as a second example, levels of resistance in hospital - acquired pathogens may change rapidly within a matter of weeks or months after changes in antimicrobial use. by contrast, studies of reductions in antimicrobial use in communities have shown slow and equivocal effects on resistance in community - acquired pathogens (55). mathematical models suggest that, in communities, the key factor driving the change in resistance levels may be the " fitness cost " of resistance, i.e., resistance will decline after a reduction in antimicrobial use if resistant organisms in untreated patients are at a disadvantage for transmission or persistence (19,50,56,57). this cost may be small in many bacteria, accounting for the slow response (55,58). in contrast, a model indicates that, in hospitals, changes in resistance may be driven primarily by the admission of new patients who often bring with them drug - susceptible flora, and this may rapidly " dilute " levels of resistance in the absence of continuing selection by antibiotics (59). if correct, this explanation suggests that the success of antimicrobial control measures should be evaluated differently for hospitals and for communities. the use of mathematical models, and more generally the attempt to predict the relative merits of different interventions, will depend on an improved understanding of the mechanisms of antibiotic selection in particular organisms. for example, two recently published models for the nosocomial spread of resistant pathogens made contrasting assumptions about whether antimicrobial treatment increased an patient s susceptibility to colonization only during treatment (60) or for a period following treatment (59), and about the importance of colonization with drug - susceptible strains in protecting against acquisition of resistant ones. as a result of these differences in assumptions, predictions differed in important ways : one model suggested that reduction of antibiotic use would be a comparatively poor intervention when endemic transmission is high and that resistant organisms could persist endemically even in the absence of input from admitted patients or antibiotic selection (60). the other model predicted rapid declines in the level of resistance when use is reduced, and a more complicated relationship between the effectiveness of interventions and the level of transmission within the hospital (59). testable predictions will permit the evaluation of different models for particular settings and provide a basis for refining the assumptions of these models. the relationship between antibiotic usage and antibiotic resistance for many types of pathogens is largely mediated by indirect effects or population - level selection. when resistant and susceptible organisms compete to colonize or infect hosts, and use of an antibiotic has a greater impact on the transmission of susceptible bacteria than resistant ones, then increasing use of the antibiotic will result in an increase in frequency of organisms resistant to that drug in the population, even if the risk for treated patients is modest. antimicrobial use and patient - to - patient transmission are not independent pathways for promoting of antimicrobial resistance, rather they are inextricably linked. study designs to assess the effect of antimicrobial use on resistance should reflect these diverse pathways of direct and indirect effects. estimates of direct effects of antimicrobial use on treated patients will be most informative if clinical cultures are combined with measurements of colonization. use of time - to - event (e.g., cox proportional hazards) models provides a natural way of controlling for the patient s length of stay when assessing the effect of treatment on acquisition of resistant organisms. analyses that control for a person 's exposure to other patients carrying resistant organisms will help to capture the effect modification because of varying transmission pressures during a study. inclusion of data on antimicrobial use by the group to which others are exposed (siblings, fellow patients on a hospital unit, total use in a community) and to individual - level data will provide one method of estimating both direct and indirect effects of antibiotic use. nonindependence of individual outcomes makes the interpretation of intervention studies problematic unless measures are taken to account for this nonindependence ; cluster - randomized studies, used in other areas of infectious disease epidemiology, are an excellent solution to this problem. we have commented elsewhere on other aspects of study design for antimicrobial resistance, notably the importance of control group selection (7,61,62). understanding in detail, for each pathogen, the mechanisms by which antimicrobial use selects for antimicrobial resistance in treated patients and in the population is of more than academic importance. for practitioners, these mechanisms matter for making well - informed decisions about the design of treatment protocols, the choice of antibiotics and doses for particular indications. for policymakers, these issues have direct bearing on the design of campaigns to encourage more rational antibiotic use and on the priorities in regulating the use of antimicrobial agents for human and animal use (63,64). | the need to stem the growing problem of antimicrobial resistance has prompted multiple, sometimes conflicting, calls for changes in the use of antimicrobial agents. one source of disagreement concerns the major mechanisms by which antibiotics select resistant strains. for infections like tuberculosis, in which resistance can emerge in treated hosts through mutation, prevention of antimicrobial resistance in individual hosts is a primary method of preventing the spread of resistant organisms in the community. by contrast, for many other important resistant pathogens, such as penicillin - resistant streptococcus pneumoniae, methicillin - resistant staphylococcus aureus, and vancomycin - resistant enterococcus faecium resistance is mediated by the acquisition of genes or gene fragments by horizontal transfer ; resistance in the treated host is a relatively rare event. for these organisms, indirect, population - level mechanisms of selection account for the increase in the prevalence of resistance. these mechanisms can operate even when treatment has a modest, or even negative, effect on an individual host s colonization with resistant organisms. |
at present a major difficulty in understanding drug action in the human brain is the ability to relate the multiple physical scales of investigation. how do the findings of molecular biology relate to cellular neurophysiology and in turn to neuroimaging and cognitive testing ? it is often only via inference and synthesis that investigators can relate complementary findings across research fields in order to construct a theory. to support such a synthesis, has sought to do this in regards to cognition and functional neuroanatomy (i.e. by quantifying the relationship between behaviour and measures of functional brain activity). at the microscopic scale there is also success in quantifying the relationship between molecular and cellular levels (e.g. by quantifying the relationship between gene expression and electrophysiology (hanna 2006)). however, integrating findings from the microscopic (single neuron) level to the macroscopic (neural population) level is underdeveloped (freeman 1992 ; breakspear and stam 2005). one potential approach is the growing area of mean field population or neural mass modelling (freeman 1975 ; lopes da silva. 1975 ; nunez 1981 ; jirsa and haken 1996 ; robinson. 1997 david and friston 2003). by spatially averaging the behaviour of neurons which constitute cytoarchitechtonically defined populations (macrocolumn), what follows is an introduction to the dynamical mean field modelling of cortical drug response, using recent findings from anaesthesia as an example. focus is placed on the physiological relevance and utility of this approach with less emphasis on the details of mathematical formulations, which are often quite involved. those readers interested in the mathematical details are directed to the cited materials below, particularly (steyn ross. the remainder of the current section will cover the applicability of mean field modelling to anaesthesia and in doing so begin to highlight the general principles of this approach. mean field modelling of neural activity will provide more details of mean field models and their biological basis. in section the neurophysiological effects of anaesthetics will be introduced along with their pharmacology (targets of anaesthetic action), followed by a review of network models of anaesthetic action in section cellular network models of anaesthetic action and a more detailed review of mean field models in section the clinical relevance of mean field models will be covered in section clinical implications of mean field population models followed by summary remarks in section the modelling of anaesthetics is an expedient test case of population models for several reasons. firstly, the clinical utility of anaesthetics has made these drugs a popular research domain, with particular focus on the sites and mechanisms of action (rudolph and antkowiak 2004). as will be discussed below this focus on the development of cellular descriptions of action greatly assist the fidelity of model parameterization. secondly, volatile anaesthetic pharmacology shows a predominate affinity for ligand - gated gamma - amino - butyric - acid (gaba) neurotransmission (franks and lieb 1994 ; rudolph and antkowiak 2004). it is therefore notable that population models incorporate fast neurotransmitter kinetics (ligand - gated ionic channels) and constitute cell populations not by diverse morphology, but by the chief functional properties of inhibition (i) and excitation (e). making the dominant cortical neurotransmitter systems (gaba (i) and glutamate (e), petroff 2002) primary interests in modelling cortical pharmacology. thirdly, as the potent influence of anaesthetics is mediated by a dominant neurotransmitter system, its resulting effects on neurophysiology and behaviour are manifestly global. for example, neocortical populations and the related cognitive faculties are progressively suppressed by anaesthetic agents (heinke and koelsch 2005). this global influence also fits with the spatial scale of mean field models. in summary, anaesthetic pharmacology is fortuitously accommodated in the biological content of mean field models of cerebral cortex. clinical benefits also arise from the development of theoretical descriptions of anaesthesia. with the growing utilization of electroencephalogram (eeg) based depth of anaesthesia (doa) monitors, the development of real - time drug monitoring in the central nervous system (cns) is taking shape. as a majority of mean field models aim to simulate the eeg, they are particularly germane to doa monitoring. for example, recently the effects of alprazolam, a benzodiazepine, were sensitively monitored (off - line) using signal processing methods derived from a population theory of eeg (liley. furthermore, these findings provided a sensitive and mechanistic description of how the benzodiazepine class of sedative anxiolytics paradoxically increase eeg beta (1525 hz) power in moderate to high concentrations (liley. this effort at theoretically driven analysis reflects biologically motivated methods, which are hoped to provide more sensitive and efficient measures than data driven approaches. in recent decades both theoretical neuroscience and cognitive science have shown a growing interest in the use of dynamical methods as a research tool (kelso 1995 ; van gelder 1998). such research utilizes the tools and vocabulary of the mathematics of dynamical systems for the modelling and empirical analysis of electrical brain activity (freeman 1992 ; stam 2005). this emerging field of neurodynamics (freeman 2000) has particularly strong ties with electroencephalography (eeg) (freeman 1975 ; basar 1980 ; nunez 1995) and magnetoencephalography (meg) (jirsa and haken 1996 ; david and friston 2003), as the time scales of these measurements (milliseconds) are more amenable to a dynamical description than bold (blood oxygen level dependent) fmri (functional magnetic resonance imaging) and pet (positron emission tomography) in addition to being more closely matched to the time scales of cognition and behaviour. oscillatory dynamics of the eeg has been a main area of research focus in neurodynamics. in general terms the theoretical methods employed to understand the dynamical genesis of the eeg can be divided into two essentially separate, though complementary, approaches. the first approach models the activity of large networks of individual model neurons imbued with a range of voltage and ligand dependent ionic conductances (koch and segev 1998). while this approach enables an arbitrary level of physiological detail to be included they are limited in as much as the eeg is a macroscopic or bulk property of populations of neurons. biophysically the eeg arises from the ionic current flowing in response to synchronised synaptic activity. a typical eeg electrode is recording the synaptic currents generated by well over 100,000 pyramidal neurons (nunez 1981) (fig. 1). thus it is simply computationally inefficient to model the eeg using this approach. a preferable approach, in which the spatial scale of the model better reflects the spatial scale of the eeg, is the continuum or mean field model (nunez 1981 ; jirsa and haken 1996 ; robinson. 1997 ; liley. it is the synaptic activity generated by spatially circumscribed populations of cortical neurons that are modelled rather than the synaptic activity of individual neurons. in general the columnar organization of cortex (e.g. the macrocolumn) is used to define this spatial scale and to subsequently define the spatial scales of neuronal interactions.fig. 1schematic representation of a cortical macrocolumn and the two major subtypes of neurons in mammalian cerebral cortex excitatory (e) and inhibitory (i). within a macrocolumn excitatory and inhibitory neurons are able to form all possible combinations of feed forward and feedback synaptic connectivity, whereas synaptic connections between cortical macrocolumns are exclusively excitatory. in human neocortex a cortical macrocolumn is estimated to contain of the order of 100,000300,000 neurons (nunez 1981). electric field lines, drawn directed along the axis of the apical dendritic trees of the two excitatory neurons on the right, are the result of inward current flow due to excitatory synaptic activity in the distal portions of the apical dendritic tree, and make the greatest contribution to the surface recordable electrocorticogram (ecog) and eeg. figure from liley and bojak (2005) schematic representation of a cortical macrocolumn and the two major subtypes of neurons in mammalian cerebral cortex excitatory (e) and inhibitory (i). within a macrocolumn excitatory and inhibitory neurons are able to form all possible combinations of feed forward and feedback synaptic connectivity, whereas synaptic connections between cortical macrocolumns are exclusively excitatory. in human neocortex a cortical macrocolumn is estimated to contain of the order of 100,000300,000 neurons (nunez 1981). electric field lines, drawn directed along the axis of the apical dendritic trees of the two excitatory neurons on the right, are the result of inward current flow due to excitatory synaptic activity in the distal portions of the apical dendritic tree, and make the greatest contribution to the surface recordable electrocorticogram (ecog) and eeg. diagram not draw to scale. figure from liley and bojak (2005) typical of these mean field theories of electrocortical activity is that of liley. this theory of eeg is capable of producing the main features of spontaneous human eeg and is able to account quantitatively and qualitatively for the effects that gaba enhancing anaesthetics and sedatives have on the eeg. it assumes that all the various types of cortical neurons can be divided into synaptically interacting excitatory or inhibitory subpopulations. each of these populations has the spatial extent of a barrel - shaped region of approximately 0.53 mm in diameter penetrating the entire thickness of cortex. locally (i.e. within a macrocolumn), excitatory and inhibitory neuronal populations interact with each other and themselves. only the excitatory populations are capable of forming connections (long range or cortico cortical) with excitatory and inhibitory populations within other nearby macrocolumns. this theory, like most other mean field theories, considers the eeg as being proportional to variations of the mean excitatory soma membrane potential he. the soma membrane is driven from its resting potential he by a weighted sum of synaptic inputs ile from excitatory (e) and inhibitory (i) populations : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \tau_e\frac{\partial}{\partial t } { \bf h}_e = h_{\rm e}^{r}-{\bf h}_e+\sum_{l = e, i } \psi_{le}\left({\bf h}_e\right){\bf i}_{le}\ ;, $ $ \end{document}with a similar equation describing the inhibitory soma membrane potential hi. in the following a bold quantity f stands for mean fields depending on position on cortex and time. we will provide here only an outline of the mathematical description, the interested reader can consult (bojak and liley 2005 ; liley and bojak 2005) for necessary details. this theory incorporates a number of key physiological properties which are parameterised in such a way that the electroencephalographic effect of changes in these properties by disease or pharmacology can be systematically studied and investigated : postsynaptic potentials and neurotransmitter kinetics : neurons are considered as passive electrical compartments into which all synaptically induced ionic current flows. the excitatory and inhibitory synaptic currents, which give rise to excitatory (epsp) and inhibitory (ipsp) post - synaptic potentials respectively, determine deviations of the mean soma membrane potential from its resting value. in particular the time course and the corresponding reversal potentials of fast ionotropic excitatory (ampa / kainate) and inhibitory (gabaa) neurotransmitter kinetics are explicitly included. the ipsp response iik to presynaptic pulses pik is 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left[\frac{\partial}{\partial t}+\gamma_{ik}\right]\left[\frac{\partial}{\partial t}+\tilde{\gamma}_{ik}\right]{\bf i}_{ik}=\upgamma_{ik}\gamma_{ik}e^{\tilde{\gamma}_{ik}\delta_{ik}}\times{\bf p}_{ik }, $ $ \end{document}and a similar equation applies to the epsps iek. mean field models of anaesthetic action below discusses the connection of and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde{\gamma}$$\end{document } to the characteristic times and, and their particular significance to the modelling of anaesthesia.fig. 2a schematic illustration of the essential parameters used to characterise the shape and amplitude of an inhibitory - postsynaptic potential (ipsp) or inhibitory postsynaptic current for both theory and experiment. the decay constant of the ipsp, ik is defined as the value at which the ipsp has decayed to approximately 1/e of its peak value. further details regarding this parameterisation can be found in liley and bojak (2005). figure from liley and bojak (2005)short - range intracortical connectivity : in addition to the reciprocal shortrange excitatory the former because they make up approximately 60% of all synaptic connections in cortex, the latter because they have been shown to be important determinants of the stability and frequency of alpha band oscillatory activity. because these connections are short - range (generally less than 3 mm) conduction delays are considered negligible. 3schematic illustration of the topology of short - range (intracortical), njj), and long - range (cortico cortical), nej, fibre systems that serve to connect excitatory (e) and inhibitory (i) neuronal populations in liley.s dynamical theory of eeg generation (liley. pjj denotes extracortical input if type j to neural population of type j. figure from liley and bojak (2005)long - range cortico cortical connectivity : as already mentioned the long range cortico cortical fibres that serve to connect distant areas of cortex with each other are, on the basis of extensive anatomic and physiological evidence, modelled as being purely excitatory. however because they traverse much larger distances than the intracortical axonal fibres their conduction delays can no longer be considered negligible. the existence of conduction delays is incorporated by ascribing a finite conduction velocity to cortico cortical connectivity. the result is a two dimensional telegraph equation3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left[\left(\frac{1}{v_{ek}}\frac{\partial}{\partial t}+\uplambda_{ek}\right)^2-\frac{3}{2}\nabla^2\right]{\bf \upphi}_{ek}=n^\alpha_{ek}\uplambda_{ek}^2{\bf s}_e, $ $ \end{document}for activity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\upphi}_{ek}$$\end{document } conducted from local excitatory firing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{s}}_e$$\end{document } to distant excitatory k = e orinhibitory k = i populations.extra-cortical input : excitatory or inhibitory thalamic input onto cortical excitatory or inhibitory is incorporated however the theory currently excludes the reciprocal thalamo - cortical connections that a number of other dynamical theories of the eeg include (robinson. 1997) because on the basis of current data their cortical effects can not be meaningfully parameterised. postsynaptic potentials and neurotransmitter kinetics : neurons are considered as passive electrical compartments into which all synaptically induced ionic current flows. the excitatory and inhibitory synaptic currents, which give rise to excitatory (epsp) and inhibitory (ipsp) post - synaptic potentials respectively, determine deviations of the mean soma membrane potential from its resting value. in particular the time course and the corresponding reversal potentials of fast ionotropic excitatory (ampa / kainate) and inhibitory (gabaa) neurotransmitter kinetics are explicitly included. the ipsp response iik to presynaptic pulses pik is 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left[\frac{\partial}{\partial t}+\gamma_{ik}\right]\left[\frac{\partial}{\partial t}+\tilde{\gamma}_{ik}\right]{\bf i}_{ik}=\upgamma_{ik}\gamma_{ik}e^{\tilde{\gamma}_{ik}\delta_{ik}}\times{\bf p}_{ik }, $ $ \end{document}and a similar equation applies to the epsps iek. mean field models of anaesthetic action below discusses the connection of and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde{\gamma}$$\end{document } to the characteristic times and, and their particular significance to the modelling of anaesthesia.fig. 2a schematic illustration of the essential parameters used to characterise the shape and amplitude of an inhibitory - postsynaptic potential (ipsp) or inhibitory postsynaptic current for both theory and experiment. the decay constant of the ipsp, ik is defined as the value at which the ipsp has decayed to approximately 1/e of its peak value. further details regarding this parameterisation can be found in liley and bojak (2005). figure from liley and bojak (2005) a schematic illustration of the essential parameters used to characterise the shape and amplitude of an inhibitory - postsynaptic potential (ipsp) or inhibitory postsynaptic current for both theory and experiment. the decay constant of the ipsp, ik is defined as the value at which the ipsp has decayed to approximately 1/e of its peak value. further details regarding this parameterisation can be found in liley and bojak (2005). figure from liley and bojak (2005) short - range intracortical connectivity : in addition to the reciprocal shortrange excitatory inhibitory connections intracortical excitatory excitatory and local inhibitory inhibitory connections are included the former because they make up approximately 60% of all synaptic connections in cortex, the latter because they have been shown to be important determinants of the stability and frequency of alpha band oscillatory activity. because these connections are short - range (generally less than 3 mm) conduction delays are considered negligible. 3schematic illustration of the topology of short - range (intracortical), njj), and long - range (cortico cortical), nej, fibre systems that serve to connect excitatory (e) and inhibitory (i) neuronal populations in liley.s dynamical theory of eeg generation (liley. 2002). pjj denotes extracortical input if type j to neural population of type j. figure from liley and bojak (2005) schematic illustration of the topology of short - range (intracortical), njj), and long - range (cortico cortical), nej, fibre systems that serve to connect excitatory (e) and inhibitory (i) neuronal populations in liley.s dynamical theory of eeg generation (liley. 2002). pjj denotes extracortical input if type j to neural population of type j. figure from liley and bojak (2005) long - range cortico cortical connectivity : as already mentioned the long range cortico cortical fibres that serve to connect distant areas of cortex with each other are, on the basis of extensive anatomic and physiological evidence, modelled as being purely excitatory. however because they traverse much larger distances than the intracortical axonal fibres their conduction delays can no longer be considered negligible. the existence of conduction delays is incorporated by ascribing a finite conduction velocity to cortico cortical connectivity. the result is a two dimensional telegraph equation3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left[\left(\frac{1}{v_{ek}}\frac{\partial}{\partial t}+\uplambda_{ek}\right)^2-\frac{3}{2}\nabla^2\right]{\bf \upphi}_{ek}=n^\alpha_{ek}\uplambda_{ek}^2{\bf s}_e, $ $ \end{document}for activity \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\upphi}_{ek}$$\end{document } conducted from local excitatory firing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\text{s}}_e$$\end{document } to distant excitatory k = e orinhibitory k = i populations. extra - cortical input : excitatory or inhibitory thalamic input onto cortical excitatory or inhibitory is incorporated however the theory currently excludes the reciprocal thalamo - cortical connections that a number of other dynamical theories of the eeg include (robinson. 1997) because on the basis of current data their cortical effects can not be meaningfully parameterised. like the majority of its counterparts (freeman 1975 ; nunez 1981 ; jirsa and haken 1996 ; robinson. 1997) this theory is cast mathematically as a set of coupled non - linear partial differential equations. this general mathematical form applies to a wide variety of phenomena in nature, such as the initiation and propagation of the action potential along an axon (the celebrated hodgkin huxley equations) and the flow of compressible and incompressible fluids (the navier stokes equations) (keener and sneyd 2001). because these equations are continuous in space it implies that the macroscopic dynamics of cortex are that of a spatially continuous excitable two dimensional sheet. this theory, and other continuum mean field theories like it, are capable of producing a range of dynamical activity bearing striking similarity to that actually seen in real eeg and ecog recordings. for example (2002) is able to produce a physiologically plausible alpha rhythm when configured by physiologically admissible parameter sets, as well as producing a rich repertoire of dynamical activity that is expected to accord with the complexity of cognitive activity (dafilis. while these mean field theories provide powerful methods for understanding a range of eeg phenomena their real physiological relevance lies in establishing a mesoscopic link between microscopic physiology and macroscopic behaviour (fig. 4). the advantage of mean field population models of cortical tissue is the ability to explore how perturbations in a range of microscopic parameters influences neural activity macroscopically. for example, exploring how impairing inhibitory neurotransmission microscopically may lead to hyperexcitability at the macroscopic level (e.g. epileptic bursting in eeg) (kiss and erdi 2002 ; lopes da silva. 4mean field theories of neural populations are best defined as mesoscopic and thus intervene between microscopic and macroscopic scales. based on the microscopic study of neurons and their interactions, experimental values are used to parameterise inhibitory and excitatory neurons (a) and their connectivity at intracortical (b), cortico cortical (c) and extracortical (d) scales. subsequently these mean values of interaction define summed physiological properties which reflect macroscopic state variables mean field theories of neural populations are best defined as mesoscopic and thus intervene between microscopic and macroscopic scales. based on the microscopic study of neurons and their interactions, experimental values are used to parameterise inhibitory and excitatory neurons (a) and their connectivity at intracortical (b), cortico cortical (c) and extracortical (d) scales. although a variety of phenomena have been explored with population models of the eeg (e.g. epilepsy, sleep, chaos), only recently have researchers highlighted the dynamical modelling of drug effects (aradi and erdi 2006). in recent times, a number of researchers have developed detailed mean field descriptions of general anaesthetic drug action (steyn - ross. these models have not only shown to be capable of simulating unexpected experimental phenomena (e.g. the paradoxical rise and fall in eeg power that occurs during anaesthetic induction know as the biphasic response (kuizenga. 1998, 2001)), but have also shown the capacity to explain a number of clinical phenomenon (e.g. anaesthetic induced seizure) (liley and bojak 2005 ; wilson. 2006a). an elementary spectral comparison of the eeg between normal resting states and deep anaesthesia shows a strong suppression of alpha (813 hz) and beta (1325 hz) power bands, and a dominance of slow wave delta / theta (0.58 hz) power (john. additionally, during deep anaesthesia the eeg signal shows periods of spindle (burst) like oscillations which are interleaved with isoelectric burst suppression (rampil 1998). these eeg phenomena follow intuitively as a psychophysiological analogue to the well know eeg characterisation of sleep stages (hobson and pace schott 2002). however, the progression from resting state to deep anaesthesia does not appear to be a simple linear depression. a number of researchers have noted that the eeg increases in alpha and/or beta frequencies with low doses of general anaesthetics, particularly propofol (kishimoto. 2004), high doses of benzodiazepines (liley. 2003 ; jensen. 2005) and high doses of dissociative analgesics (rampil. 1998 ; tsuda. 1998, 2001), where band limited amplitude increases and decays with anaesthetic induction, and reverses with emergence. thus, a set of clear quantitative eeg (qeeg) phenomena exist for empirical comparison with theoretical simulation of anaesthesia (john. furthermore, these empirical phenomenon highlight the dependence of qeeg doa monitoring upon the capacity to detect the nuance of anaesthetic drug effect, in order to claim sensitivity. anaesthesia is generally defined by loss of consciousness, immobility, amnesia and lack of response to noxious stimuli (e.g. incision) (ishizawa 2007), brought about by a diverse range of neuropharmacological action. however, the predominant pharmacology of general anaesthetics in cerebral cortex is thought to be agonism of gabaa ionotropic receptor function (krasowski and harrison 1999). specifically, anaesthetics varyingly potentiate the amplitude and time course of inhibitory post - synaptic potentials (ipsp) via positive allosteric modulation of the gabaa receptor (hemmings. 2005). this positive influence, in a simplified sense, imposes a net increase in inhibitory neurotransmission (increased cl conductance) and thus a depression of the cns (rudolph and antkowiak 2004). having a heteropentameric structure the gabaa receptor contains a large subunit / subtype diversity (fig. recent findings in molecular biology have suggested that particular subunit compositions of the gabaa receptor mediate general anaesthesia and its sedative / hypnotic influence (nelson.. in particular benzodiazepines may be sensitive to and subtypes, whilst anaesthetics differ in affinity relative to subtype (rudolph and antkowiak 2004).fig. 5gaba ionotropic receptors (gabaa and gabac) can be formed via combinations of (16), (14), (13), (1), (1), (1), (1) and (13) subunits (sieghart and sperk 2002). predominately gabaa receptors are of the 212212 isoform, as shown above (mckernan and whiting 1996 ; johnston. resulting in a cl specific ion channel that is gated by gaba, shown above as g. benzodiazepines modulate gabaa receptors via a different binding location, represented as b. anaesthetics show preference for the 2 subunit (campagna - slater and weaver 2007) gaba ionotropic receptors (gabaa and gabac) can be formed via combinations of (16), (14), (13), (1), (1), (1), (1) and (13) subunits (sieghart and sperk 2002). predominately gabaa receptors are of the 212212 isoform, as shown above (mckernan and whiting 1996 ; johnston. resulting in a cl specific ion channel that is gated by gaba, shown above as g. benzodiazepines modulate gabaa receptors via a different binding location, represented as b. anaesthetics show preference for the 2 subunit (campagna - slater and weaver 2007) in modelling anaesthetic action, investigators need to pay attention to these differences in agent affinity, receptor composition and postsynaptic physiological influence. in the case of current mean field models it is mainly the shape and amplitude of the ipsp which is parametrically altered to model anaesthetic effects (see fig. 2). due to the considerable diversity of channel composition and lack of empirical data, individual receptor kinetics can not be comprehensively modelled at present. in the future more detailed experimental investigations however, differences between anaesthetic agents and their gabaa receptor subtype affinity may well be accounted for sufficiently by their precise impact on the ipsps alone. as will be highlighted in section clinical implications of mean field population models, minor differences in ipsp shape can lead to significant macroscopic effects. before exploring some of the details of mean field models of anaesthesia, a brief summary of the more discrete cellular and network models will provide some of the principles necessary to parameterise drug action. the chief aim of cellular approaches is to successfully create discrete models of spiking neurons which can be subject to analytical or numerical exploration (arhem. 2003). it is through the coupling of neurons (nodes) that larger cell assemblies / networks can be explored in a similar fashion. a key example of the cellular / network approach to modelling anaesthesia is the work of traub. (1999) applied their work on hippocampal gamma oscillations to anaesthetics, benzodiazepines and pro - convulsants (thiopentone, diazapam and bicuculline respectively). simulations of highly coupled inhibitory networks (96 all - to - all connected interneurons), simulated empirical findings obtained from hippocampus. specifically, the authors found that the interneuron network peak field potential frequency (predominantly gamma) was sensitive to the parameter gaba(a), the decay time constant of hyperpolarising gabaa receptor - mediated conductance. increases in gaba(a) modelled benzodiazepine and anaesthetic action, consistent with postsynaptic hyperpolarisation and lowered network local field potential frequency. conversely, the reduction of gaba(a) reflected those agents which block or otherwise antagonise the gabaa receptor (pro - convulsants), leading to hyperexcitability and increased network frequency. one interesting development of these findings relates to capacity of high dose benzodiazepines to increase eeg beta power. (1999) speculated that increased beta activity after the ingestion of benzodiazepines may be due to the frequency of background gamma power being lowered, more synchronous and larger in amplitude thus making it more easily detectable at the scalp. these simulation studies may prove to be helpful in understanding similar phenomena induced by low dose intravenous anaesthetics and high dose dissociative anaesthetics (e.g. propofol and ketamine respectively). in their studies faulkner. explored the influence of anaesthetics upon rat hippocampal slices, both empirically and computationally. in the first of two studies faulkner. explored the influence of anaesthetic and analgesic agents (thiopental, diazepam / temazepam, morphine and ketamine) on elicited synchronous gamma (40 hz) oscillations in rat hippocampus (ca1) complementary computational simulations used a neural network of individual excitatory and inhibitory layers (each being 30 20 nodes). nodes were then coupled with 50 excitatory and 49 inhibitory inputs, and a subsection (slice) of the array selected for simulation (6 20 ; 120 pyramidal ; 120 interneurons). therefore from a model topology which approximated the ca1 hippocampal morphology, a subsection of this model (slice) could then be compared with the results obtained from experimental hippocampal slice preparations. baseline oscillatory activity was created though tonic excitatory driving of both cell groups, with inhibitory drug effects modelled by modifying gabaa conductance.. showed strong similarities between slice recordings and computational simulations of thiopental s suppressive influence on gamma synchrony. the authors postulated that decreased gamma synchrony lowered distributed microscopic communication in the hippocampus, providing a putative amnestic mechanism. extrapolations of this mechanism therefore suggests that anaesthesia results from wide spread loss of high frequency synchronized network activity. are sensitive to changes in the specific subunit composition of the gabaa receptor. in a recent investigation ujfalussy. (2007) combined experimental and computational methods to explore gabaa receptor subunit sensitivity of two positive allosteric modulators (zolpidem and l-838417). computational models were constructed to simulate temporal pacemaker properties of the septo - hippocampal system. (2007) assumed that the maximal synaptic conductance of all gabaa ionotropic receptor channels in hippocampus (ca1) and medial septum would increase. therefore, either the inhibitory time (decay) constant or amplitude of the ipsp was modified to increase the net charge transfer (synaptic current). in contrast, the other agent modelled, l-838417, had specific influence on hippocampal pyramidal neurons and was therefore modelled by lowering the driving phasic input of septo - hippocampal projecting neurons. results showed that the simulated effect of zolpidem and l-838417 produced differing patterns of spike and oscillatory behaviour, however both reduced the commonly observed theta oscillation of the septo - hippocampal circuit (ujfalussy. as zolpidem binds to 1gabaa receptors and l-838417 to 2/3/5 gabaa receptors, these methods can assist in drug development. in this particular case, the authors suggest that some of the side effects of the broadly binding benzodiazepines (e.g. diazepam) may be avoided by compounds with more specific anxiolytic gabaergic targets (ujfalussy. this study highlighted the capacity to not only simulate drug action by specific pharmacological parameterisation, but also through altering specific cell group connectivity (i.e. the tonic driving strength of excitatory pyramidal neurons). over the past 510 years a small number of research groups have focused on developing mean field population models of anaesthetic action, which sceniak and maciver (2006) referred to as anaesthesia in silico in a recent editorial. these mesoscopic models are complimentary to single neuronal network based methods, as they allow collective properties of neural tissue dynamics to be simulated on much larger scales. thus it can be said that while discrete approaches to macroscopic cortical modelling are impractical, so too are the application of population field models to the modelling of microscopic circuits. the first reported exploration of anaesthetic action in a mean field population model was that of steyn - ross. (1998) to a set of equations which facilitated analytical exploration using a range of established stochastic methods. with this new set of equations steyn - ross. (1999) aimed to simulate the effects of anaesthesia by systematically modulating parameters that correspond to the known pharmacological targets associated with anaesthetic action. in particular, of the many system parameters steyn - ross. (1999) specifically modulated the inhibitory neurotransmitter rate constant (i), which quantifies the shape of the ipsp. as highlighted above, general anaesthetics typically exert their influence by positive allosteric modulation of the gabaa receptor, increasing negative charge transfer and prolonging the tail of the unitary ipsp. thus reducing i, which increases the time scale of inhibitory neurotransmission, models the increasing effects of anaesthetics. therefore, anaesthesia was parameterised by scaling the inhibitory neurotransmitter rate constant relative to empirical findings of ipsp prolongation by anaesthetics (franks and lieb 1994). by observing how the state variables equivalent to the eeg (excitatory mean soma membrane potential ; he) changed with modelled increases in drug concentration, the effect of anaesthesia on the eeg was simulated. (1999) found that through the modulation of the inhibitory neurotransmitter rate constant, the model showed three stages of analytical equilibrium which they termed : coma (i), wakefulness (ii) and epilepsy (iii) (fig. 6d). each of these three system states reflected a progressive reduction of anaesthetic concentration. high levels of modelled anaesthesia (large prolonging of ipsp) produced a state neurophysiologically equivalent to coma or very deep anaesthesia. subsequently, approximate zero levels of simulated anaesthetic effect produced wakefulness, which in turn could be perturbed to an epileptic like state when anaesthetic influence became negative (equivalent to simulating a pro - convulsant compound which antagonises the gabaa ipsp, e.g. bicuculline). (1999) also were able to model a number of important empirical eeg phenomena associated with anaesthetic action, including the biphasic effect (fig. as indicated above when comparing normal resting levels with deep anaesthesia the eeg shows strong slow wave (delta / theta) dominance. however, the transition between these two states shows an unexpected rise and fall of band limited (1115 hz) amplitude, hence the term biphasic response (kuizenga. (1999) found that modelled eeg spectra also showed a clear biphasic response during simulated anaesthesia.fig. (2004) modelled the abrupt changes in eeg spectral power that have been commonly observed to occur during anaesthetic induction and emergence. (a) the abrupt biphasic response in total modelled eeg spectral power during simulated anaesthetic induction. anaesthetic action was modelled as a prolongation of the duration of the gabaergic ipsp (). (c) actual eeg recordings of anaesthetic induction and emergence with the intravenous anaesthetic agent propofol which illustrate clearly the biphasic variations in total eeg power as anaesthetic concentration is increased and decreased. propose that anaesthetic induced variations in eeg spectral power can be explained theoretically by a stochastically driven system switching between two stable states via a saddle node bifurcation caused by anaesthetic induced variations in ipsp duration. (2004) modelled the abrupt changes in eeg spectral power that have been commonly observed to occur during anaesthetic induction and emergence. (a) the abrupt biphasic response in total modelled eeg spectral power during simulated anaesthetic induction. anaesthetic action was modelled as a prolongation of the duration of the gabaergic ipsp (). (c) actual eeg recordings of anaesthetic induction and emergence with the intravenous anaesthetic agent propofol which illustrate clearly the biphasic variations in total eeg power as anaesthetic concentration is increased and decreased. propose that anaesthetic induced variations in eeg spectral power can be explained theoretically by a stochastically driven system switching between two stable states via a saddle node bifurcation caused by anaesthetic induced variations in ipsp duration. 2004), with permission the primary conclusion of steyn - ross. (1999) was that the empirical biphasic response could be understood as a parametrically induced phase transition between two dynamically distinct states. (1999) went on to further develop their phase transition description of anaesthesia, drawing analogy to the phase transitions commonly seen in a range of thermodynamic systems, such as the sudden transition of a liquid to a gas during heating (steyn - ross. (2001b) developed an excitation parameter which reflected the inverse of anaesthetic effect, and therefore constituted the analogue of heat. it was through a thorough analytical analysis of changes in excitation (heat) that steyn - ross. the authors justify the physiological relevance of decreases in entropy after the phase transition to unconsciousness (low excitation and strong anaesthetic effect), by pointing to empirical findings of lowered metabolic state (stullken. 1977) and lowered eeg spectral entropy during anaesthesia. (1999) had used a simplified and spatially lumped version of the liley. (1998) mean field continuum theory, some of the qualitative characteristics of eeg dynamics were not preserved. therefore in order to begin exploration of the spatial character of the dynamic changes induced by anaesthesia, steyn - ross. (2003) constructed a one - dimensional cortex of linked neural masses (cortical rod). this theoretical construction allowed for rudimentary comparison of spatial inhomogeneities during anaesthetic induction (i.e. the theoretical comparison of two separated monopolar electrodes, with a common reference) (steyn - ross. (2003) related the findings of their analytical work to recent discoveries in anaesthesia research, highlighting their theoretical findings of increased spatial covariance as being supported by those empirical results showing increased inter - electrode coherence at the points of loss and recovery of consciousness (i.e. the points of proposed phase transition) (john. (2004) posited that the transition from consciousness to unconsciousness occurs as a consequence of a phase transition in cortical neural population dynamics brought about due to the increasing inhibitory effects of modelled anaesthetic concentration. during this transition there is a surge in band limited eeg power which is of greater magnitude during emergence than during induction, with this hysteresis being observed empirically. furthermore, the phase transition is also characterised by increased spatial and temporal correlation, for both induction and emergence, with cortical entropy being lowered during unconsciousness. on this basis the authors argue that anaesthetics induce a sharp rise in eeg power that produces a rapid shift into a state of slowed and more ordered eeg rhythmicity, which is reversed, though not symmetrically, with emergence. while the results of steyn - ross. were able to account for a number of important macroscopic eeg properties associated with anaesthetic action, they did so in a simplified model that was unable to produce normal resting oscillatory behaviour, such as alpha. in order to remedy this bojak. (2002) equations to model the effects of anaesthetics on the eeg. unlike the original investigations of steyn - ross. (2002) equations, rather the full set of equations where numerically solved on a spatio - temporal scale that would more accurately approximate observed eeg. in doing so, the influence of microscopically defined parameter modification could be observed on a macroscopic scale that is comparable to empirical eeg. (2004) were also able to qualitatively reproduce the biphasic response through only the modulation of the inhibitory decay constant (ipsp prolongation). a further development in bojak and liley (2005) was the reparametrisation of postsynaptic potentials to more accurately fit experimental data. alpha form had been used, i.e., for an incoming presynaptic pulse at time t = 0 the assumed psp response at t 0 was:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_\alpha(t)=\upgamma\gamma\exp(1)\times t \exp\left(-\gamma t\right), $ $ \end{document } as illustrated in fig. 2 for an ipsp, this response has a maximum amplitude at rise time 1/. unfortunately the psp decay time for which the response falls again to /exp(1) is for the alpha form fixed at 3.1462. anaesthetic agents often potentiate gabaa induced currents and/or attenuate glutamate mediated ionic currents (rudolph and antkowiak 2004), thereby selectively prolonging the inhibitory ik without affecting the inhibitory rise time ik. hence bojak and liley (2005) introduced eq. 2 yielding a response of the form5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r(t)=\upgamma\gamma \exp\left(\tilde{\gamma}\delta\right)\times \frac{\exp\left(-\gamma t\right)-\exp\left(-\tilde{\gamma}t\right)}{\tilde{\gamma}-\gamma }, $ $ \end{document}with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta\equiv\hbox{ln}(\tilde{\gamma}/\gamma)/(\tilde{\gamma}-\gamma).$$\end{document } this bi - exponential has the same maximum amplitude r(t =) =. however, the additional parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde{\gamma}$$\end{document } can be used to adjust the decay time freely. this allowed bojak and liley (2005) to directly incorporate into their model experimental data on the effect of isoflurane on psps (maciver. 1996 ; banks and pearce 1999 ; nishikawa and maciver 2000), and to predict the eeg under anaesthesia by calculating the corresponding changes to the excitatory mean soma membrane potential he, see eq. 1. bojak and liley (2005) were able to show systematic changes in modelled eeg as a function of isoflurane concentration. an additional selection criteria was then imposed upon these sets of data, which aimed at extracting sets producing a clear biphasic response (bojak and liley 2005). the outcomes of bojak and liley s (2005) explorations showed some difference to that of steyn - ross. it is particularly worth highlighting that while both theories describe changes in macroscopic dynamics during anaesthesia, steyn - ross. (2004) suggest a phase transition (e.g. boiling water to steam), whereas bojak and liley (2005) posit a continuum transition. bojak and liley (2005) highlight that the sharp rise in neural firing rate required for steyn - ross.s (2004) phase transition is not supported by empirical findings of smooth suppression of action potential frequency with progressive anaesthesia. (2007) sought to further develop the work of steyn - ross. (2004) and bojak and liley (2005), by implementing a model parameterisation of slow voltage dependent mechanisms on excitatory firing rate dynamics. the common sigmoidal relationship between mean soma membrane potential and mean firing rate was modified to included the influence of slow shifts in membrane potential. this voltage dependant slow ionic leak, was motivated by the empirically observed switching (i.e. up and down states) in firing rates of neurons during anaesthesia and non - rem sleep, thought to be controlled by cation conductance. (2007) explored progressive increases in simulated desflurane anaesthesia, showing up state dominance during waking levels, up / down state switching (delta) at moderate anaesthesia and down state predominance during strong anaesthetic effect (fig. this switching in firing rate between bursting up states and quiescent down states relates biophysically to the synchronised delta power local field potential predominately seen during deep anaesthesia and stage 3/4 sleep. these findings further promote the ability to theoretically relate cellular experimental observations with bulk macroscopic cortical dynamics via mean field theoretics.fig. (2007) simulated eeg (mean soma membrane potential he) at various concentrations of simulated anaesthesia by assuming that anaesthetics, in addition to increasing the duration of the ipsp, also cause slow adaptive changes in the shape of the mesoscopic firing rate function which relates mean soma membrane potential to mean population firing rate. (2) actual eeg recorded in children undergoing anaesthesia with the volatile halogenated agent desflurane. (a) 0 mac (minimum alveolar concentration), (b) 1 mac, (c) > 1 mac and (d) 2 mac. both real and simulated data show that increasing anaesthetic effect is associated with enhanced total eeg power and reductions in mean frequency. (2007), with permission (1) molaee - ardekani. (2007) simulated eeg (mean soma membrane potential he) at various concentrations of simulated anaesthesia by assuming that anaesthetics, in addition to increasing the duration of the ipsp, also cause slow adaptive changes in the shape of the mesoscopic firing rate function which relates mean soma membrane potential to mean population firing rate. (2) actual eeg recorded in children undergoing anaesthesia with the volatile halogenated agent desflurane. (a) 0 mac (minimum alveolar concentration), (b) 1 mac, (c) > 1 mac and (d) 2 mac. both real and simulated data show that increasing anaesthetic effect is associated with enhanced total eeg power and reductions in mean frequency. anaesthesia is generally defined by loss of consciousness, immobility, amnesia and lack of response to noxious stimuli (e.g. incision) (ishizawa 2007), brought about by a diverse range of neuropharmacological action. however, the predominant pharmacology of general anaesthetics in cerebral cortex is thought to be agonism of gabaa ionotropic receptor function (krasowski and harrison 1999). specifically, anaesthetics varyingly potentiate the amplitude and time course of inhibitory post - synaptic potentials (ipsp) via positive allosteric modulation of the gabaa receptor (hemmings. 2005). this positive influence, in a simplified sense, imposes a net increase in inhibitory neurotransmission (increased cl conductance) and thus a depression of the cns (rudolph and antkowiak 2004). having a heteropentameric structure the gabaa receptor contains a large subunit / subtype diversity (fig. recent findings in molecular biology have suggested that particular subunit compositions of the gabaa receptor mediate general anaesthesia and its sedative / hypnotic influence (nelson. in particular benzodiazepines may be sensitive to and subtypes, whilst anaesthetics differ in affinity relative to subtype (rudolph and antkowiak 2004).fig. 5gaba ionotropic receptors (gabaa and gabac) can be formed via combinations of (16), (14), (13), (1), (1), (1), (1) and (13) subunits (sieghart and sperk 2002). predominately gabaa receptors are of the 212212 isoform, as shown above (mckernan and whiting 1996 ; johnston. resulting in a cl specific ion channel that is gated by gaba, shown above as g. benzodiazepines modulate gabaa receptors via a different binding location, represented as b. anaesthetics show preference for the 2 subunit (campagna - slater and weaver 2007) gaba ionotropic receptors (gabaa and gabac) can be formed via combinations of (16), (14), (13), (1), (1), (1), (1) and (13) subunits (sieghart and sperk 2002). predominately gabaa receptors are of the 212212 isoform, as shown above (mckernan and whiting 1996 ; johnston. resulting in a cl specific ion channel that is gated by gaba, shown above as g. benzodiazepines modulate gabaa receptors via a different binding location, represented as b. anaesthetics show preference for the 2 subunit (campagna - slater and weaver 2007) in modelling anaesthetic action, investigators need to pay attention to these differences in agent affinity, receptor composition and postsynaptic physiological influence. in the case of current mean field models it is mainly the shape and amplitude of the ipsp which is parametrically altered to model anaesthetic effects (see fig. 2). due to the considerable diversity of channel composition and lack of empirical data, individual receptor kinetics can not be comprehensively modelled at present. in the future more detailed experimental investigations however, differences between anaesthetic agents and their gabaa receptor subtype affinity may well be accounted for sufficiently by their precise impact on the ipsps alone. as will be highlighted in section clinical implications of mean field population models before exploring some of the details of mean field models of anaesthesia, a brief summary of the more discrete cellular and network models will provide some of the principles necessary to parameterise drug action. the chief aim of cellular approaches is to successfully create discrete models of spiking neurons which can be subject to analytical or numerical exploration (arhem. it is through the coupling of neurons (nodes) that larger cell assemblies / networks can be explored in a similar fashion. a key example of the cellular / network approach to modelling anaesthesia is the work of traub. (1999) applied their work on hippocampal gamma oscillations to anaesthetics, benzodiazepines and pro - convulsants (thiopentone, diazapam and bicuculline respectively). simulations of highly coupled inhibitory networks (96 all - to - all connected interneurons), simulated empirical findings obtained from hippocampus. specifically, the authors found that the interneuron network peak field potential frequency (predominantly gamma) was sensitive to the parameter gaba(a), the decay time constant of hyperpolarising gabaa receptor - mediated conductance. increases in gaba(a) modelled benzodiazepine and anaesthetic action, consistent with postsynaptic hyperpolarisation and lowered network local field potential frequency. conversely, the reduction of gaba(a) reflected those agents which block or otherwise antagonise the gabaa receptor (pro - convulsants), leading to hyperexcitability and increased network frequency. one interesting development of these findings relates to capacity of high dose benzodiazepines to increase eeg beta power. (1999) speculated that increased beta activity after the ingestion of benzodiazepines may be due to the frequency of background gamma power being lowered, more synchronous and larger in amplitude thus making it more easily detectable at the scalp. these simulation studies may prove to be helpful in understanding similar phenomena induced by low dose intravenous anaesthetics and high dose dissociative anaesthetics (e.g. propofol and ketamine respectively). in their studies faulkner. explored the influence of anaesthetics upon rat hippocampal slices, both empirically and computationally. in the first of two studies faulkner. explored the influence of anaesthetic and analgesic agents (thiopental, diazepam / temazepam, morphine and ketamine) on elicited synchronous gamma (40 hz) oscillations in rat hippocampus (ca1). complementary computational simulations used a neural network of individual excitatory and inhibitory layers (each being 30 20 nodes). nodes were then coupled with 50 excitatory and 49 inhibitory inputs, and a subsection (slice) of the array selected for simulation (6 20 ; 120 pyramidal ; 120 interneurons). therefore from a model topology which approximated the ca1 hippocampal morphology, a subsection of this model (slice) could then be compared with the results obtained from experimental hippocampal slice preparations. baseline oscillatory activity was created though tonic excitatory driving of both cell groups, with inhibitory drug effects modelled by modifying gabaa conductance.. showed strong similarities between slice recordings and computational simulations of thiopental s suppressive influence on gamma synchrony. the authors postulated that decreased gamma synchrony lowered distributed microscopic communication in the hippocampus, providing a putative amnestic mechanism. extrapolations of this mechanism therefore suggests that anaesthesia results from wide spread loss of high frequency synchronized network activity. are sensitive to changes in the specific subunit composition of the gabaa receptor. in a recent investigation (2007) combined experimental and computational methods to explore gabaa receptor subunit sensitivity of two positive allosteric modulators (zolpidem and l-838417). computational models were constructed to simulate temporal pacemaker properties of the septo - hippocampal system. (2007) assumed that the maximal synaptic conductance of all gabaa ionotropic receptor channels in hippocampus (ca1) and medial septum would increase. therefore, either the inhibitory time (decay) constant or amplitude of the ipsp was modified to increase the net charge transfer (synaptic current). in contrast, the other agent modelled, l-838417, had specific influence on hippocampal pyramidal neurons and was therefore modelled by lowering the driving phasic input of septo - hippocampal projecting neurons. results showed that the simulated effect of zolpidem and l-838417 produced differing patterns of spike and oscillatory behaviour, however both reduced the commonly observed theta oscillation of the septo - hippocampal circuit (ujfalussy. as zolpidem binds to 1gabaa receptors and l-838417 to 2/3/5 gabaa receptors, these methods can assist in drug development. in this particular case, the authors suggest that some of the side effects of the broadly binding benzodiazepines (e.g. diazepam) may be avoided by compounds with more specific anxiolytic gabaergic targets (ujfalussy. this study highlighted the capacity to not only simulate drug action by specific pharmacological parameterisation, but also through altering specific cell group connectivity (i.e. the tonic driving strength of excitatory pyramidal neurons). over the past 510 years a small number of research groups have focused on developing mean field population models of anaesthetic action, which sceniak and maciver (2006) referred to as anaesthesia in silico in a recent editorial. these mesoscopic models are complimentary to single neuronal network based methods, as they allow collective properties of neural tissue dynamics to be simulated on much larger scales. thus it can be said that while discrete approaches to macroscopic cortical modelling are impractical, so too are the application of population field models to the modelling of microscopic circuits. the first reported exploration of anaesthetic action in a mean field population model was that of steyn - ross. (1998) to a set of equations which facilitated analytical exploration using a range of established stochastic methods. with this new set of equations steyn - ross. (1999) aimed to simulate the effects of anaesthesia by systematically modulating parameters that correspond to the known pharmacological targets associated with anaesthetic action. (1999) specifically modulated the inhibitory neurotransmitter rate constant (i), which quantifies the shape of the ipsp. as highlighted above, general anaesthetics typically exert their influence by positive allosteric modulation of the gabaa receptor, increasing negative charge transfer and prolonging the tail of the unitary ipsp. thus reducing i, which increases the time scale of inhibitory neurotransmission, models the increasing effects of anaesthetics. therefore, anaesthesia was parameterised by scaling the inhibitory neurotransmitter rate constant relative to empirical findings of ipsp prolongation by anaesthetics (franks and lieb 1994). by observing how the state variables equivalent to the eeg (excitatory mean soma membrane potential ; he) changed with modelled increases in drug concentration, the effect of anaesthesia on the eeg was simulated. (1999) found that through the modulation of the inhibitory neurotransmitter rate constant, the model showed three stages of analytical equilibrium which they termed : coma (i), wakefulness (ii) and epilepsy (iii) (fig. 6d). each of these three system states reflected a progressive reduction of anaesthetic concentration. high levels of modelled anaesthesia (large prolonging of ipsp) produced a state neurophysiologically equivalent to coma or very deep anaesthesia. subsequently, approximate zero levels of simulated anaesthetic effect produced wakefulness, which in turn could be perturbed to an epileptic like state when anaesthetic influence became negative (equivalent to simulating a pro - convulsant compound which antagonises the gabaa ipsp, e.g. bicuculline). (1999) also were able to model a number of important empirical eeg phenomena associated with anaesthetic action, including the biphasic effect (fig. as indicated above when comparing normal resting levels with deep anaesthesia the eeg shows strong slow wave (delta / theta) dominance. however, the transition between these two states shows an unexpected rise and fall of band limited (1115 hz) amplitude, hence the term biphasic response (kuizenga. (1999) found that modelled eeg spectra also showed a clear biphasic response during simulated anaesthesia.fig. (2004) modelled the abrupt changes in eeg spectral power that have been commonly observed to occur during anaesthetic induction and emergence. (a) the abrupt biphasic response in total modelled eeg spectral power during simulated anaesthetic induction. anaesthetic action was modelled as a prolongation of the duration of the gabaergic ipsp (). (c) actual eeg recordings of anaesthetic induction and emergence with the intravenous anaesthetic agent propofol which illustrate clearly the biphasic variations in total eeg power as anaesthetic concentration is increased and decreased. propose that anaesthetic induced variations in eeg spectral power can be explained theoretically by a stochastically driven system switching between two stable states via a saddle node bifurcation caused by anaesthetic induced variations in ipsp duration. (2004) modelled the abrupt changes in eeg spectral power that have been commonly observed to occur during anaesthetic induction and emergence. (a) the abrupt biphasic response in total modelled eeg spectral power during simulated anaesthetic induction. anaesthetic action was modelled as a prolongation of the duration of the gabaergic ipsp (). (c) actual eeg recordings of anaesthetic induction and emergence with the intravenous anaesthetic agent propofol which illustrate clearly the biphasic variations in total eeg power as anaesthetic concentration is increased and decreased. propose that anaesthetic induced variations in eeg spectral power can be explained theoretically by a stochastically driven system switching between two stable states via a saddle node bifurcation caused by anaesthetic induced variations in ipsp duration. 2004), with permission the primary conclusion of steyn - ross. (1999) was that the empirical biphasic response could be understood as a parametrically induced phase transition between two dynamically distinct states. (1999) went on to further develop their phase transition description of anaesthesia, drawing analogy to the phase transitions commonly seen in a range of thermodynamic systems, such as the sudden transition of a liquid to a gas during heating (steyn - ross. (2001b) developed an excitation parameter which reflected the inverse of anaesthetic effect, and therefore constituted the analogue of heat. it was through a thorough analytical analysis of changes in excitation (heat) that steyn - ross. (2001b) constructed a dynamical mean field description of anaesthesia. as with thermodynamic phase transitions, steyn - ross. the authors justify the physiological relevance of decreases in entropy after the phase transition to unconsciousness (low excitation and strong anaesthetic effect), by pointing to empirical findings of lowered metabolic state (stullken. (1999) had used a simplified and spatially lumped version of the liley. (1998) mean field continuum theory, some of the qualitative characteristics of eeg dynamics were not preserved. therefore in order to begin exploration of the spatial character of the dynamic changes induced by anaesthesia, steyn - ross. (2003) constructed a one - dimensional cortex of linked neural masses (cortical rod). this theoretical construction allowed for rudimentary comparison of spatial inhomogeneities during anaesthetic induction (i.e. the theoretical comparison of two separated monopolar electrodes, with a common reference) (steyn - ross. (2003) related the findings of their analytical work to recent discoveries in anaesthesia research, highlighting their theoretical findings of increased spatial covariance as being supported by those empirical results showing increased inter - electrode coherence at the points of loss and recovery of consciousness (i.e. the points of proposed phase transition) (john. (2004) posited that the transition from consciousness to unconsciousness occurs as a consequence of a phase transition in cortical neural population dynamics brought about due to the increasing inhibitory effects of modelled anaesthetic concentration. during this transition there is a surge in band limited eeg power which is of greater magnitude during emergence than during induction, with this hysteresis being observed empirically. furthermore, the phase transition is also characterised by increased spatial and temporal correlation, for both induction and emergence, with cortical entropy being lowered during unconsciousness. on this basis the authors argue that anaesthetics induce a sharp rise in eeg power that produces a rapid shift into a state of slowed and more ordered eeg rhythmicity, which is reversed, though not symmetrically, with emergence. while the results of steyn - ross. were able to account for a number of important macroscopic eeg properties associated with anaesthetic action, they did so in a simplified model that was unable to produce normal resting oscillatory behaviour, such as alpha. in order to remedy this bojak. (2002) equations to model the effects of anaesthetics on the eeg. unlike the original investigations of steyn - ross. (2002) equations, rather the full set of equations where numerically solved on a spatio - temporal scale that would more accurately approximate observed eeg. in doing so, the influence of microscopically defined parameter modification could be observed on a macroscopic scale that is comparable to empirical eeg. (2004) were also able to qualitatively reproduce the biphasic response through only the modulation of the inhibitory decay constant (ipsp prolongation). a further development in bojak and liley (2005) was the reparametrisation of postsynaptic potentials to more accurately fit experimental data. alpha form had been used, i.e., for an incoming presynaptic pulse at time t = 0 the assumed psp response at t 0 was:4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r_\alpha(t)=\upgamma\gamma\exp(1)\times t \exp\left(-\gamma t\right), $ $ \end{document } as illustrated in fig. 2 for an ipsp, this response has a maximum amplitude at rise time 1/. unfortunately the psp decay time for which the response falls again to /exp(1) is for the anaesthetic agents often potentiate gabaa induced currents and/or attenuate glutamate mediated ionic currents (rudolph and antkowiak 2004), thereby selectively prolonging the inhibitory ik without affecting the inhibitory rise time ik. hence bojak and liley (2005) introduced eq. 2 yielding a response of the form5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ r(t)=\upgamma\gamma \exp\left(\tilde{\gamma}\delta\right)\times \frac{\exp\left(-\gamma t\right)-\exp\left(-\tilde{\gamma}t\right)}{\tilde{\gamma}-\gamma }, $ $ \end{document}with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta\equiv\hbox{ln}(\tilde{\gamma}/\gamma)/(\tilde{\gamma}-\gamma).$$\end{document } this bi - exponential has the same maximum amplitude r(t =) =. however, the additional parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde{\gamma}$$\end{document } can be used to adjust the decay time freely. this allowed bojak and liley (2005) to directly incorporate into their model experimental data on the effect of isoflurane on psps (maciver. 1996 ; banks and pearce 1999 ; nishikawa and maciver 2000), and to predict the eeg under anaesthesia by calculating the corresponding changes to the excitatory mean soma membrane potential he, see eq. 1. bojak and liley (2005) were able to show systematic changes in modelled eeg as a function of isoflurane concentration. an additional selection criteria was then imposed upon these sets of data, which aimed at extracting sets producing a clear biphasic response (bojak and liley 2005). the outcomes of bojak and liley s (2005) explorations showed some difference to that of steyn - ross. it is particularly worth highlighting that while both theories describe changes in macroscopic dynamics during anaesthesia, steyn - ross. (2004) suggest a phase transition (e.g. boiling water to steam), whereas bojak and liley (2005) posit a continuum transition. bojak and liley (2005) highlight that the sharp rise in neural firing rate required for steyn - ross.s (2004) phase transition is not supported by empirical findings of smooth suppression of action potential frequency with progressive anaesthesia. (2007) sought to further develop the work of steyn - ross. (2004) and bojak and liley (2005), by implementing a model parameterisation of slow voltage dependent mechanisms on excitatory firing rate dynamics. the common sigmoidal relationship between mean soma membrane potential and mean firing rate was modified to included the influence of slow shifts in membrane potential. this voltage dependant slow ionic leak, was motivated by the empirically observed switching (i.e. up and down states) in firing rates of neurons during anaesthesia and non - rem sleep, thought to be controlled by cation conductance. (2007) explored progressive increases in simulated desflurane anaesthesia, showing up state dominance during waking levels, up / down state switching (delta) at moderate anaesthesia and down state predominance during strong anaesthetic effect (fig. this switching in firing rate between bursting up states and quiescent down states relates biophysically to the synchronised delta power local field potential predominately seen during deep anaesthesia and stage 3/4 sleep. these findings further promote the ability to theoretically relate cellular experimental observations with bulk macroscopic cortical dynamics via mean field theoretics.fig. (2007) simulated eeg (mean soma membrane potential he) at various concentrations of simulated anaesthesia by assuming that anaesthetics, in addition to increasing the duration of the ipsp, also cause slow adaptive changes in the shape of the mesoscopic firing rate function which relates mean soma membrane potential to mean population firing rate. (2) actual eeg recorded in children undergoing anaesthesia with the volatile halogenated agent desflurane. (a) 0 mac (minimum alveolar concentration), (b) 1 mac, (c) > 1 mac and (d) 2 mac. both real and simulated data show that increasing anaesthetic effect is associated with enhanced total eeg power and reductions in mean frequency. (2007), with permission (1) molaee - ardekani. (2007) simulated eeg (mean soma membrane potential he) at various concentrations of simulated anaesthesia by assuming that anaesthetics, in addition to increasing the duration of the ipsp, also cause slow adaptive changes in the shape of the mesoscopic firing rate function which relates mean soma membrane potential to mean population firing rate. (2) actual eeg recorded in children undergoing anaesthesia with the volatile halogenated agent desflurane. (a) 0 mac (minimum alveolar concentration), (b) 1 mac, (c) > 1 mac and (d) 2 mac. both real and simulated data show that increasing anaesthetic effect is associated with enhanced total eeg power and reductions in mean frequency. the volatile general anaesthetic enflurane, among others, has been shown to paradoxically promote epileptiform activity (modica. this pro - convulsant phenomenon is clearly at odds with the suppressive nature of anaesthetic compounds and lacks any clear physiological explanation. liley and bojak (2005) explored the pro - convulsant properties of enflurane by parameterising the ipsp with data from in vivo studies. these findings suggested that a distinguishing factor between enflurane and other volatile anaesthetics (e.g. isoflurane) was a significant reduction in ipsp amplitude (banks and pearce 1999) even though both agents increase net inhibition. because of the early developments in ipsp parameterisation liley and bojak (2005) were able to systematically explore how independent modulation of the ipsp amplitude influences system dynamics. it was found that anaesthetic agents, such as enflurane, that reduce ipsp amplitude more than other anaesthetics, were more likely to produce eeg activity characteristic of epilepsy (liley and bojak 2005). again liley and bojak (2005) used biologically constrained parameter sets which displayed suitable resting eeg spectra. it was found using a bifurcation analysis of a spatially homogeneous model, that isofluranes minimal effect on ipsp amplitude did not produce any progression to system instabilities. however, in over a third of the parameter sets the lower amplitude enflurane ipsps were shown to induce a strong shift from low firing, low amplitude resting state to an epileptic - like regime (fig. 8liley and bojak (2005) showed that systematically modulating ipsp shape and amplitude could induce high firing large amplitude activity in a mean field model parameterised to produce electroencephalographically plausible activity (1/f low frequency activity and strong alpha resonance). (b) it is predicted that anaesthetics that more rapidly reduce the amplitude of the ipsp (ik) in addition to prolonging it (ik), are more likely to produce high firing large amplitude activity electroencephalographic characteristic of epileptiform activity. 5 and 11 of liley and bojak (2005) liley and bojak (2005) showed that systematically modulating ipsp shape and amplitude could induce high firing large amplitude activity in a mean field model parameterised to produce electroencephalographically plausible activity (1/f low frequency activity and strong alpha resonance). (b) it is predicted that anaesthetics that more rapidly reduce the amplitude of the ipsp (ik) in addition to prolonging it (ik), are more likely to produce high firing large amplitude activity electroencephalographic characteristic of epileptiform activity. figure adapted from figs. 5 and 11 of liley and bojak (2005) at approximately the same time wilson. similar to the work of liley and bojak (2005), wilson. (2006a) showed too that it was the sensitivity towards ipsp amplitude which made enflurane unique in its effects when compared with isoflurane. an additionally interesting suggestion put forward on the basis of these findings was that the anticonvulsant thiopentone, which has been shown to be proepileptic during administration with low doses of enflurane, may enhance enflurane s influence because of its negligible effect on ipsp amplitude (wilson. (2006a) was the suggestion that the apparent sensitivity of macroscopic dynamics upon the magnitude of the drug induced peak ipsp amplitude may reflect a general phenomenon in which epileptic - like behaviour may be produced by interference with the integrative properties of dendrites. more generally, mean field models have an immediate potential to assist in the development of physiologically meaningful methods for monitoring anaesthetic depth using eeg. the use of biophysical models of cerebral cortical dynamics in parameterising or otherwise motivating eeg analysis methods for doa monitoring have the potential to more sensitively detect clinically relevant changes in the eeg given that they are derived from quantitative physiological descriptions of the anaesthetic process. at present most monitoring techniques are based on the construction of discriminating statistical functions from the quantitative analysis of eeg, in large populations of normal and clinical groups undergoing a range of anaesthetic procedures, in order to capture the generalised quantitative changes in the eeg that show systematic variations with anaesthetic action. despite the early promise of such black - box approaches, recent evidence suggests that they do not provide the clinician with any additional information regarding hypnotic state. in a recent study involving 2,000 patients avidan. (2008) found that the use of bispectral index (bis) monitoring was not associated with a reduction in the incidence of awareness during anaesthesia when compared to other more standard techniques for monitoring anaesthetic depth. this is significant as the bis is clinically the most widely used black - box eeg approach to monitoring anaesthetic depth. furthermore, the bis and other heuristic eeg monitoring approaches have been reported to be blind to the effects of a number of anaesthetic agents which are in routine clinical use, including nitrous oxide and the opioids (e.g. rampil. 1998 ; anderson and kakobsson 1992). by using methods of analysis derived from physiological descriptions of the anaesthetic process, quantitative indexing of the eeg for doa monitoring can perhaps be more robust to the variety of macroscopic physiological consequences of anaesthetic action as observed in the eeg. in an attempt to understand the bulk dynamical properties of cortical tissue, researchers have found mean field theories of particular utility due to their ability to incorporate a large range of important physiological phenomena in a relatively tractable mathematical formulation. through developing biologically plausible models of cortical tissue that span a number of traditionally relevant scales, a quantitative link can be made theoretically between a range of empirical measurements. theoretically spanning this explanatory gap between molecular / cellular drug action and macroscopic population dynamics assists in dealing with the often inscrutable complexities of brain dynamics. essential to the success of this approach is the biological plausibility of not only the models but also the parameterisation of drug action. as has been discussed above, some of the first attempts at using such an approach have already been fruitful in the study of anaesthesia. these encouraging results add to the growing utility of dynamical systems theory in the analysis and conceptualisation of neural activity. central to these limitations is the trade off between model utility and model complexity, where maintaining tractability is a serious technical challenge. firstly, mean field models provide a tractable way of simulating the eeg on large spatial scales, but due to limited physiological and anatomical data often do so by assuming spatially homogeneous parameterisation. given the well know heterogeneity of neocortical architectonics, improving the spatial plausibility (topology) of these models, and comparing their differences, is a central issue (e.g. qubbaj and jirsa 2007). additionally, the inclusion of more biophysical details to these models often incurs a disproportionate increase in model complexity. therefore, as part of the improvements to neural field models more generally (biophysical / topological accuracy), attempts should be made to simplify current models where possible in order to limit redundancy and improve parsimony (e.g. kim and robinson 2007). more immediate future developments in mean field or neural mass modelling of cerebral cortex include the extension of the current literature of simulated anaesthesia, especially the exploration of dissociative anaesthetic agents such as ketamine, nitrous oxide and xenon whose pharmacological action is the antagonism of the excitatory glutamatergic nmda receptor (hirota 2006). such developments naturally lead into other domains of brain function, such as learning and memory in the general case of nmda receptor functioning. indeed, mean field models of anaesthesia have been extended into sleep (e.g. wilson. 2006b), where the similarities of the eeg spectrum and behavioural state suggest some shared putative mechanisms. development of these models requires some judicial extension as their already large degrees of freedom must be constrained by empirical data. some constraints can be provided by utilising mean field models for eeg doa monitoring as mentioned above, where clinical requirements insist on rapid online assessment of eeg dynamics and therefore limit computational complexity. together these theoretical and practical improvements may produce a better understanding not only of the quantitative changes in the eeg during anaesthesia but also the physiological processes underlying conscious states. | a great explanatory gap lies between the molecular pharmacology of psychoactive agents and the neurophysiological changes they induce, as recorded by neuroimaging modalities. causally relating the cellular actions of psychoactive compounds to their influence on population activity is experimentally challenging. recent developments in the dynamical modelling of neural tissue have attempted to span this explanatory gap between microscopic targets and their macroscopic neurophysiological effects via a range of biologically plausible dynamical models of cortical tissue. such theoretical models allow exploration of neural dynamics, in particular their modification by drug action. the ability to theoretically bridge scales is due to a biologically plausible averaging of cortical tissue properties. in the resulting macroscopic neural field, individual neurons need not be explicitly represented (as in neural networks). the following paper aims to provide a non - technical introduction to the mean field population modelling of drug action and its recent successes in modelling anaesthesia. |
in situ hybridization and immunostaining were performed to analyze expression of relevant gene transcripts and protein products, respectively. targeted knockout mice were used to analyze the consequence of loss in embryo of tbx6 and/or sox2 enhancer n1, and transgenic mice to monitor enhancer n1 activity or to ectopically express sox2. whole mount in situ hybridization was done as previously described by wilkinson26 with the following modifications : proteinase k treatment was done at 5 g / ml (e8 embryos) or 10 g / ml (e9 embryos) for 5min ; incubation with glycine after proteinase k treatment was omitted ; digoxigenin - labeled probes were used at 0.5 g / ml ; and anti - digoxigenin antibody was reacted in 1.5% blocking reagent (roche). the stained embryos were photographed in 80% glycerol / pbt, then embedded in paraffin and sectioned. the probes used : tbx613, pax627, uncx4.128, foxf129, pax230, foxa131, wnt8a32, fgf833, fgf434, sox1 3 utr (stui - xhoi fragment), sox2 (sacii - acci coding sequence fragment), sox3 (627 - 1128 bp, genbank nm_009237), pax3 (hindiii - psti coding sequence fragment), mox1 (1688 - 2235 bp, genbank nm_010791), egfp (full coding sequence), and wnt3a (a combination of probes for wnt3a 3utr35 and the wnt3a coding sequence). embryos were fixed overnight with 4% pfa in pbs, immersed in 15% and 25% sucrose in pbs in sequence for 2 - 3 hr each step, and embedded in oct compound. cryosections with thickness of 10 m were prepared, and treated at 105 c for 15 min in antigen unmasking solution (vector laboratories) using an autoclave, which also quenched venus fluorescence. the sections were reacted with 10% normal donkey serum for 30 min, then with the primary antibodies overnight at 4 c. the primary antibodies, used at 1:200 dilutions, were : rabbit anti - laminin (l9393 sigma), goat anti - sox2 (af2018 r&d systems), rabbit anti - pax6 (prb-278 covance), and rat anti - ha (1867423 roche). after several washes, the samples were incubated with fluorescent dye - coupled secondary antibodies for 1 hr at ambient temperature. the following donkey antibodies were used at 1:200 dilutions : alexa fluor 488-labeled anti - goat igg (a-11055 molecular probes), alexa fluor 555-labeled anti - rabbit igg (a-31572 molecular probes), and cf 633-labeled anti - rat igg (20137 biotium). after several washes the samples were stained with 0.5 g / ml hoechst33342, and mounted in permafluor (thermo scientific). transgene dna constructs were linearized by digestion with restriction enzyme, freed from vector sequences. injection of dna (4 ng/l) into the male pronucleus of fertilized eggs and lacz enzyme staining were done using the standard procedures36. n1-tkegfp were used to establish transgenic lines, while other transgenes were used in primary transgenic embryos. the integration of transgenes into embryo genomes was determined by pcr amplification of the lacz or egfp / venus sequences from yolk sac dna. embryo culture and mesodermal transfection with pcaggs - cnoggin4 the targeting vector used to create the enhancer n1 mutation was constructed by using a 15 kb mouse dna fragment, which including enhancer n1, derived from the bac clone rp23 - 274p9 (bacpac resource center, children s hospital oakland research institute, ca, usa). the stneob cassette38 was inserted 3 of enhancer n1, loxp sequences were inserted to flank these sequences, and a dt - a cassette39 was inserted at the 3 terminus of the vector. the linearized vector was electroporated into r1 es cells, which had been engineered to have ires - egfp immediately downstream of the sox2 orf. after germ - line transmission from chimeras, heterozygous mice were crossed with caggs - cre mice40 to obtain the n1-deleted allele of sox2. a schematic of the procedure full length tbx6 (436 a.a.) was synthesized using a tnt system (promega), and emsa analysis was performed as described previously41 using 0.1 g/l poly[di - dc ] as a non - specific competitor. | the classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. however, recent cell lineage tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast (cle)1,2. tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm3 must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. here we demonstrate that tbx6-dependent regulation of sox2 determines the fate of axial stem cells. in wild - type embryos, enhancer n1 of the neural primordial gene sox2 is activated in the cle, and the cells staying in the superficial layer sustain n1 activity and activate sox2 expression in the neural plate4 - 6. in contrast, the cells destined to become mesoderm activate tbx6 and turn off enhancer n1 before migrating into the paraxial mesoderm compartment. in tbx6 mutant embryos, however, enhancer n1 activity persists in the paraxial mesoderm compartment, eliciting ectopic sox2 activation and transforming the paraxial mesoderm into neural tubes. an enhancer n1-specific deletion mutation introduced into tbx6 mutant embryos prevented this sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that tbx6 regulates sox2 via enhancer n1. tbx6-dependent repression of wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. paraxial mesoderm - specific misexpression of a sox2 transgene in wild type embryos resulted in ectopic neural tube development. thus, tbx6 represses sox2 by inactivating enhancer n1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells. |
hellp syndrome represents a severe form of preeclampsia, presenting with hemolysis, elevated liver enzymes, and low platelet count. visual symptoms can be present in up to 25% of patients with preeclampsia, usually in the form of decreased visual acuity. retinal detachment is a rare, but the well - document cause of visual loss in patients with preeclampsia and eclampsia. it is observed in < 1% of patients with preeclampsia, specially in the patients with severe hypertension. in women with preeclampsia and eclampsia, coexistence of hellp syndrome point - of - care ocular ultrasonography allows for bedside detection of ocular pathologies such as retinal detachment, globe rupture, lens dislocation, and vitreous hemorrhage. here, we report a case or retinal detachment in a patient with hellp syndrome, which was detected at the bedside by an intensivist, using ocular ultrasonography. a 26-year - old female, para 2 and gravida 3, presented with the history of 2 days of swelling of whole body, headache, and blurring of vision associated with decreased urine output. her liver enzymes and lactate dehydrogenase were raised, platelet count was 55,000 per ml, and urine examination revealed 4 plus albuminuria and hematuria. emergency lower segment cesarean section was performed, and the patient was transferred to the intensive care unit. an intensivist, trained in bedside focused ultrasonography, performed ocular ultrasound (using high frequency linear probe ; frequency range of 613 mhz ; micromaxx ; sonosite, usa), which revealed a linear hyperechoic membrane floating off the posterior globe, with the medial end attached to the margin of optic nerve head, which was suggestive of retinal detachment [figure 1 ]. she was started on injection glyceryl trinitrate, along with amlodipine 10 mg 12 hourly, prazosin 2.5 mg 12 hourly, metoprolol 50 mg 12 hourly, clonidine 100 mcg 12 hourly, and methyldopa 500 mg 6 hourly. with these drug combinations, blood pressure was controlled (130/80 mm hg) and glyceryl trinitrate was tapered and stopped after 24 h. the patient underwent four sessions of hemodialysis for acute kidney injury. renal function and liver function gradually improved. a linear hyperechoic membrane floating off the posterior globe (marked by white arrow), with the medial end attached to the margin of optic nerve head, suggestive of retinal detachment the patient was explained about the favorable prognosis. with the supportive care, ocular ultrasound repeated after 7 days showed regression of retinal detachment [figure 2 ]. after another 1 week, she had complete recovery of vision. resolving retinal detachment (marked by white arrow). retinal vascular changes, usually in the form of spasm and narrowing of retinal vessels, occur in up to 40%100% of patients with preeclampsia / eclampsia syndrome. arteriolar vasospasm affecting the retinal pigment epithelium leads to breakdown of blood - retinal barrier. subsequent leakage of protein and fluids from the capillaries, into the subretinal space, causes retinal detachment. the combination of microangiopathic hemolysis, hypoalbuminemia, and severe hypertension further contributes to the pathophysiology of retinal detachment. bedside ultrasound by a trained physician has been shown to be reliable and accurate for the diagnosis of retinal detachment.. the incidence of retinal detachment in these conditions can be much higher than actually detected. in our patient, early initiation of aggressive supportive care was coupled with favorable prognostication of visual outcome to the patient. the progressive improvement of vision correlated with both the ophthalmoscopic evidence of resolution of retinal detachment and sonographic appearance of resolution. to conclude, bedside ocular sonography can be a valuable and easily available tool for early detection of retinal detachment in patients with preeclampsia / eclampsia / hellp syndrome, presenting with visual symptoms. | retinal detachment is a rare, but well - known cause of visual impairment in patients with hemolysis, elevated liver enzymes, and low platelet count (hellp) syndrome. with supportive care, patients usually improve, with complete recovery of vision. bedside ultrasonography of the orbit can be helpful for early detection of retinal detachment in these patients. here, we present a case of hellp syndrome presenting with severe visual symptoms. retinal detachment was detected with point - of - care ocular sonography, which was confirmed with ophthalmoscopic examination. the patient was reassured of the favorable prognosis. early initiation of aggressive supportive care was followed by progressive improvement of vision, which correlated with sonographic evidence of resolution of detachment. her vision recovered completely in 2 weeks. |
transition state theory (tst) describes the rates of chemical reactions in terms of the free energy barrier for the process. although the theory was developed mainly to describe reaction dynamics in the gas phase, it has been applied to activated processes in condensed - phase environments using one of the following forms:1 in eq 1, is the reaction coordinate, and the dynamical factor || corresponds to the equilibrium ensemble average of the absolute crossing velocity, d/dt, evaluated at the transition state (ts), where =. the reaction - coordinate velocity is typically described as the velocity of a free particle : || = (2/meff), where = (kbt), in which kb is boltzmann s constant and t is the temperature, and meff specifies the effective (or reduced) mass for motion along the reaction coordinate. q is the reduced classical phase - space density at the dividing hypersurface along the reaction coordinate, and qrs is the classical partition function for the reactant state (rs). the ratio q / qrs is equivalent to the ratio of the equilibrium populations of the system in the ts and the rs, ()/ () d, and represents the probability density that the system will reach. specifically, the integration in the denominator of the last term is over values of the reaction coordinate corresponding to the rs. it is this probability ratio, or activation factor, that dominates the rate constant and, most importantly, its temperature dependence. the potential of mean force (pmf), w(), is the classical - mechanical free energy profile along the reaction coordinate averaged over all other (orthogonal) degrees of freedom. essential to this theory is the activated complex located at the dividing surface between reactants and products (i.e., the ts ensemble). this activated complex is presumed to be in quasi - equilibrium with the reactant molecules (i.e., the complexes concentration is unaffected by the rate of transformation to products or the products concentration) and is calculated by equilibrium theory. in most flavors of traditional tst, a distinct reaction coordinate is separated out from all other degrees of freedom, and the motion of the system along the reaction coordinate leading to the ts is assumed to be separable from these other degrees of freedom at. if the motion along the reaction coordinate is slow relative to the response of the remaining degrees of freedom, equilibrium solvation is assumed. moreover, tst treats barrier crossing at the ts as a free translational motion, and nuclear quantum effects (nqes) such as tunneling are ignored. these inherent limitations of traditional tst are well - known and have been reviewed extensively. the missing ingredients in tst may be added as correction terms in the generalized version of tst, thus in principle providing the true rate constant. indeed, prefactors accounting for ts recrossing and nqes have been added with great success. however, the basic assumptions hidden within the tst framework, such as equilibrium solvation and free - particle behavior at the ts, might directly influence the nature of the activated complex. it is not clear that correction terms relying on the tst activated complex are sufficient to obtain the correct rate constant. moreover, finding an optimal reaction coordinate is extremely challenging when a large number of degrees of freedom is involved in the reaction, such as reactions in water and in enzymes. indeed, the choice of reaction coordinate can greatly influence the computed rate constant within tst. interestingly, a recent work by peters and co - workers suggested that it might be impossible to remove the recrossing phenomenon even in a fairly simple system such as ion dissociation in water. the above - mentioned question regarding the nature of the activated complex obtained from tst - based approaches becomes particularly acute in complex systems such as enzymes. although our current understanding of enzyme catalysis relies on decades of groundbreaking work within the tst framework, this understanding assumes in most cases that the environment influences the reaction by means of equilibrium solvation. moreover, the role of dynamics in condensed - phase reactions is still under intense debate. in this paper, we scrutinize the nature of the activated complex obtained from standard simulation methods employed in conjunction with tst. in particular, we use umbrella sampling (us) and the string method (sm) with various reaction coordinates defined on the basis of the reacting species. the us and sm simulations yield minimum - free - energy paths along a set of reaction coordinates in which the environment provides equilibrium solvation (i.e., the effect of the degrees of freedom orthogonal to the reaction coordinate is treated as a boltzmann - averaged influence on the reaction progress). typically, us and sm employ some form of biasing potentials to allow barrier crossings. these standard methods in computational enzymology are compared with simulations using transition - path sampling (tps), which yields a collection of reactive dynamics trajectories. using tps, one considers only trajectories connecting the reactant and product basins. as a result of the bias introduced with this requirement, configurations on the transition pathways therefore, configurations with low weight in the equilibrium ensemble might have a much larger weight in the transition - path ensemble if they belong to regions that must be traversed to cross from reactants to products. however, the dynamics of the barrier crossings are unperturbed and independent of the definition of the reaction coordinate. thus, the present work provides an important benchmark comparing condensed - phase reaction simulations using tst - like methods (i.e., us and sm) and dynamics - based methods (i.e., tps). the model system for the current study is the enzyme dihydrofolate reductase from escherichia coli (ecdhfr), which serves as an important test system in enzymology. the hydride transfer in ecdhfr (scheme 1) has been studied by numerous researchers, both experimentally and computationally. the current results show that ts ensembles obtained from classical simulations for ecdhfr using standard us or sm techniques with various reaction coordinates are substantially different from those obtained using tps. in particular, the observed donor acceptor distances (dads) at the ts are considerably longer for tps than us / sm. the present discrepancy between the two sets of approaches warrants further studies to understand the extent to which reaction coordinate dynamics, nonequilibrium solvation, and biasing potentials affect the computed rate constants and kinetic isotope effects. the initial coordinates used to build the model for the present study were based on the crystal structure of a complex of ecdhfr with folate, nadp, and water molecules (pdb i d code 1rx2(54)), where the met20 loop is in the closed conformation. the original ligands in this structure were exchanged with n5-protonated 7,8-dihydrofolate (henceforth h3folate) and nadph to form a model of the reactive michaelis complex. of the 159 amino acid residues, all of the ionizable residues were treated as bearing protonation states corresponding to neutral ph. in particular, asp27 was modeled as deprotonated, and the specific protonation states of the histidine residues were determined on the basis of hydrogen - bonding interactions. this model was soaked in a pre - equilibrated 65 65 65 cubic water box and thereafter neutralized by adding 14 sodium ions to allow evaluation of electrostatic interactions using the ewald summation scheme. the potential energy surface (pes) in the current study was described by a hybrid quantum mechanics / molecular mechanics (qm / mm) hamiltonian. the qm region consisted of 69 atoms, including portions of the substrate and coenzyme in proximity to the reaction center along with two link atoms. this part was described by a modified am1 semiempirical hamiltonian in which the specific reaction parameters (srps) were optimized to treat model reactions involving various derivatives of nicotinamide and pterin compounds (denoted am1-srp). the mm part contained the protein, the remaining substrate and coenzyme atoms not described by qm, waters, and ions. the mm atoms were treated using the charmm36 force field with grid - based energy correction maps (cmap) for peptide dihedral angles, and water molecules were represented by the three - point - charge tip3p model. qm / mm interactions were treated by electrostatic embedding, wherein the mm partial atomic charges were included in the one - electron hamiltonian. the qm / mm interaction energies between the reacting fragments (qm) and the protein (mm) were fine - tuned by modifying the van der waals parameters of the qm hydrogen atoms. this combined potential energy was shown to yield accuracy comparable to that of density functional theory (dft), giving accurate results for the hydride transfer reaction in ecdhfr. periodic boundary conditions were applied, and the ewald method was employed for reciprocal - space summations between mm sites as well as for the qm / mm interactions (64 64 64 fft grid, = 0.340). a 13.0 group - based cutoff was applied for van der waals and electrostatic interactions. all atoms were gradually relaxed at the mm level of theory to remove close contacts in the initial protein ligand isobaric (npt) ensemble was employed at 298 k and 1 atm using the extended pressure / temperature (cpt) algorithm with the hoover thermostat. newton s equations of motion were integrated with a time step of 1 fs, and the shake algorithm was applied to constrain all mm bonds involving hydrogen atoms. the system was heated in a stepwise fashion from 48 k to 298 k over 25 ps and thereafter equilibrated at the target temperature (298 k) over the course of 1 ns at the mm level of theory, with a further 200 ps of equilibration using the qm(am1-srp)/mm potential. further details of the molecular dynamics (md) simulations are available in ref (48). the classical - mechanical pmf was determined using the us technique in order to sample the high - energy regions of the pes. the reaction coordinate () was defined geometrically as the difference between the lengths of the breaking (c4nnadph h4n) and forming (h4n c6h3folate) bonds (henceforth denoted as 1dasym). a total of 17 discrete regions along the reaction coordinate (windows) were defined with uniform spacing of 0.25. each simulation was performed with the addition of a biasing potential (roughly the negative of the computed pmf) and a harmonic restraint centered at each window. the sm simulations were carried out using the implementation by ovchinnikov. in charmm. the first one used two distances (the forming and breaking bond distances), and the second one used three distances (the forming and breaking bond distances and the dad). for each set of cvs, the entire path (called the string) connecting the reactant basin to the transition state and to the product basin was represented by 48 discretized images, with one md replica assigned to each image. for example, image 0 represented the reactant - state md replica and image 47 the product - state md replica. first, starting from an initial path generated by the us simulation, an iterative path optimization was carried out for 200 ps. each iteration was composed of a short (0.5 ps) md simulation, during which the force on each cv was evaluated, and an update of the cvs. the md simulation was carried out with a harmonic restraining potential applied to each cv with a force constant of 250 kcal mol. the cv update was carried out by evolving the path in the direction of the negative gradient of the pmf and then reparametrizing the cvs to enforce approximately equal arc lengths between neighboring images. then the final pmf was evaluated over a 200 ps md simulation without updating the cvs. during the final pmf simulation, the error of the final pmf values was computed by block - averaging (50 ps) of the entire 200 ps results (figure s1 in the supporting information). the tps simulations followed the implementation by the group of schwartz and co - workers. the initial step in the tps algorithm is to define the reactant and product basins. herein we defined the reactant basin as h3folate + nadph and the product basin as h4folate + nadp (scheme 1). the reactive bond lengths were employed as order parameters for each basin, and a bond was considered formed if the distance between the donor or acceptor carbon and the transferring hydride was 1.5. an order parameter of 1.3 was also tested and had no qualitative impact. initially, a single reactive trajectory was generated using a biasing potential with a gentle harmonic restraint centered at the ts, which was obtained from us simulations. this resulted in a 500 fs reactive trajectory connecting the reactant and product basins. subsequently, a random point along this trajectory was chosen as a seed point for a new trajectory. the momenta of all of the atoms in the system were perturbed by a small amount to generate a new set of velocities. the perturbations were chosen from a zero - mean gaussian distribution multiplied by a scaling factor in the range 0.100.25. the perturbation was then rescaled to ensure that the total energy was conserved and that the system did not acquire net linear or angular momenta. the random seed point with the new momenta was then propagated forward and backward in time for 250 fs, yielding a 500 fs trajectory. if the new trajectory connected the reactant and product basins, it was selected as a new reactive trajectory. if the trajectory was not reactive, it was rejected and a new point along the previous reactive trajectory was chosen. in this way, we simulated eight separate tps runs until each yielded 400 reactive trajectories, giving a total of 3200 reactive trajectories. following the generation of the reactive trajectories, we then turned to locating the ts along each trajectory using committor analysis. the dividing surface was defined as a point in phase space with equal probabilities of ending in the reactant basin and the product basin. for each point along the reactive trajectory, a set of activated dynamics simulations with random initial velocities chosen from a maxwell boltzmann distribution were initiated, and these trajectories were followed as they settled into the reactant or product basins. the numbers of trajectories settling in the reactant basin (nr) and the product basin (np) were then collected. in practice, we performed a bracketing search in the vicinity of the minimum dad for each trajectory and initiated 30 activated dynamics runs. to determine the momentum distribution of the transferring hydride atom at the dividing surface, we employed the quantum classical open - chain path integral (qcopi) method. the tss were defined on the basis of the pmf in the case of the us simulations and the committor analysis in the case of the tps trajectories. the open paths were represented by 33 beads, and the simulations were performed with isotropic sampling. approximately 10 classical configurations and 100500 monte carlo staging steps were employed in computing the momentum distribution for the ts ensembles. a representative tps reactive trajectory is presented in figure 1. the reactive event is followed by tracing the distances between the transferring hydride and the donor and acceptor atoms as well as by monitoring the dad. as the reaction occurs, the c4n h4n distance increases while the c6h4n distance is reduced. toward the ts the reactive c h vibration develops large amplitudes, similar to those of the product c h shortly after the reaction. this is indicative of thermally hot vibrations, which lie at the edge of the maxwell boltzmann kinetic energy distribution and only dissipate after hundreds of femtoseconds. concomitant with these changes, a dip in the dad curve is observed, which is typical for h / h / h transfer reactions (also see the comparison of rs and ts in table 1). each chemical event is rapid and occurs within the time frame of a single donor acceptor symmetric vibration (i.e. on the order of a few hundred fs). it is therefore unlikely that the current tps simulations are biased toward short transitions. the very fast hydride transfer is facilitated by favorable reactive 6n - dimensional phase - space states (3n for the configuration and 3n for the corresponding momentum, i.e., dynamics). during such fast chemical events it is unlikely that the enzyme environment has sufficient time to fully relax, and quasi - equilibrium is not obeyed. illustrative geometric features from a reactive event during a transition - path sampling trajectory. to quantify the convergence of the tps simulations, we used the minimum dad during the reactive event. employing this metric, we followed eight independent trajectories as they evolved in trajectory space. the results are displayed in figure 2. as is readily clear from the figure, the trajectory search converged after ca. this suggests that considerable trajectory searches are required until enzyme states that are optimal for hydride transfer are found. the final 50 trajectories of each of the eight independent tps runs were employed in the ts analysis. minimum donor acceptor distances (dads) from eight separate tps reactive trajectory series. the block - averaged (ba) minimum dads are shown in black. in table 1 we have collected the average values of key geometric parameters for the rs and ts obtained from the tps simulations. these are compared with values obtained from us and sm simulations following previously described approaches. further details of the sm results are available in figure s1 in the supporting information. analysis of the geometrical parameters of the ts ensembles revealed significant differences between the tps simulations on the one hand and the us and sm simulations on the other hand. for simplicity, in the following we compare only the tps and us results, as the us and sm results are similar. at the ts, the average c4n h4n distance from tps is 1.37 0.05, while the average c6h4n distance is 1.41 0.04. the tps average value for the antisymmetric stretch coordinate (i.e., rc4n h4n rc6h4n), which is often employed as a reaction coordinate in standard us simulations, is 0.04 0.07. the corresponding values obtained from us (1dasym) are 1.30 0.03, 1.40 0.03, and 0.10 for rc4n h4n, rc6h4n, and the antisymmetric stretch coordinate. in our previous work, we defined a rehybridization coordinate and applied it in multidimensional free energy simulations of several enzyme - catalyzed reactions involving a hydrogen transfer step. this coordinate quantifies the difference between the orbital hybridization states of the acceptor and donor carbons in geometric means and is independent of the position of the transferring hydrogen. the average ts value of the rehybridization coordinate obtained from tps is 0.09 0.12, compared to 0.10 0.20 from us. these values suggest that the ts obtained using tps is similar to that obtained using us. however, the average tps dad is 2.75 0.06, which is considerably longer than that obtained with us (2.64 0.06). the dad distributions from us and tps, which are presented in figure s2 in the supporting information, show a wider distribution in the tps simulations than in the us simulations. in addition, the tps ts is more linear, with an average c4n h4n c6 angle of 164.2 6.4, while an angle of 159.8 7.8 was obtained with us. we note that an in vacuo model bimolecular ts complex calculated with the same am1-srp hamiltonian has a saddle - point angle of 162.4. the van der waals contact / proximity between the sulfur atom of the met20 side chain and the n5 and n7n positions of the reactive complex allows the lone pair of the sulfur to maintain interactions with the bound ligands via either hydrogen bonding or dative bonding. together with the hydrogen bonding between the n7n atom and the carbonyl oxygen of ile14, these support the nicotinamide ring in its binding site toward the catalytic event. according to the present tps simulations, the interaction between the pterin n5 nitrogen and the met20 sulfur atom is slightly weakened during the course of the reaction because the positive charge on the nitrogen is neutralized as the hydride transfer occurs. this latter finding is in contrast to that of luk and co - workers, who suggested a stronger interaction with met20 in the ts on the basis of us simulations. indeed, this subtle difference was also not observed in the current us simulations. the hydrogen bonds of the nadph / nadp amide group with the backbones of ala7 and ile14 are shortened upon reaching the ts, suggesting tighter binding of the ts according to the tps simulations. again, this trend was not fully observed in our us simulations. finally, the tps simulations predict the angle between the amide moiety and the pyridine ring of the nicotinamide to be 1520 in the rs, while it is ca. 6 at the ts (similar to that observed in the crystal structure of the dhfr : folate : nadp ternary complex), significantly increasing the overlap between the systems within the nicotinamide. thus, tps seems to predict that dhfr is an enzyme designed to bind the ts more tightly while enhancing orbital conjugation, although differences between many of the structural parameters are within the standard deviations. we further note that the classical recrossing transmission coefficients () computed using us and tps are considerably different (figure 3). using us with an antisymmetric stretch coordinate, we obtained a recrossing factor of 0.63, corresponding to a free energy error of 0.3 kcal / mol in the barrier height. in contrast, using tps we obtained a transmission coefficient of 0.82, as there is much less recrossing. similarly, using a 3d coordinate, we obtain transmission coefficients of 0.76 and 0.87 with us and tps, respectively. time - dependent transmission coefficients, (t), for the hydride transfer reaction in ecdhfr computed on the basis of trajectories produced with one- and three - dimensional umbrella sampling simulations with an antisymmetric stretch and collective reaction coordinates (us 1d and 3d, gray) and from transition - path sampling simulations (tps 1d and 3d, black). in the tps simulations, the transition states were determined using a committor analysis and (t) was computed using the 1d and 3d coordinates as a metric. we note that using a three - dimensional reaction coordinate composed of an antisymmetric stretch, the dad, and a rehybridization coordinate gave nearly identical ts geometries as the simple one - dimensional antisymmetric stretch coordinate. additionally, sm with an additional collective reaction coordinate gave similar results as us, albeit with a slightly longer dad. we also note that employing an environmental reaction coordinate based on potential energy in conjunction with a standard antisymmetric stretch coordinate yielded results similar to the current us results. taken together, the results of the present work suggest that simulations incorporating tst - like assumptions are unable to properly sample long dads, whereas the dynamics - based tps method accesses long dads during barrier crossing. finally, we compared the quantum momentum distributions (obtained using the recently developed qcopi method) for the transferring hydride at the us and tps dividing surfaces. the momentum distribution is a highly sensitive reporter of the potential experienced by a particle. in figure 4 we compare the momentum distributions obtained for dividing - surface configurations from us simulations restrained to different dads with the ts obtained from tps simulations. it is clear from inspection of the us curves in figure 4 that as the dad increases at the ts, the momentum distribution becomes increasingly narrow, reflecting a wider position distribution. interestingly, the momentum distribution obtained for the tps dividing surface is significantly different from all of those attained with us. careful inspection of the tails of the momentum distributions suggests that the potential experienced by the hydride using us resembles a single - well potential, whereas the potential using tps corresponds to a small double - well potential. this suggests that the potentials experienced by the transferring hydride at the ts obtained using different techniques are rather different. momentum distributions for the transferring hydride at transition states obtained using umbrella sampling at different donor tps simulations have been applied to various enzyme systems, although presently it is not a household method in computational enzymology. in the present work, we performed a comparison between the activated complexes obtained for the enzyme dhfr using the tps method and two other widely used approaches, us and sm. we found that the tss obtained using the two families of methods are significantly different. in particular, the tss obtained using tps have longer dads and are more linear than those observed using us and sm. additionally, the classical recrossing transmission coefficients using us and tps were found to be significantly different. with us there is considerable recrossing of the ts, whereas with tps there is much less recrossing. in the current study, we did not compare the free energy profiles associated with the transition - path ensemble. this is not a trivial task, and additional information is required to determine reaction probabilities. however, the free energy profiles from tps are not expected to be significantly different from the us and sm profiles because the reaction barrier is less sensitive to the exact location of the ts. indeed, the free energy surface for dhfr is rather flat in the ts region with respect to the dad. a related careful comparison between us and tps on the rotational barrier in a disaccharide in vacuo showed similar results with the two methods, although additional pathways were found using tps. however, certain kinetic parameters such as the kinetic isotope effect and dynamic recrossing at the ts are sensitive to the geometry of the ts. in particular, the nqes responsible for the kinetic isotope effect are highly sensitive to the fine details of the ts. it is not clear that simple corrections to the rate constant via various prefactors can rectify the errors introduced into the computation of kinetic isotope effects using an incorrect ts ensemble. we stress that the difference between the us / sm and tps approaches is not merely one of computational strategy. instead, it is likely that underlying physical assumptions inherent to the methods, such as equilibrium solvation and the nature of the reaction coordinate and its motion, give rise to the different results. the traditional tst - based us and sm techniques assume that the barrier - climbing process is slow relative to the relaxation of the environment, and therefore, at every value of the reaction coordinate the environment is assumed to be fully relaxed to achieve thermodynamic equilibrium throughout the entire system for all degrees of freedom. in contrast, the actual barrier crossing is very rapid in tps, and the environment is largely unchanged during the reaction. this is consistent with a reaction dynamics model where the system spends most of its time searching for a reactive configuration and set of momenta. once the proper 6n - dimensional state (3n for the configuration and 3n for the momentum, where n is the number of atoms in the system) suitable for reaction is attained, the reaction occurs rapidly. in this case, if certain protein dynamics are coupled with the chemical step, the effects of such dynamics are naturally taken into account in tps, whereas they are ignored in the tst - based methods. an additional fundamental difference between the two families of methods concerns the motion along the reaction coordinate. in us and sm, the motion along the reaction coordinate is unphysical because of the applied bias. the reaction coordinate is assumed to be separable from the other degrees of freedom, and the velocity of the reactive mode is assumed to approach that of a free particle. on the other hand, the reactive trajectories produced from tps correspond in principle to a true barrier - climbing process. indeed, in tps simulations there is a kinetic energy associated with the barrier crossing, similar to what one would expect in the true chemical step. this motion along the reaction coordinate, combined with the lack of time for environmental reorganization during hydride transfer, generates a friction force that influences the nature of the activated complex in multidimensional systems. we note that additional differences in the details of the current simulations exist. the us and sm simulations were performed in the isothermal isobaric (npt) ensemble using a thermostat, while the tps simulations employed a microcanonical (nve) ensemble (albeit conforming with nvt conditions). however, us in the nve ensemble yielded dads identical to those in the npt simulations, so this is unlikely to be of significance here. we believe that it could be of interest to compare biased and unbiased simulations for other activated processes and to explore the validity of the equilibrium solvation assumption inherent to traditional tst as well as the effects of nonequilibrium dynamics on the reaction. additionally, more advanced tools for analysis of the dividing surface could be employed. we further note that a quantum - dynamical analogue of classical tps simulations should be used to identify the ts more precisely. finally, as noted above, direct rates or kinetic isotope effects were not computed with tps in the current work. a direct comparison of computed and experimental absolute rates and kinetic isotope effects and their temperature dependence will be necessary to further scrutinize the current tps and us results. | the rate expression of traditional transition state theory (tst) assumes no recrossing of the transition state (ts) and thermal quasi - equilibrium between the ground state and the ts. currently, it is not well understood to what extent these assumptions influence the nature of the activated complex obtained in traditional tst - based simulations of processes in the condensed phase in general and in enzymes in particular. here we scrutinize these assumptions by characterizing the tss for hydride transfer catalyzed by the enzyme escherichia coli dihydrofolate reductase obtained using various simulation approaches. specifically, we compare the tss obtained with common tst - based methods and a dynamics - based method. using a recently developed accurate hybrid quantum mechanics / molecular mechanics potential, we find that the tst - based and dynamics - based methods give considerably different ts ensembles. this discrepancy, which could be due equilibrium solvation effects and the nature of the reaction coordinate employed and its motion, raises major questions about how to interpret the tss determined by common simulation methods. we conclude that further investigation is needed to characterize the impact of various tst assumptions on the ts phase - space ensemble and on the reaction kinetics. |
aldehyde dehydrogenases (aldh) comprise a superfamily of enzymes that catalyze the nad(p)-dependent oxidation of aldehydes to their corresponding carboxylic acids. enzymes in this superfamily exhibit diversity in their specificity for substrates. detrimental changes in their contributions to specific metabolic pathways lead to several disease states, including sjgren larsson syndrome, type ii hyperprolinemia, hyperammonemia, and alcohol flushing disease as well as cancer. using known structural and catalytic attributes for several of these family members has led to the discovery and characterization of some selective chemical modulators for aldh2 and aldh1/3 as well as broad - spectrum modulators. our prior work with a broad - spectrum inhibitor demonstrated that the enzyme catalyzed production of a vinyl - ketone intermediate that inhibited aldh1a1, aldh2, and aldh3a1 through the formation of a covalent adduct with their catalytic cysteine residue. however, to achieve selective inhibition of particular isoenzymes, molecules that do not rely solely on common mechanistic features may be more desirable. therefore, this study looks to further that work by characterizing a class of inhibitors that utilize a common mechanistic feature but that can achieve selectivity through elaboration of the common functional group, indole-2,3-dione. we report here the kinetic and structural characterization of a diverse group of substituted indole-2,3-diones, from which selective inhibitors for aldh1a1, aldh2, and aldh3a1 may be derived. recently, we reported a class of compounds identified during a high - throughput screen for modulators of aldh2 that showed nonselective covalent inhibition of aldh isoenzymes. to achieve a more selective inhibition of aldh isoenzymes, we reasoned that reliance on mechanistic features common to aldh family members was not desirable. consequently, we re - evaluated the original high - throughput screening results for compounds that might demonstrate better isoenzyme selectivity. re - examination of these screens led to the identification of four aldh2 inhibitors with structural similarity to five aldh3a1 inhibitors, some of which showed excellent selectivity toward aldh3a1. to characterize this group of compounds further, we obtained an additional 33 structurally similar analogues from chemdiv and chembridge and evaluated their ability to inhibit aldh1a1, aldh2, and aldh3a1 using nad(p)-dependent aldehyde oxidation to measure activity. the compounds in this study are all derived from the indole-2,3-dione parent compound, but three distinct structural groupings can be created on the basis of the nature of the substitutions to the indole-2,3-dione ring system and their ability to inhibit selected aldh isoenzymes. these were the least selective between aldh isoenzymes and exhibited low micromolar ic50 values for aldh2 and middle - to - high nanomolar ic50 values for aldh1a1 and aldh3a1 (table 1). compounds in group 2 are characterized by the addition of a benzyl moiety via an alkyl chain linker attached to the indole ring nitrogen atom with and without halogen substitutions at the 5-position of the indole ring. however, the nature of the substitutions can shift the potency 380-fold in favor of aldh1a1 or 40-fold in favor of aldh2 (1-pentyl-2,3-dihydro-1h - indole-2,3-dione (compound 3) vs 5-bromo-1-(2-phenylethyl)-1h - indole-2,3-dione (compound 8), table 1). in general, longer alkyl - chain linkers favor aldh1a1 and aldh3a1 inhibition. halogens at the 5-position improve potency toward aldh2, but 5-bromo - substitutions on the indole ring reduce the potency toward aldh1a1. substitution of either a 5-chlorine or 5-bromine on the indole ring severely reduces potency toward aldh3a1 (1-(2-phenylethyl)-1h - indole-2,3-dione (compound 6) vs 8, table 1). the addition of a double bond to the linker between the indole and benzyl rings almost eliminates potency toward aldh2 (1-(3-phenyl-2-propen-1-yl)-1h - indole-2,3-dione (compound 10)), but introduction of the 5-chloro group to the same molecule restores potency (5-chloro-1-[(2e)-3-phenylprop-2-en-1-yl]-2,3-dihydro-1h - indole-2,3-dione (compound 11)). group 3 compounds possess either a piperazine, morpholine, or imidazolidine nonaromatic ring linked to the indole nitrogen (table 1). these compounds tend to be the most selective for haldh3a1 and show little if any inhibition of aldh2. only the compound with a 5-bromo substitution on the indole ring (1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-5-bromo-1h - indole-2,3-dione (compound 21)) was a poor inhibitor of haldh3a1 (table 1). to understand the mechanism of inhibition for these compounds better, compounds these inhibitors exhibited noncompetitive mixed - type inhibition with respect to varied coenzyme, and they exhibited competitive inhibition with respect to varied aldehyde substrate for all three aldh isoenzymes (tables 2 and 3). consistent with the kinetic data, the crystal structure of aldh2 with 3 and 1 bound shows that the compounds bound within the aldehyde substrate binding site with the 3-keto group sandwiched between the active - site cysteine residues 301 and 303 (figures 1 and 2). the distance of interaction in these models suggest that both cysteine residues are interacting equivalently on either face of the carbonyl carbon but do not appear to have formed formal adducts. interestingly, the orientation of their indole-2,3-dione rings are flipped such that the opposing faces are interacting with cys301 and cys303 in the 1 versus 3 structures. the 5-methyl substituent of 1 forms hydrophobic interactions with the side chains of trp177, leu173, and met174, whereas the 7-bromo substituent is oriented toward the solvent - exposed exit of the substrate - binding site. in contrast, the 5-position of 3 is oriented toward the solvent, and the 1-benzyl substituent is tucked tightly into the substrate binding side adjacent to the side chain of phe465, which is displaced from the position found in all other aldh2 crystal structures. there is an inverse correlation between ordered positioning of the 1-n - benzyl group and that of phe465, suggesting that one or the other is mobile in this complex. the close contacts between these aromatic rings is likely responsible for the relatively high ic50 exhibited by aldh2 for 3. despite the flipped orientations of their common ring system, the indole-2,3-diones of 1 and 3 maintain the same aromatic -stacking interactions with the side chains of phe170 and phe459. (a) side view of the protein surface containing compound 1 (left) and (b) top view with the original unbiased figure of merit, a - weighted, 2fo fc (blue ; contoured at one standard deviation of the map) and fo fc electron density map (green ; contoured at 2.5 standard deviations of the map) for 1 prior to its inclusion in the model superimposed on the final refined model. (a) side view of the protein surface containing compound 3 (left) and (b) top view with the original unbiased figure of merit, a - weighted, 2fo fc (blue ; contoured at one standard deviation of the map) and fo fc electron density map (green ; contoured at 2.5 standard deviations of the map) for 3 prior to its inclusion in the model superimposed on the final refined model. in contrast, the crystal structure of aldh3a1 in a complex with 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (compound 20) (figure 3) shows the 3-keto group of the indole-2,3-dione ring bound within the substrate - binding site and forming an adduct with the active - site nucleophile (cys243 in aldh3a1). the distance between cys243 and the carbonyl - carbon as well as the out - of - plane displacement of the carbonyl oxygen is consistent with the formation of an adduct between these two reactive groups. that this covalent bond is reversible is supported by the fact that addition of dithiothreitol to the reaction solution after preincubation restores the enzymatic activity. there is sufficient electron density to model the indole-2,3-dione and the n - piperazine moiety, but insufficient electron density is present to model the terminal benzyl - dioxol moiety. computational placement of the benzyl - dioxol moiety onto the crystallographically observed partial structure suggests that the benzyl - dioxol lies at the interface between the exit of the substrate - binding site and bulk solvent, where it can apparently adopt multiple conformations. (a) side view of the protein surface containing the crystallgraphically observed portion of compound 20. (b) top view with the original unbiased figure of merit, a - weighted, 2fo fc (blue ; contoured at one standard deviation of the map) and fo fc electron density map (green ; contoured at 2.5 standard deviations of the map) prior to its inclusion in the model superimposed on the final refined model. there is insufficient electron density to fit and refine properly the terminal benzyldioxol group in the structure. consequently, we have placed and refined only that portion that can be accounted for by the available electron density. there are 19 members of the aldh superfamily in the human genome, with many of the family members participating in defined metabolic pathways, such as proline, valine, retinal, folate, and gaba metabolism. in contrast, several members have less well - defined substrate preferences and constitute a cellular defense system against endogenously and exogenously generated aldehydes. in particular, aldh1a1, aldh2, and aldh3a1 demonstrate broad and overlapping substrate specificities and are frequently expressed in the same tissues or cell types. for instance, aldh1a1 and aldh3a1 have both been shown to be biomarkers for cancer as well as cancer stem cells. aldh1a1 overexpression is indicative of high - grade ductal carcinoma, multiple myeloma, and acute myeloid leukemia. aldh3a1 is generally found in stratified squamous epithelium, and both aldh3a1 and aldh1a1 confer resistance to the cell - killing effects of cyclophosphamide. however, aldh2 has primary contributions to the elimination of acetaldehyde and to the bioactivation of nitroglycerin. thus, selective inhibitors of these general aldehyde - oxidizing enzymes could provide important research tools for the assessment of their individual contributions to their common substrates or cellular functions. toward this end, we have examined derivatives of indole-2,3-diones for their ability to inhibit differentially these three forms of aldh. members of this class of compounds were identified in separate inhibitory screens on aldh2 and aldh3a1, although the diversity of the substitutions on the indole rings of these initial hits suggested that selectivity could be achieved through optimization of those substitutions. that this level of selectivity could be achieved is further supported by the differences in the chemical and surface topologies of their respective substrate - binding sites. aldh2 and aldh3a1 share only 30% sequence identity, and despite sharing the common catalytic residues they have distinct substrate - binding - site characteristics (figure 4). similarly, aldh2 and aldh1a1 share 68% sequence identity, and, although their substrate - binding sites are both largely hydrophobic in nature, the middle and exterior regions of the site in aldh1a1 are considerably wider because of smaller amino acid side chains at positions 124 and 459. it is in this context that the structure activity relationships for these substituted indole-2,3-diones should be interpreted. comparison of aldib interactions with active - site cysteines in aldh2 and aldh3a1. (a) crystal structure of aldh2 with 1 (gray) has been overlaid with that of 3 (magenta) to show how the steric hindrance of the surrounding hydrophobic residues determines the mode of binding. (b) surface map of 3 (magenta) bound to aldh2 (cyan) aligned to the crystallographically observed partial structure of 20 (orange) bound to aldh3a1 (yellow). the kinetic data is consistent with these inhibitors binding in a manner that is competitive with respect to aldehyde binding. although the details of the interactions between the aldib inhibitors and the enzymes differ in the precise details, all compounds occupy the substrate - binding site in a manner that requires their displacement prior to productive binding of substrate aldehydes. their mixed - type noncompetitive inhibition patterns with respect to varied coenzyme is consistent with the largely ordered bi bi mechanism followed by aldh family members that have been characterized in this manner. the distinct differences in the nature of the interactions between the indole-2,3-dione rings and aldh3a1 and aldh2 highlight the structural and functional differences of these distantly related isoenzymes. consistent with this level of sequence identity, a structural alignment yields a rmsd of 2.1 for 395 similarly positioned c atoms. the amino acids lining their respective substrate - binding sites are both largely hydrophobic, but the differences in their positioning and identity create unique topographical features (figures 3 and 4). the manner in which 20 is bound within the aldh3a1 substrate site also provides key information with respect to the manner in which aldh3a1 selectivity is achieved when compared to the substrate - binding sites of aldh1a1 and aldh2 (figure 3). it is these structural and functional differences that underlie the distinct way these substituted indole-2,3-dione molecules bind to these aldh active sites. the 3-keto group in these inhibitory molecules is a well - known electrophile, so it was not surprising that this group would seek a reactive nucleophile within the aldh active site. however, it was surprising that only in aldh3a1 did these compounds form an adduct with the catalytic nucleophile, as the active site of aldh2 is generally thought to contain the stronger cysteine nucleophile. aldh2 generally exhibits low to submicromolar km values for aldehydes, whereas aldh3a1 exhibits km values at least 2 orders of magnitude higher. why then does 20 bind to the catalytic nucleophile in aldh3a1 but neither 1 nor 3 does so in aldh2 ? an alignment of the respective active sites demonstrates that in order for either 1 or 3 to bind to cys302 the side chain of trp177 has to move, but its position is restricted by the side chains of leu173, met174, and leu477 (figure 4). this explains why the indole-2,3-dione ring can not bind productively to cys302 in aldh2 and why the sar for aldh2 was so difficult to understand before the structures of 1 and 3 were available. the most difficult features of the aldh2 sar to understand were the favorability of larger halogens at the 5-position and how the changes in inhibition strength varied as the alkyl linker between the indole ring and a benzene substituent on the n1 position was lengthened. if the indole ring bound similarly to that in aldh3a1, then we would expect the sar on halogens at the 5-position to follow that of aldh3a1 ; namely, that larger halogens are detrimental to inhibitory potency (table 1, 8 vs 5-chloro-1-(2-phenylethyl)-1h - indole-2,3-dione (compound 7) vs 6, 20 vs 21). however, this is not the case for aldh2, as increasing the size of the halogen at the 5-position either has little effect (1-benzyl-5-bromo-1h - indole-2,3-dione (compound 5), 1-benzyl-5-chloro-2,3-dihydro-1h - indole-2,3-dione (compound 4), and 3, table 1) or increases potency (8 vs 7 vs 6, 10 vs 11, table 1). in addition, lengthening the alkyl linker to the benzene ring had no real impact on potency, which is opposite that seen with aldehyde substrates where the longer and more hydrophobic the alkyl chain, the better the substrate. however, the presence of a double bond in this linker virtually abolishes inhibitory potency (10 vs 1-(3-phenylpropyl)-1h - indole-2,3-dione (compound 9), table 1), whereas addition of a 5-chloro group to 10 (11) restores potency. this data suggests that the binding modes for the halogen versus non - halogen - substituted compounds differ. we suggest that the binding mode observed for 3 in aldh2 is maintained for group 2 compounds lacking halogens at the 5-position (table 1), as the ability of the longer alkyl chains to adopt new conformations necessary to exit the active site past the position of cys302 requires a flexible linker. however, this binding mode is inconsistent with the increased potency of halogen substitution at the 5-position because the 5-position of 3 is within van der waals contact to val120, met124, and phe296. consequently, the sar would suggest that 5-halogen - substituted group 2 compounds adopt the position observed for 1, where the 5-methyl substituent will approximate the position of the 5-chloro or 5-bromo substituents and the indole nitrogen is now pointing toward the solvent - exposed exit of the substrate - binding site. in addition, the aldh2 crystal structures presented here show that cys302 is pointing in the direction of the cofactor pocket and away from the substrate pocket, which has been referred to as the resting conformation. lang. suggested that this resting conformation contributes to cys301/303 having the primary role in stimulation of the cys302 thio - carbonyl adduct formation, lowering the acidity of cys302. furthermore, comparison of aldh2 with other enzyme family members suggests that cys303 interacts with substrates via hydrogen bonding or by an ion the crystal structures of 1 and 3 presented here suggest that cys301/303 may contribute to substrate binding through a trapping mechanism and possibly by serving to dehydrate the hydrated aldehydes that form in solution. weiner and colleagues proposed that elements of the enzyme s proton - relay system performed this function, but this proposal preceded structure determination by 12 years. the proton - relay system is identical in aldh1a1 and aldh3a1, but the two residues immediately surrounding the catalytic nucleophile in the active - site loop are not. in this regard, aldh1a1 has ile at 301 and a single additional cys residue at 303, whereas aldh3a1 lacks cys residues at either equivalent position (thr and val, respectively), which correlates with their decreased catalytic efficiency for small aliphatic aldehydes with the greatest level of hydration. in contrast to the complex sar for aldh2, selectivity for aldh3a1 is more simply achieved through the addition of a nonaromatic ring system linked by a single carbon atom to the indole nitrogen (table 1). this addition to the indole ring alone abrogates any potency toward aldh2 and severely diminishes potency toward aldh1a1. addition of hydrophobic substituents at the 4-position of the piperazine ring further improves potency up to 4-fold (1-(4-morpholinylmethyl)-1h - indole-2,3-dione (compound 12) versus 20) while still maintaining a 40-fold selectivity over aldh1a1. although the benzyl - dioxol substituent is disordered in our crystal structure, presumably because of the multiple binding modes near the exit of the substrate - binding site, these additional hydrophobic substituents likely interact with the side chains of trp233 and met237 (figure 3). substitution of the central nonaromatic ring with an aromatic ring reduces potency by about 6-fold (3 versus 12). this is likely due to the generation of an unfavorable steric contact between the aromatic ring and met237 (figure 3). addition of one or more additional linking carbon atoms between the indole and aromatic rings improves potency by more than 15-fold, presumably by bypassing the restrictive space occupied by met237 (3 vs 6 or 9, table 1). however, these changes come at the cost of selectivity for aldh3a1 as neither 6 nor 9 are selective inhibitors for aldh3a1. another unique characteristic of aldh3a1 is the negative impact that substitution of large halogen atoms at the 5-position of the indole ring has on potency. this effect can be explained by the close proximity of the side chains of phe401, tyr412, and his413 (figure 3). substitution of anything larger than a hydrogen atom would crowd this location. with the exception of the compounds in group 3, most aliphatic or aromatic n - substituted indole-2,3-diones are potent inhibitors of aldh1a1 (table 1). only the substitution of a nonaromatic ring linked to the indole nitrogen or the presence of a 5-bromo group is detrimental to aldh1a1 potency. consistent with its ability to oxidize retinaldehyde with high efficiency, aldh1a1 has the largest and most hydrophobic substrate - binding site, and substituents on the indole nitrogen that enhance hydrophobic interactions improve the potency of these compounds toward this isoenzyme. the structure activity data for aldh1a1 is consistent with a binding mode for the substituted indole-2,3-diones that is similar to that for 20 in aldh3a1. this data is supported by presence of gly124 in aldh1a1, rather than met124 in aldh2 ; this substitution provides some additional room for a 5-chloro group but is still insufficient for a 5-bromo substitution, which would still clash with trp177. the aim of this work was to develop further a new class of small molecules that can be adapted to selectively inhibit aldh enzymes. as such, several compounds were identified that show reasonable selectivity (> 40-fold) toward aldh1a1 (3), aldh2 (8), or aldh3a1 (20 or 1-{[4-(4-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (compound 16)) based on the size and chemical characteristics of each isoenzyme s substrate - binding site. the competition assays show that these inhibitors are noncompetitive with respect to the coenzyme - binding site and are competitive toward the substrate - binding site. x - ray crystallography data confirms inhibition occurs through direct interactions between the 3-keto group of the indole ring and conserved cysteine residues within the active site. selective inhibition can be achieved through substitutions at the c5 and n1 positions of the indole ring system. these compounds may act as useful tools in elucidating the contributions of individual aldh isoenzymes toward specific, as well as overlapping, metabolic pathways. compounds used for the inhibitor screen were found using a 80% structure similarity search against 5-methyl-1h - indole-2,3-dione using the pubchem project. the following compounds were purchased from chembridge for further in vitro assay screening : 5301889, 1-benzyl-5-bromo-1h - indole-2,3-dione (5) ; 5192630, 1-(2-phenylethyl)-1h - indole-2,3-dione (compound 6) ; 7196590, 5-chloro-1-(2-phenylethyl)-1h - indole-2,3-dione (7) ; 6047303, 5-bromo-1-(2-phenylethyl)-1h - indole-2,3-dione (8) ; 6378722, 7-bromo-5-methyl-1h - indole-2,3-dione (1) ; 6433626, 1-(3-phenylpropyl)-1h - indole-2,3-dione (9) ; 6997087, 1-(3-phenyl-2-propen-1-yl)-1h - indole-2,3-dione (compound 10) ; 6505720, 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (20) ; 8918814, 1-{[4-(4-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (16) ; 8918815, 1-{[4-(3-chlorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (compound 18) ; 8918818, 1-{[4-(2-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (compound 15) ; 8918819, 1-{[4-(3-fluorobenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (compound 17) ; 8919812, 1-{[4-(3-methoxybenzyl)-1-piperazinyl]methyl}-1h - indole-2,3-dione (compound 19) ; 6989645, 1-{[4-(1,3-benzodioxol-5-ylmethyl)-1-piperazinyl]methyl}-5-bromo-1h - indole-2,3-dione (21) ; 5250099, 1,1-[1,3-imidazolidinediylbis(methylene)]bis(1h - indole-2,3-dione) (compound 22) ; 5115436, 1-(4-morpholinylmethyl)-1h - indole-2,3-dione (12) ; 5260280, 1-[(4-methyl-1-piperazinyl)methyl]-1h - indole-2,3-dione (compound 13) ; and 5115499, 1-[(4-benzyl-1-piperazinyl)methyl]-1h - indole-2,3-dione (compound 14). the following compounds were purchased from chemdiv for further screening : 0764 - 0625, 1-pentyl-2,3-dihydro-1h - indole-2,3-dione (compound 2) ; 0764 - 0628, 1-benzyl-2,3-dihydro-1h - indole-2,3-dione (3) ; and 5353 - 0854, 1-benzyl-5-chloro-2,3-dihydro-1h - indole-2,3-dione (4). on the basis of the sar data collected, compound bbv-138984, 5-chloro-1-[(2e)-3-phenylprop-2-en-1-yl]-2,3-dihydro-1h - indole-2,3-dione (11), was purchased from molport as proof of the selective - inhibition scheme. the nmr spectra confirming > 95% compound purity provided by the vendors is included in the supporting information. human aldh1a1 (haldh1a1), aldh2 (haldh2), and aldh3a1 (haldh3a1) were expressed and purified as described elsewhere. crystals of aldh3a1 at 3 mg / ml in 10 mm hepes, ph 7.5, were grown from solutions containing 0.2 m potassium acetate and 18% peg3350 at 25 c. enzyme complexes with inhibitors were generated through direct - soaking experiments by first equilibrating the crystals overnight in a solution containing 2% dmso, which was then supplemented with 100 m compound 20. crystals of aldh2 were grown from protein solutions containing 8 mg / ml of aldh2 in 100 mm aces, ph 6.26.8, 100 mm guanidine - hcl, 10 mm mgcl2, and 1419% peg 6000. the aldh2 complexes with inhibitors were generated through direct - soaking experiments by first equilibrating the crystals overnight in solutions containing 2% dmso, which was then supplemented with 100 m of either compound 1 or 3. all diffraction data were collected at a wavelength of 0.9869 and at 100 k. all diffraction data were indexed, integrated, and scaled using the hkl3000 program suite. the structure of the aldh3a1 complex with 20 was solved by molecular replacement using the aldh3a1 apo structure (pdb code 3sza). the presence of inhibitor was determined after inspection of the initial -a weighted fo fc electron density maps. the structures of aldh2 complexed with 1 and 3 were solved using the coordinates of the refined aldh2 in the p21 space group after removal of solvent and ligands (1cw3). the aldh2 complex with 1 demonstrates high - occupancy binding in subunits a and h and insufficient electron density for unequivocal assignment in the remaining subunits. the aldh2 complex with 3 shows high - occupancy binding in subunits a, b, e, and h and insufficient electron density for unequivocal assignment in the remaining subunits. ic50 inhibition curves for the inhibitors were measured using the activity of haldh2, haldh1a1, and haldh3a1 as described elsewhere. in short, the enzyme was incubated with the inhibitor and coenzyme for 2 min prior to initiation of the reaction with aldehyde substrate. the inhibition curves were fit to the four - parameter ec50 equation using sigmaplot (version 11, statsys). inhibition of aldh activity was measured using the activity of haldh2, haldh1a1, and haldh3a1 to determine the kinetic mode of inhibition versus varied coenzyme or aldehyde substrate. coenzyme competition was determined by measuring aldh activity for various concentrations of compound while varying the concentration of nad for haldh2 and haldh1a1 and nadp for haldh3a1. likewise, substrate competition was determined for different concentrations of each compound while varying propionaldehyde concentration for haldh2 and haldh1a1 and benzaldehyde concentration for haldh3a1. in each experiment, the concentration of the nonvaried substrate was set to saturating levels, and the varied substrate concentration ranged at least 10-fold spanning the calculated km value. similarly, the concentration range for the inhibitors was varied a minimum of 5-fold, exclusive of the control (no inhibitor) reactions, which spanned the calculated ki values. the kinetic mode of inhibition was determined by fitting data to the competitive, noncompetitive, and uncompetitive inhibition equations and evaluating the goodness of fit to each equation. data fitting and analysis was performed using sigmaplot (version 11.0) with the enzyme kinetics module (version 1.3). | aldehyde dehydrogenases (aldh) participate in multiple metabolic pathways and have been indicated to play a role in several cancerous disease states. our laboratory is interested in developing novel and selective aldh inhibitors. we looked to further work recently published by developing a class of isoenzyme - selective inhibitors using similar indole-2,3-diones that exhibit differential inhibition of aldh1a1, aldh2, and aldh3a1. kinetic and x - ray crystallography data suggest that these inhibitors are competitive against aldehyde binding, forming direct interactions with active - site cysteine residues. the selectivity is precise in that these compounds appear to interact directly with the catalytic nucleophile, cys243, in aldh3a1 but not in aldh2. in aldh2, the 3-keto group is surrounded by the adjacent cys301/303. surprisingly, the orientation of the interaction changes depending on the nature of the substitutions on the basic indole ring structure and correlates well with the observed structure activity relationships for each aldh isoenzyme. |
our study enrolled 40 consecutive patients with a traumatic or foreign body removal - related corneal abrasion (occurring within 24 hours of the beginning of the study and not caused by thermal, radiant, or caustic agents) that spared the bowman membrane and the limbus, and involved 4 mm). however, there were concerns over their infection rates, costs, and need for follow up.20 collagen shields have also proven to be more comfortable than patching, but they are not used in clinical practice.21 nevertheless, the most recently published survey on corneal abrasion management conducted in an accident and emergency department in the uk has confirmed that eye patching is still widely used in clinical practice.2 during the wound repair process, the use of a lubricant is always advisable because hydration and lubrication smooth out irregularities in the corneal epithelium, thus protecting the ocular surface from damage due to friction and desiccation. for this reason, we evaluated the effect of a lubricant eye gel in patients with corneal abrasion sparing the bowman membrane and involving less than 50% of the cornea. this gel is available in some eu countries (xanternet) in preservative - free unidoses containing two natural biopolymers, sodium hyaluronate and xanthan gum, in addition to aminoglycoside netilmicin. sodium hyaluronate and xanthan gum are high molecular weight polysaccharides with water binding and mucoadhesivity properties, providing effective hydration and protection of the cornea.2226 in addition, sodium hyaluronate is well known to promote wound healing by stimulating corneal epithelial cell migration and proliferation.911 netilmicin is a broad spectrum, third generation aminoglycoside with excellent in vitro activity against the most common bacterial ocular pathogens, including pseudomonas spp.14,2729 netilmicin is noncytotoxic and does not interfere with physiological healing, and is therefore able to provide good protection against bacterial infections during the wound repair process.12,13,15 in the present study, we compared routine patching (48 to 72 hours) with short patching (12 hours), followed by a treatment with the eye gel in patients with simple corneal erosion. corneal defects were photographed, stained with fluorescein, and measured at each control visit, allowing an objective evaluation of the wound healing. other parameters, such as the extent of punctate erosions, conjunctival, hyperemia, lid edema, pain, and other subjective symptoms of ocular discomfort, were also evaluated throughout the study. after 3 days, the residual corneal defect and all other parameters were not statistically different. the major limitation of the study is the trial design ; the two groups of patients were not fully comparable because of the different uses of the patching regime. nevertheless, our data suggest that although a reduction of the duration of patching followed by the topical administration of xanternet eye gel does not affect the healing of the corneal defect, it does improve patient compliance. additional clinical data with more patients comparing a group treated with the eye gel with a nonpatched group are required to recommend the eye gel as unique tool in the management of traumatic corneal abrasions. | purposethe aim of this study was to investigate the effects of an ophthalmic gel containing sodium hyaluronate and xanthan gum in addition to the antibiotic netilmicin in the management of traumatic corneal abrasions.patients and methodspatients with traumatic corneal abrasions were randomly treated as follows : group a (n = 20) with an occlusive patching for 12 hours plus one drop of an eye gel containing 0.15% sodium hyaluronate, 1% xanthan gum and 0.3% netilmicin qid for 5 days ; and group b (n = 20) with an occlusive patching for 23 days plus one application of 0.3% netilmicin ophthalmic ointment qid for 5 days. all patients were evaluated after the third and seventh day by slit - lamp examination, fluorescein staining, and corneal defect photograph in order to assess corneal re - epithelialization. conjunctival hyperaemia, lid oedema, subjective symptoms of discomfort, and conjunctival swabs were also evaluated.resultsno statistically significant difference was observed between the groups in terms of the extent of corneal healing after 3 days of treatment. both treatments were also highly effective in decreasing the erosion score and the conjunctival hyperemia (p < 0.0001, p < 0.005, respectively) without any significant difference between the two types of treatment. subjective symptoms of discomfort and conjunctival swabs were also evaluated.conclusionin the management of traumatic corneal abrasions, the administration of an eye gel containing sodium hyaluronate and xanthan gum is able to reduce the length of occlusive patching. in addition, the presence of netilmicin guarantees good antibiotic prophylaxis during the wound repair process. |
there exists sufficient evidence which links the diseased state of atherosclerosis to elevated levels of total cholesterol (tc), low - density lipoproteins cholesterol (ldl - c), very low - density lipoprotein cholesterol (vldl - c), and triglycerides (tg) (14, 18, 24). current research suggests that atherosclerosis is linked to the frequent and chronic elevation of postprandial lipemia (14, 29). an increase in postprandial triglycerides (ppt) is complicated by the increased daily ingestion of multiple fatty meals ; as is commonly seen in first - world populations (14). moderate - intensity walking at 60% and 50% vo2max for 90min has shown to be an effective tool in decreasing ppt (6, 9, 18, 24, 29). this decrease in ppt can be seen among both trained and untrained individuals, supporting the theory that the increase in tg clearance is independent of anthropomorphic changes (14). acute exercise creates a clearance of existing intramuscular cholesterol via the skeletal muscle which is replenished as skeletal muscle uptakes plasma vldl in the postprandial state (16). the decrease in circulating vldl allows for less competition of lpl activity and thereby allowing an increase in tg hydrolysis (14, 16). additionally, there is an increase in skeletal muscle lpl activity with exercise, which aides in the attenuation of plasma tg concentrations (14). exercise effects on ppt are short lived and are insignificant 2448 hours post - exercise (14, 16). current research has determined the optimal time to measure ppt following an oral fat tolerance tests (oftt) is 1216 hours post - exercise (14, 24, 28). exercise benefits measured directly prior to, or directly after, a high - fat meal show a decrease in effectively reducing ppt when compared to measured exercise benefits at 12 hours prior to the high - fat meal (30). research concerning lipid metabolism commonly utilizes a younger population (2050 years of age), creating a scarcity in data corresponding to older adults. there is a presumed decrease in lpl activity with age, causing an increase in plasma tg (16). the mechanisms and parameters by which tg can be naturally reduced through exercise are of specific concern to an older population. therefore, the purpose of this study was to discover whether 90 minutes of moderate - intensity exercise attenuates ppt among older adults. subjects consisted of men (1) and women (7) of various activity levels with a minimum age of 50 years and a mean age of 588. subjects bmi was relatively homogeneous with a 26.54.2 average. a health history questionnaire as well as an informed consent advising them of procedures, risks, and benefits of the present study was completed. subjects with the diseased conditions of known cardiovascular disease, hyperlipidemia, or diabetes were excluded from the present study. subjects with signs or symptoms of cardiovascular, pulmonary, or metabolic disease, as defined by the american college of sports medicine (acsm), were also not allowed to participate (1). subjects currently taking medication to monitor lipid metabolism, carbohydrate metabolism, or insulin therapy were also excluded ; as were smokers or those partaking in recreational drugs. subjects with increased resting values prior to the exercise or control trial were also excluded ; those values were as follows : blood glucose was over 125mgdl, blood pressure greater than 160/100 mm hg, or total cholesterol over 240mgdl. activity levels ranged from moderately active (walking 3 days per week), to sedentary (non - strenuous work activities). this homogeneously active group has been selected to eliminate the synergistic effects of endurance training on ppt clearance (29). subjects participated in two, two - day, oftt trials and underwent the exercise trial first, followed by the control trial, with a minimum of seven days between trials. on day one of the two - day protocol, the oftt took place on day two of the two - day protocol among both control and exercise trial. both trials underwent the same protocols for data collection ; however, exercise was omitted in the control trial and replaced with 90 minutes of rest (seated none activity). prior to data collection on day one and two subjects rested in a supine position for 15 minutes ; at the conclusion of which, resting hr, bp, lipid profile, and blood glucose were collected utilizing the alere cholestech ldx (alere north america llc, orlando fl). subjects were advised to abstain from alcohol for a 24-hour period prior to day one and continue to abstain until the conclusion of day two. each subject kept a food journal, recording what they ate for the 24 hours prior to day one and concluding with the day two data collection. this food journal was used to replicate the subject s diet prior to each oftt. subjects were advised to abstain from exercise for 48 hours prior to day one and concluding with the day two data collection. on day one, between the hours of 18002000, the exercise trial participated in a 90-minute walk of moderate intensity. water intake was measured and replicated during the 90 minute rest period of the control trial. subjects returned on day two between the hours of 07300800 after participating in a 1216 hour overnight fast. after the resting vitals were obtained, subjects were given a 20 minute period in which to consume an oftt. the oftt consisted of a standardized dennys milkshake containing 490 kcal, 92 g fat, 146 g cho, and 24 g protein. a dose of 9000pcc lactase units was given in conjunction with the milkshake to ensure that the meal was easily digested. hr, bp, lipid profile, and blood glucose was collected at 0 (pre - oftt), 1, 2, 3, and 4 hours postprandially. two - way repeated measures anova - s were used to analyze the difference in tg, ldl, hdl, and glucose over time for the control and exercise trials. a tukey post hoc was utilized to examine the differences within the data, when appropriate. an alpha level of p < 0.05 was considered statistically significant for all comparisons. subjects consisted of men (1) and women (7) of various activity levels with a minimum age of 50 years and a mean age of 588. subjects bmi was relatively homogeneous with a 26.54.2 average. a health history questionnaire as well as an informed consent advising them of procedures, risks, and benefits of the present study was completed. subjects with the diseased conditions of known cardiovascular disease, hyperlipidemia, or diabetes were excluded from the present study. subjects with signs or symptoms of cardiovascular, pulmonary, or metabolic disease, as defined by the american college of sports medicine (acsm), were also not allowed to participate (1). subjects currently taking medication to monitor lipid metabolism, carbohydrate metabolism, or insulin therapy were also excluded ; as were smokers or those partaking in recreational drugs. subjects with increased resting values prior to the exercise or control trial were also excluded ; those values were as follows : blood glucose was over 125mgdl, blood pressure greater than 160/100 mm hg, or total cholesterol over 240mgdl. activity levels ranged from moderately active (walking 3 days per week), to sedentary (non - strenuous work activities). this homogeneously active group has been selected to eliminate the synergistic effects of endurance training on ppt clearance (29). subjects participated in two, two - day, oftt trials and underwent the exercise trial first, followed by the control trial, with a minimum of seven days between trials. on day one of the two - day protocol, the oftt took place on day two of the two - day protocol among both control and exercise trial. both trials underwent the same protocols for data collection ; however, exercise was omitted in the control trial and replaced with 90 minutes of rest (seated none activity). prior to data collection on day one and two subjects rested in a supine position for 15 minutes ; at the conclusion of which, resting hr, bp, lipid profile, and blood glucose were collected utilizing the alere cholestech ldx (alere north america llc, orlando fl). subjects were advised to abstain from alcohol for a 24-hour period prior to day one and continue to abstain until the conclusion of day two. each subject kept a food journal, recording what they ate for the 24 hours prior to day one and concluding with the day two data collection. this food journal was used to replicate the subject s diet prior to each oftt. subjects were advised to abstain from exercise for 48 hours prior to day one and concluding with the day two data collection. on day one, between the hours of 18002000, the exercise trial participated in a 90-minute walk of moderate intensity. the subjects wore polar heart rate monitors (model ft7, polar electro inc., water intake was measured and replicated during the 90 minute rest period of the control trial. subjects returned on day two between the hours of 07300800 after participating in a 1216 hour overnight fast. after the resting vitals were obtained, subjects were given a 20 minute period in which to consume an oftt. the oftt consisted of a standardized dennys milkshake containing 490 kcal, 92 g fat, 146 g cho, and 24 g protein. a dose of 9000pcc lactase units was given in conjunction with the milkshake to ensure that the meal was easily digested. hr, bp, lipid profile, and blood glucose was collected at 0 (pre - oftt), 1, 2, 3, and 4 hours postprandially. two - way repeated measures anova - s were used to analyze the difference in tg, ldl, hdl, and glucose over time for the control and exercise trials. a tukey post hoc was utilized to examine the differences within the data, when appropriate. an alpha level of p < 0.05 was considered statistically significant for all comparisons. there exists no significant difference in triglycerides when comparing the exercise effect over time, and control effect over time. there does exist a significant difference when examining the effect of time on ppt (p < 0.05). this difference lies in the one, two, three, and four hours postprandial over the pre - prandial (baseline) measurement. ppt increases along a predictable curve among both groups with a slight decrease among the exercise trial (figure 1). there is a significant difference (p < 0.05) of time at all points when compared against pre - feeding values (figure 2). there exists a significant difference of time at the two and three hour points over the pre - feeding value (figure 3). no significant difference among glucose values were found between the exercise and control groups over time. there was a significant difference (p < 0.05) of time at the one, two, and three hour postprandial values over the pre - feeding time point (figure 4). the main findings of this investigation is an acute bout of moderate - intensity exercise preformed 1216 hours prior to ingesting a high - fat meal does not attenuate ppt in older adults. there was no difference in ppt when comparing the exercise group over time with the non - exercise group over time. there exists a significant difference over time at all time points beyond the pre - feeding value. this difference is to be expected among any population and originates from the natural process by which the body metabolizes food and experiences a rapid increase in tg. previous examinations have discovered that a chronic exercise intervention is required to effect change in ldl and hdl, or exercise vigorous enough to elicit a deficit of 1100 kcal in one bout. the decreased exercise influence in the present study is likely not sufficient to influence ldl and hdl values (14, 23). conversely, a change in tg has been observed when eliciting a deficit of between 350700 kcal based on fitness (lower fitness requiring lower kcal expenditure) (14). tc experienced an increase upon feeding, and glucose followed the normal metabolic pattern of feeding, having an initial peak, followed by a decline to near pre - feeding values. the findings of the present study are not consistent with similar investigations which have found a significant difference following a 90-minute moderate - intensity exercise bout and 1216 hour fast. a decrease in ppt has been found when walking for 90 minutes at 50% vo2max, and 60% vo2max followed by a 1216 hour overnight fast in non - smoking males and females, trained and untrained, ages 2143 (3, 6, 10, 29). exercise intensity as low as 30% vo2peak for 100 minutes has been shown to reduce ppt among eight young women, age 27 1 (18). the mean age in this present study was 58 8 ; therefore, age may be an indicator of a decrease in lpl activity with age, leading to an impaired ability to metabolize tg in older adults. minimal research has been performed examining the effects of lpl activity on older adults ; however, reduced lpl activity in rodent models has shown defective fatty acid utilization and tg lipolysis leading to cardiac complications in older mice, including cardiac fibrosis (2). increased lpl activity has been linked to reduced all - cause mortality ; likely attributed to a decrease in circulating cholesterol over time (27). increased circulating vldl and chylomicrons can penetrate the intima and perpetuate the diseased state of atherosclerosis ; therefore, it can be speculated that mutations in lpl production with age can decrease the exercise effect on tg. an estimate of hr max was used in the present study, as it is more likely to appeal to an older population who may be unwilling (or unable) to perform a vo2max test. the utilization of maximal heart rate by way of measuring exercise intensity has yielded successful investigations and is friendlier to sedentary populations (as examined in the present investigation) having not met the exercise guidelines as defined by acsm (1, 19). the exercise intensity employed in the present study was 60% of estimated heart rate max which is similar to other investigations that have found a significant changes in ppt utilizing younger populations and exercise bouts of 90 minutes (6, 9, 10, 29). the oftt used in the present study contained 92 g of fat and is similar to that of other investigations which have found a significant difference among exercise trials utilizing a fat content of 40 g, 80 g, 87 g, 98 g, and 140 g (4, 6, 11, 26). researchers have had success catering the fat content to the lean mass or body mass of the participant ; this may be a more effective measure by which the fatty meal is delivered (5, 7, 10, 17, 19, 20, 29). the present study used a standardized dennys in an effort to simulate a meal that is ingested daily by restaurant patrons. the wide range of fat content and quality among researchers suggests the necessity for a standardized oftt (24). future researchers should consider incorporating a young population, as well as the older population, in an attempt to determine whether age is the divergent factor in tg metabolism. additional limitations include the scheduled exercise trial prior to the control trial ; future studies should consider utilizing a randomized cross - over design, thus decreasing the risk of participants being conscience of their activity and eating habits leading up to the trial. the subjects in the present study self - monitored their hr throughout the exercise trial and reported their heart rate verbally at 15 minute intervals ; future studies should consider constant researcher monitoring of hr. in conclusion, the results of the present study demonstrate that 90 minutes of moderate - intensity exercise 1216 hours prior to a fatty meal does not influence the rate of ppt clearance in subjects 50 years of age. the present study has sought to determine whether the exercise response on tg among older adults is similar to that of younger populations ; whom have experienced an attenuation of tg under similar parameters (6, 9, 10, 29). further research is needed on lpl activity among older adults and its influence on tg clearance. | to determine whether 90 minutes of moderate - intensity exercise, prior to a high fat meal, attenuates postprandial triglycerides (ppt) in older adults. eight sedentary older adult volunteers (mean sd age = 58 8 years, bmi 26.5 4.2) ; completed two trials consisting of exercise and a no - exercise control. exercise trials involved 90 minutes of moderate - intensity exercise 60% heart rate reserve (hrr). following exercise, an overnight fast of 1216 hours was performed. participants were given a high fat meal that consisted of 146 grams of cho, and 92 grams of fat and instructed to rest. lipid levels were collected at pre - feeding, 1, 2, 3, and 4 hours post feeding. the control trial involved no exercise, performed an overnight fast of 1216 hours, and was given the high fat meal followed by four hours of rest and data collection. there was no difference in ppt between the control and exercise trials (p < 0.05). triglycerides (tg) increased in both trials over pre - feeding values (pre - feeding 123.13 65.03 con. 111 53.9 ex., 1hr 161.50 83.77 con. 149 71.03 ex., 2hrs 208.25 120.69 con. 177 97.29 ex., 3hrs 228 146.99 con. 147.25 87.64 ex., 4hrs 211.75 140.15 con. 169.5 68.14 ex). no difference in triglycerides over time was observed among older adults between the exercise and control trials. |
about 65% of all people with a visual impairment are 50 years old and older, and this age group currently comprises about 20% of the world s population. with an increasing elderly population worldwide, more people will be at risk of visual impairment due to chronic eye diseases and ageing processes1. serious concern has emerged about the primary medical and nursing care treatment of individuals with disabilities due to their increasing severity and complexity along with their increasing cost to society. in japan, while the number of patients blinded by diabetic retinopathy is decreasing, the percentage of those receiving dialysis in connection with diabetes increased by 0.7% from fy2009 to 35.8% in 20152. this converts to 2,320 people per year. in the rural japanese community of kusatsumachi in gunma prefecture, which was the focus of this study, the rate of dialysis treatment for those who are visually impaired was 3% out of a total population of 7,1203. additionally, the management of lifestyle diseases for those with vision impairment is inadequate. indeed, compared to sighted people, visually impaired people have limited ability to obtain information necessary to learn about exercise and dietary habits for personal health maintenance. along with advances in wearable devices, instructional video technology supporting physical activities has been on a rise4, and the number of healthy people utilizing it is increasing. however, blind or visually impaired individuals will have inadequate access to such information technology because of the long time it takes to universalize or convert its content to auditory information that would be able to support the physical activities. these studies investigated physical activities in competitive sports through analyzing athletes movements5 or clinical fear, a neglected causal factor regarding the training of activities of daily living (adl) that many people complain of during movement. one possible reason for this is that there are no provisional methods to diagnostically define clinical fear, and such reports will not lead to the development of evaluative methods to predict performance. with a focus on recent and interesting theories of cognitive behavioral science, we aimed to develop a clinical diagnostic index as a method to diagnostically define clinical fear. additionally, using the presence or absence of a sense of danger as a standard, we examined whether the fear experienced during movement acquisition in adults with visual impairment was classifiable. experimenters focused on the presence of an observable freeze response, which is believed to be a sign of the participant sensing danger and used as the criterion for determining the presence or absence of a sense of danger. additionally, fear was divided into two categories, fear 1 and fear 2, which from the perspective of early developmental criteria become innate fear and learned fear. from the perspective of full maturity, these could respectively be referred to asnon - dangerous fear, and dangerous fear. accordingly, based on whether a corresponding relationship exists between danger and fear, the following four classifications were considered and utilized to classify fear preceding the movement : type 1 = danger with fear ; type 2 = danger without fear ; type 3 = no danger with fear ; and type 4 = no danger and no fear. this typological configuration is known as a dual - process model6,7,8, which has recently been equivalently, expressed, as a 2-systems model9, 10. the present pilot study aimed to classify the fear that occurs when movement acquisition is impaired based on the interviews and the results of autonomic functional analyses conducted during the process of squat - like movement acquisition undergone for individual health maintenance by adults with visual impairment who had never received aerobic exercise instruction. it further sought to clarify which instructional content was perceived as safe and effective at alleviating fear for participants. subjects were fifteen participants, 45 years old and older, selected randomly from a pool of 72 adults with visual impairment who were independently performing activities of daily living (adl) in kusatsumachi, gunma, japan. the study was conducted at the kusatsumachi welfare center from march through august 2015. participant recruitment was done by our staff who explained the study intent to the society for the visually impaired (which has approximately 72 members in total) and the social welfare council, both of which were already involved in providing activity support to visually impaired individuals in kusatsumachi, gunma prefecture. subsequently, we recruited potential research subjects based on verbal communication with relevant parties. discontinuance criteria specified situations that arise in which there is a legitimate reason for a participant to withdraw from the study (e.g., changes in the home environment, aggravation of disease, declines in physical function). ethics review and approval was obtained from the gunma paz college ethics committee (no. a desk and chair were prepared for observation and listening and a 30 cm force plate (pasco ps2142 force plate, usa) was placed on the floor to measure ground reaction force (grf). the participants were asked to stand on the plate, and their condition was uncontiuuonsly observed during the daytime from 10 a.m. until 5 p.m. while they performed squat - like movements following verbal instruction. grf measurement and the protocol assessed the level of comprehension of the pointers used in movement execution. in the protocol for movement instruction, the assessor provided verbal instruction to the participant, saying, to squat, lower your hips towards your feet, bend your knees as far as you can without straining them, and then raise your hips up again. participants were then prompted with, now, to your own ability, let s try a quick and easy set of five repetitions. participants were then told, if at any time you feel afraid that you will fall, please call out. i can support you so that you wo nt fall, but please do your best to complete the exercise by yourself. in provision of verbal support, the assessor also provided feedback when the movement was performed consistently by saying, you re doing a good job of maintaining your form throughout the repetitions. to assess the comprehension of the pointers used in movement execution, the assessor interviewed the participants and asked about impressions regarding their understanding of movement orientation, the effects of pointers on movement speed, the use of movement repetition timing cues, and pointers regarding spatial location information. ground reaction force (grf) was measured on the floor where the participants stood. the presence or absence of subjective fear assessed the presence of anecdotal expressions of fear immediately prior to the squat - like movement on the force plate. presence or absence of objective fear as thru an anxiety index measures the ratio of low and high frequency components (lf / hf ratio) of heart rate variability spectra (cm / hz) based on resting rate (rr) intervals (actigraph wgt3x - bt, usa). autonomic hyperactivity was defined as over 2.0 (lf / hf ratio), indicating that objective fear was occurring. statistical analysis first included a typological analysis for which a dual - process theory was adapted to classify clinical fear based on the relationships between danger and subjective and objective fear. the fear types included perception of danger with freeze response that one was in a position to fall from a top a platform, type 1 is danger with anxiety, type 2 is danger without anxiety, and type 3 is no danger with anxiety, type 4 is no fearless. danger and no anxiety. we then sought the frequency of occurrence of each of these clinical fear types. a fisher s exact test was conducted to examine the relationships among evaluated fear types and expected types (p<0.05) using ibm - spss ver. 20. participants were asked to stand on a ground reaction force (grf) plate placed on the floor. subsequently, they were evaluated on aerobics state, or their movements were evaluated the presence of an observable freeze response (table 1table 1.ground reaction force plate on the floor utilized by participants (persons with visual impairment)idgenderbody height (cm)body weight (kg)vertical impulsehorizontal impulsemean of vertical impulsemean of horizontal impulse1male15349.19,727 163 1,945 33 2male15763.424,096 340 4,819 68 3male16557.518,522 326 3,704 65 4female1153418,522 326 3,704 65 5male16854.57,485 220 1,497 44 6male149.549.729,753 109 5,951 22 7male16565.416,275 281 3,255 56 8female1415933,353 80 6,671 16 9male161.554.922,125 151 4,425 30 10female14040.57,502 29 1,500 6 11male16961.66,816 77 1,363 15 12male17066.18,887 152 1,777 30 13female13841.310,670 207 2,134 41 14male167557,252 132 1,450 26 15female14252.919,443 141 3,889 28 impulse : five repetitions of integrated force (ns) of ground reaction force (grf), sampling frequency 200 hz.pasco 2-axis force platform ps2142). impulse : five repetitions of integrated force (ns) of ground reaction force (grf), sampling frequency 200 hz. pasco 2-axis force platform ps2142 the relationships between a sense of danger and subjective / objective (autonomic arousal) fear during movement were evaluated, and the results are shown in tables 1 and 2. all 15 participants were able to execute the squat - like movements until the end of the session. the relationships of sensing and not sensing danger with the presence or absence of freeze the fear types were able to divide into four types reflecting combinations of danger and objective fear factors. the clinically categorized subjective fear was noted for two (type 1 = danger with fear), seven (type 3 = no danger with fear), six (type 2 = danger without fear), and zero (type 4 = no danger and no fear) persons for each type (tables 2table 2.cross tabulation of sense of danger with freeze and subjective fear in people with visual impairment (n=15) congenital (7) or acquired blindness (8)acquired or congenitalno fearfeartotalacquired blindness feeling danger with freeze (n)303no feeling of danger with freeze (n)415total718congenital blindnessfeeling danger with freeze (n)303no feeling of danger with freeze (n)224total527a fischer s exact test in acquired blindness vs. congenital blindness by no fear vs fear : n.s. and 3table 3.cross tabulation of sense of danger with freeze and objective anxiety as objectiveover or under the set point of lf / hf(2.0)no fearfeartotalunderfeeling danger with freeze (n)011no feeling of danger with freeze (n)101total112overfeeling danger with freeze (n)606no feeling of danger with freeze (n)527total11213fear (lf / hf) in people with visual impairment (n=15). fischer s exact test with different of set point of lf / hf by no fear vs. fear : n.s.). a fischer s exact test in acquired blindness vs. congenital blindness by no fear vs fear : n.s. fischer s exact test with different of set point of lf / hf by no fear vs. fear : n.s. the frequencies of occurrence of clinically categorized subjective fear were two, seven, six, and zero persons for types 1, 3, 2, and 4, respectively. when looking at cases in which there was a sense of danger, the relationship of congenital and acquired blindness with the frequency of occurrence of the four fear types was non - significant (table 4table 4.cross tabulation of fear and subjective classification in persons with visual impairment (n=15)acquired or congenitalclassificationno fearfeartotalacquired blindness subjective (feeling based) fearfeeling danger (n)classification 1415no feeling danger (n)classification 2000subjective (feeling based) no fearfeeling danger (n)classification 3303no feeling of danger (n)classification 4000total718congenital blindnesssubjective (feeling based) fearfeeling danger (n)classification 1112subjective (feeling based) no fearfeeling danger (n)classification 2112subjective (feeling based) fearno feeling of danger (n)classification 3303subjective (feeling based) no fearno feeling of danger (n)classification 4000total527fischer s exact test of acquired blindness vs. congenital blindness by classifications : n.s.). the clinically categorized frequency of objective fear for those with innate blindness was three, six, zero, and six people in types one, 3, 2, and 4, respectively (table 5table 5.cross tabulation of relationship in acquired or congenital blind, observation fear / no fear, feeling danger, classifications of persons with visual impairment (n=15)acquired or congenitalclassificationno fearfeartotalacquired blindness observation (feeling based) fearfeeling danger (n)classification 1011no feeling of danger (n)classification 2044observation (feeling based) no fearfeeling danger (n)classification 3000no feeling of danger (n)classification 4303total358congenital blindnessobservation (feeling based) fearfeeling danger (n)classification 1022observation (feeling based) no fearfeeling danger (n)classification 2022observation (feeling based) fearno feeling of danger (n)classification 3000observation (feeling based) no fearno feeling of danger (n)classification 4303total347a fischer s exact test in the matrix cells, acquired or congenital blind vs. classifications : linear - by - linear association, p=0.011 ; 1, linear - by - linear association, p=0.019). when looking at cases in which there was a sense of danger, a significant relationship was found between the occurrence frequency of the four fear types and innate people with blindness (tables 4 and 5). fischer s exact test of acquired blindness vs. congenital blindness by classifications : n.s. a fischer s exact test in the matrix cells, acquired or congenital blind vs. classifications : linear - by - linear association, p=0.011 ; 1, linear - by - linear association, p=0.019 the findings suggest a possible feasibility of fear classification based on examining the process of a squat - like movement of persons with visual impairment. our results indicated that a sense of fear and the presence or absence of a sense of danger affected movement learning. all 15 participants in this study were able to execute the squat - like movements until the end of the experiment. of these, the lf / hf ratio of hrv was measurable in eight participants until the end of the experiment. when we controlled for objective fear, the relationships among congenital blindness, sense of danger, and fear were significant but when adapting for subjective fear these relationships were non - significant. that our classification method adopted for objective fear (autonomic hyperactivity) found significant relationships independently between congenital blindness, sense of danger, and fear, and did not find significant relationships when adapted to subjective fear, which can be taken as an indication of its effectiveness of optimize subjective fear as follow as types by 2-systems model9, 10. in corroboration with a report by shibasaki. in which autonomic functional determinants were used in prognosis estimations of elderly persons and exercise function, the circumstances of stable physical function are thought to be predictable and have constant applicability. although almost objective fear corresponds to subjective fear, congenitally - blind people do not feel fear when they do not sense danger which may explain why significant independent relationships emerged for clinical fear classifications when autonomic function was included to discriminate objective fear. innate fear is similar to physiological fear, and it should be manifested in a manner opposite to that of psychological fear. thus, the existence of danger, which can not be learned, may suggest the existence of fear that can not be verified. if so - called type 1 was a cognitive process possessed by everyone. the present study confirmed cases that were classifiable as type 2 fear in those with congenital blindness who sensed neither subjective fear nor danger, a previously unobtainable result in dual - process theory - based classification of fear in sighted people. it is thought that the present study is the first in the world to successfully verifiably demonstrate the lack of fear manifestation in correspondence with a 2-systems model. type 2 is re - defined as the predictable anxiety that can be described no - orally before exercising in response to an unprecedented event that requires the subject to perform repetitive tasks. on the other hand, type 3 is able to defined that can not be described orally before exercising in response to an unprecedented event that requires the subject to perform repetitive tasks. furthermore, through our experiment, we confirmed that type 2 shows the changes in autonomic nerve functions. therefore, in reference to the principles of behavioral science, it is possible to express as danger without fear. additionally, while type1 and type 2 reflect subjective anxiety, we defined dangerous conditions as those that are evident in subjects before exercising as presence of danger and clearly seen by third parties. when there is clear danger that can be seen objectively, such as the subject s foot being off the base, this is defined as danger. in other cases, it is possible to refer to as no danger. these operative definitions are possible to adopt to test interactions between fear and danger. the motive behind this study is to find a clue to the problem of not being able to acquire certain levels of aerobics activities in a given period compared to able - bodied subjects using these results of oral expression data. this study conducted this research to quantitatively measure the anxiety that goes along with exercise / movements to make circumstantial judgments. the novel finding in the present study identifies shortcomings in our prior understanding of fear response and dual - process models that could not have been explained based on 2-systems models11. our new theory hypothesizes that explanations of fear and danger by conventional dual - process models, which do not consider the effects of physical movement, are limited to static conditions and that fear, when accompanied by conditions that demand the acquisition of movement and the mastery of exercise training, is expressed by physiological phenomena12. thus, even if complaints of subjective fear are not exhibited when providing support for aerobic exercise movement acquisition in adults with visual impairment, an exercise physiologist and physical therapist could give them a better safety in particular conduct by this classification of clinical fear like a human factors. the limitations of this study include the difficulty of statistical estimates due to the small sample size. the present study was limited in terms of insufficient measurement due to the inability to continuously monitor subjective fear over a sufficient length of time. moreover, our results lack generalizability, as we can not rule out the possibility of bias based on the unique characteristics of the community examined. the procedure is used to classify fear might be increased safety feasible during the process of squat - like movement by persons with visual impairment. unconscious fear does not show to be exist, but the classification of clinical fear based on physiological alterations in autonomic activity could be of significance for the development of instructional methods to support the health of the elderly with visual impairments. | [purpose ] this study aimed to classify the fear that occurs during the process of a squat - like movement by persons with visual impairment. [subjects and methods ] fifteen persons with a visual impairment, 45 years and older, who were independent in daily living were recruited for the study. the study utilized a field experiment design. the setting was the kusatsumachi welfare center, and the study period was from march through august 2015. outcome measures included the presence or absence of fear during attempt of a squat - like movement on a force plate. metrics included the ratio of low and high frequency components of heart rate variability spectra (cm2/hz) based on resting rate intervals (actigraph wgt3x - bt, usa). autonomic hyperactivity was defined as the occurrence of objective fear with lf / hf ratio of over 2.0. statistics included fischer s exact test. [results ] four fear types included the combination of danger and objective fear factors. the frequencies of occurrence of clinically categorized subjective fear across the four fear types were two (danger, fear), seven (no danger, fear), six (danger, no fear), and zero (no danger, no fear) persons. [conclusion ] this procedure of classifying fear might be feasible during evaluation of a squat - like movement by persons with a visual impairment. |
the dichotomy of having both generalists and specialists is omnipresent in many fields and is not restricted to physician occupations. one can observe that a growing number of health care professions, such as nurses and pharmacists, are increasingly becoming subdivided into specific specialties.1,2 this type of generalist specialist dichotomy is also a common phenomenon outside the field of medicine, such as in finance, journalism, or information technology. the generalist some specialists may complain of generalists not doing enough from their own specialty s perspective. a number of studies have demonstrated that the outcomes of certain conditions were better under the care of specialists, as opposed to generalists,3,4 while others contested these claims.5,6 on the other end of the spectrum, generalists may not approve of the way specialists see their patients, as they sense that specialists focus only on specific diseases or organs, and not on the patients as a whole.7 friedrich engels and karl marx wrote in their book, the communist manifesto : society as a whole is more and more splitting up into two great hostile camps, into two great classes, directly facing each other : bourgoisie and proletariat.8 of course, one hopes that the relationship between generalists and specialists in medicine is not as hostile as the picture depicted by engels and marx ; however, the concept of splitting up into two groups, the concept of dichotomy nurturing tension, or a sort of class struggle most likely exists in the field of medicine as well. but what does this ultimately lead to ? does nt this type of mutual criticism simply confuse patients, who may sense the tension between the two, often competing, contrarieties ? this kind of detrimental dichotomy may potentially jeopardize the quality of health care as a whole, since it is the patients who suffer most from the disconnections and misunderstandings between generalists and specialists. to overcome this problem, the author herein proposes a third pathway, namely, the concept of the genecialist. the genecialist refers to a hybrid comprising elements inherent to both generalists and specialists. he / she is a physician who can function as both a primary care physician (generalist) and as a specialist. the author proposes the integration and harmonization of both groups as a potential solution to overcome the numerous issues posed by the generalist specialist dichotomy. the concept of the genecialist was conceptualized by referencing noteworthy philosophers in recent history who developed the process of dialectics, illustrated by georg wilhelm friedrich hegel s aufheben, which articulated the tension between thesis and antithesis.9 the concept of the genecialist was born in a manner similar to aufheben by exhaustive inquest into both generalists and specialists. representation, by definition, is a declaration of being a member of a faction. generalists represent themselves as members of general medicine and specialists represent themselves as members of a given specialty. the declaration makes one isolated from others, who do not belong to that particular faction. factionalization is the true enemy that must be slain in order to effectively nurture a mutual understanding between ostensibly polar contrarieties (eg, men and women, west and east, white and black, doctors and patients, the examples are endless). this concept of dichotomy is detrimental to achieving mutual understanding, sympathy and empathy to others. thus, in this novel depiction of generalists and specialists, the difference is to a certain extent relative. but are generalists and specialists really different ? or, a generalist is classically thought of as doing and knowing something about everything, but not everything about something. the classical image of a generalist is, therefore, a wide, short rectangle, covering a broad range of knowledge, but few in great depth. one might call this the lake okeechobee effect, referring to a large inland lake in florida with extensive surface area, but which is at no point more than 2 meters deep. on the other hand, the classical depiction of a specialist is a rather tall, but narrow rectangle, covering only one field of specialty, yet with a very high level of expertise. staying with the aquatic analogy, this might be represented by oregon s crater lake, with a relatively small surface area but a great depth of up to 2000 meters. in reality however, the author believes that the typical generalists and specialists adhere to neither of these analogies. rather, most generalists i know can better be described as a triangle in terms of shape. in general, they are more than capable in many senses to function as primary care physicians, but they also often have in - depth capabilities in a specified field, which also makes them special. this can be for a specific disease entity, or subspecialty such as expertise in rheumatologic diseases or infectious diseases. it can also refer to a particular setting of their respective duties, such as an emergency department, house calls, rural medicine, or occupational medicine, to name a few. they may also excel at some cross sectional entities, such as evidence based medicine, narrative medicine, medical ethics, palliative care, clinical reasoning, or medical education. many generalists do have something, and therefore the rectangular analogy fails to do them justice ; instead these physicians are more closely represented by a triangular geometry. although not enough to be called generalist perhaps, they do have some width and learn certain things related to their original specialty. some are good in the management of postsurgical infections, nutrition, pain management, and so on. they also fit more of a triangle description, rather than rectangles, as described above for generalists. the difference between these two groups is merely in the width and length, which is comparatively relative, and not definitive. some cardiologists devote themselves only to cardiac catheterization and do not bother doing echocardiograms, stress tests, or even electrocardiogram reading. these are super - specialists, who are even better than ordinary specialists on select topics. now, if you consider the three distinct triangles constructed to depict the generalist, specialist, and super - specialist, you will find a relative gradient, not showing any definite dichotomy. this is similar to what you will find when looking at people with differing heights and weights. there may be a generalist with a very, very wide coverage of a broad range of topics with relatively short height on his / her triangle. for example, the majority of general internists do not see trauma patients, pregnancy - related problems, or pediatric cases. a frequent topic discussed by generalists is what is the minimum requirement to be considered a generalist ? some might say you must see pregnant women, or you need skills in trauma care. personally, i feel that such lines of questioning are irrelevant since the difference in the width of the triangle is merely relative. if one caters to a greater number of gay patients, hiv may be a more common entity. some may work at a veteran s affair medical center where most of the patients are men and smokers with many tobacco - related comorbidities. are generalists in these settings specialists, covering a much narrower spectrum of patients ? it probably depends on how you categorize them but this in itself is an arbitrary process. structuralism, founded by ferdinand de saussure, claude lvi - strauss, and others explained that every categorization is rather arbitrary and not definitive,10 which is not unlike attempting to define the number of colors of a rainbow. if so, why separate them ? why do we not handle them collectively as one entity, to avoid the unnecessary and often confounding factionalization ? pickering described a very similar concept in the 1970s.11 he stated : as th huxley said, know something about everything and everything about something. as a physician i hold a similar view. in my youth i became an expert on peripheral vascular disease. i remember with great satisfaction the patients i saved from vascular surgery, for example, hysteria, multiple sclerosis, prolapsed intervertebral disc. what chiefly terrifies me about medicine in the us is the danger to the patient of falling into the hands of a subspecialist, particularly one who uses questionnaires, for he starts with a presumed diagnosis and the patient is almost certain to become a disease and cease to be a person. genecialists are those who have completed training in both general medicine and a subspecialty. but this does not have to be a typical subspecialty such as cardiology or nephrology. for instance, i know of one primary care physician who is very knowledgeable about evidence - based medicine. he wrote a number of books on this topic1214 and is widely respected for his expertise. prominence in one topic in which one knows (almost) everything is a condition to make the topic special to oneself, as well as to others., he is a prime example of a genecialist. knowing (almost) everything about something helps you in many ways. by penetrating through a certain field, you will paradoxically realize how much you do not know about many things involved in daily patient care. suppose we have 20 specialties in medicine (we have more of course, but this is merely for the sake of a thought experiment). if you are specialized in one of these 20 fields, because there are 19 more specialties in which you have no specialist expertise, you realize that the ratio of things you know to those that you do not know is 1:19, which means that the things you do not know outweigh the things that you do know by a factor of 19. you realize that there are so many things that you do not know because you have deep knowledge and skills only in a specific subject. without specialist expertise, you will never understand the potential height that each specialty truly embodies. without generalist expertise it is only when you have enough height and width in your respective triangle that you can truly come to the realization that you have many things that you have yet to learn. this encourages you to learn more, listen to others, and respect others, so that you can improve yourself. again, the true value of becoming a genecialist does not reside in the knowledge you have. it is the awareness of knowing what you do not have that is the true value of being a genecialist. by being a specialist, you notice the slight differences among similar cases, since you see many cases accumulated by consultation. at a glance, patient but they differ from a specialist s view ; a and b are treated differently, since they really are different. the judgment to differentiate between these two pneumonia cases is again arbitrary according to structuralism. these subtle differences go unnoticed to the uninitiated, who do not have enough experience, namely nonspecialists. i think that john has an unusual pneumonia, which might be a little difficult to treat. why do nt we consider extended treatment for this patient ? this happens often in my infectious diseases consultation service, but this kind of contingent judgment may not be described in a textbook or on pubmed. i have noticed that some generalists mock a specialist because he / she did not follow evidence or guideline in a particular case. however, there are things that evidence alone might not be able to tell you. a high level, sophisticated, randomized, controlled clinical trial may be useful in general, but may not be applicable if the patient in front of you is different from the ones enrolled in the trial, and it should be noted that this happens frequently. overreliance on evidence and ignoring one s experience is not evidence - based medicine and is more like evidence - biased medicine. by being specialists and by seeing various types of patients, you are more likely to notice these subtle differences. this also leads to the appreciation of other specialists. for the sake of argument, this individual may not give beta blockers to a particular patient with a myocardial infarction, because there may be an underlying condition to preclude their use that he / she sees, which i do not have knowledge of. i may not be able to acknowledge the difference myself, but i will know the difference exists and this is what is important. for example, one patient you have known for years may present with chest pain. the generalist may sense this pain is different from others in the past because, as a generalist, he / she knows the patient well and will be able to pick up on subtleties that i can not. becoming a genecialist is a very effective way to realize the existence of such eyes and avoid any unnecessary or otherwise confounding miscommunications. having something special is good because you are somebody who can be relied upon. i acknowledge that sometimes primary care physicians feel understandably uncomfortable about not having something special about which others ask your opinion a medical forte, if you will. this might produce a relationship with a sort of laterality, since there will be a dichotomy of one entity calling upon the other, but by the same token not being called upon. thorsen expressed the general practitioners frustration at their dialogue being rather asymmetrical.15 should individuals become genecialists, the one - way communications hierarchy will be effectively lifted, resulting in individuals calling upon others and having the very same gesture reciprocated. the attributes of family physicians according to rakel are shown in table 1.7 there is a cautionary statement below this list, which states : these characteristics are desirable for all physicians, but are of greatest importance for the family physician.7 now, what rakel may be implying is that idealistically all physicians should but in reality do not embody these characteristics. i think these attributes should be of great importance to all physicians and personally can not imagine a physician who is exempt from having a strong sense of responsibility, compassion, empathy, curiosity, and so on. if the specialist leaves these attributes only to the family physician (or any generalist), it implies that the specialist is, will be, and must be, inferior to others in terms of those attributes. will certainly enrich their practice, allow them to take better care of their patients and will result in less conflict with so - called generalists. with the acquisition of these virtues described by rakel, i firmly believe that all specialists can be categorized as generalists. i know a number of so - called specialists who do possess all of these attributes described by rakel. i know an american specialist in pulmonary medicine who has cared for her asthma patients for years, in a very compassionate and comprehensive way, who has gone out of her way to even provide instruction to her patients on how to buy an appropriate carpet or how to wash their pillowcases. there is also a japanese rheumatologist whose care was thoroughly compassionate and never ceased to impress his patients. after years of dedicated care at one hospital, he decided to leave the institution and start a new practice in a distant locality. some of his patients actually moved closer to this individual, simply to seek his care. as an infectious diseases doctor, they are not merely a subset of patients that must be constantly observed for cd4s and viral loads. we have to extend our care to their anxiety, the lack of understanding by their family members, the fear of discrimination, cultural conflicts, concurrent problems such as the use of illicit drugs, or the problems of unsafe sexual practices. compassion, dedication, comprehensiveness, continuity, and many other virtues described by rakel are a must in hiv / aids care. there need be no distinction between a classic generalist and specialist in one s approach to hiv / aids care. in fact, our patients desperately need the watchful eyes of both generalists and specialists. meza and passerman propose the integration of narrative medicine and evidence - based medicine.16 they do not consider these to be antagonistic concepts. a good narrative guides physicians and patients to ask appropriate questions to resolve the patients narrative dilemma. this is the first step towards entering into evidence - based medicine. unless you ask clinically appropriate questions that closely relate to the patient, a high quality search for evidence and detailed evaluation of articles will not result in the benefit or happiness of the patient. integrating all dichotomies will harmonize separate concepts as one, which will benefit all of the parties concerned. i would like to propose the same notion here with the concept of the genecialist to preemptively resolve any potential conflicts between generalists and specialists. one is interested in harmonization and integration, and the other is interested in specialization and disintegration.17 either view is possible and arbitrary, as explained in structuralism. more than likely, it is integration, rather than disintegration that has a greater affinity with the field of medicine. karl marx tried to overcome the dichotomy and class struggle between the bourgeoisie and proletariats, rather unsuccessfully. he foresaw the latter would overcome and destroy the former in the class struggle, but that prediction turned out to be untrue. i would like to propose a better way to overcome our dichotomy in the field of medicine : the coalescence of both entities and aufheben to nurture a new concept of the genecialist, for better patient care. knowing (almost) everything about something helps you in many ways. by penetrating through a certain field, you will paradoxically realize how much you do not know about many things involved in daily patient care. suppose we have 20 specialties in medicine (we have more of course, but this is merely for the sake of a thought experiment). if you are specialized in one of these 20 fields, because there are 19 more specialties in which you have no specialist expertise, you realize that the ratio of things you know to those that you do not know is 1:19, which means that the things you do not know outweigh the things that you do know by a factor of 19. you realize that there are so many things that you do not know because you have deep knowledge and skills only in a specific subject. without specialist expertise, you will never understand the potential height that each specialty truly embodies. without generalist expertise it is only when you have enough height and width in your respective triangle that you can truly come to the realization that you have many things that you have yet to learn. this encourages you to learn more, listen to others, and respect others, so that you can improve yourself. again, the true value of becoming a genecialist does not reside in the knowledge you have. it is the awareness of knowing what you do not have that is the true value of being a genecialist. by being a specialist, you notice the slight differences among similar cases, since you see many cases accumulated by consultation. at a glance, patient but they differ from a specialist s view ; a and b are treated differently, since they really are different. the judgment to differentiate between these two pneumonia cases is again arbitrary according to structuralism. these subtle differences go unnoticed to the uninitiated, who do not have enough experience, namely nonspecialists i think that john has an unusual pneumonia, which might be a little difficult to treat. why do nt we consider extended treatment for this patient ? this happens often in my infectious diseases consultation service, but this kind of contingent judgment may not be described in a textbook or on pubmed. i have noticed that some generalists mock a specialist because he / she did not follow evidence or guideline in a particular case.. however, there are things that evidence alone might not be able to tell you. a high level, sophisticated, randomized, controlled clinical trial may be useful in general, but may not be applicable if the patient in front of you is different from the ones enrolled in the trial, and it should be noted that this happens frequently. overreliance on evidence and ignoring one s experience is not evidence - based medicine and is more like evidence - biased medicine. by being specialists and by seeing various types of patients, you are more likely to notice these subtle differences. this also leads to the appreciation of other specialists. for the sake of argument, let s say i have a colleague who is a cardiologist. this individual may not give beta blockers to a particular patient with a myocardial infarction, because there may be an underlying condition to preclude their use that he / she sees, which i do not have knowledge of. i may not be able to acknowledge the difference myself, but i will know the difference exists and this is what is important. for example, one patient you have known for years may present with chest pain. the generalist may sense this pain is different from others in the past because, as a generalist, he / she knows the patient well and will be able to pick up on subtleties that i can not. becoming a genecialist is a very effective way to realize the existence of such eyes and avoid any unnecessary or otherwise confounding miscommunications. having something special is good because you are somebody who can be relied upon. i acknowledge that sometimes primary care physicians feel understandably uncomfortable about not having something special about which others ask your opinion a medical forte, if you will. this might produce a relationship with a sort of laterality, since there will be a dichotomy of one entity calling upon the other, but by the same token not being called upon. thorsen expressed the general practitioners frustration at their dialogue being rather asymmetrical.15 should individuals become genecialists, the one - way communications hierarchy will be effectively lifted, resulting in individuals calling upon others and having the very same gesture reciprocated. the attributes of family physicians according to rakel are shown in table 1.7 there is a cautionary statement below this list, which states : these characteristics are desirable for all physicians, but are of greatest importance for the family physician.7 now, what rakel may be implying is that idealistically all physicians should but in reality do not embody these characteristics. i think these attributes should be of great importance to all physicians and personally can not imagine a physician who is exempt from having a strong sense of responsibility, compassion, empathy, curiosity, and so on. if the specialist leaves these attributes only to the family physician (or any generalist), it implies that the specialist is, will be, and must be, inferior to others in terms of those attributes. will certainly enrich their practice, allow them to take better care of their patients and will result in less conflict with so - called generalists. with the acquisition of these virtues described by rakel, i firmly believe that all specialists can be categorized as generalists. i know a number of so - called specialists who do possess all of these attributes described by rakel. i know an american specialist in pulmonary medicine who has cared for her asthma patients for years, in a very compassionate and comprehensive way, who has gone out of her way to even provide instruction to her patients on how to buy an appropriate carpet or how to wash their pillowcases. there is also a japanese rheumatologist whose care was thoroughly compassionate and never ceased to impress his patients. after years of dedicated care at one hospital, he decided to leave the institution and start a new practice in a distant locality. some of his patients actually moved closer to this individual, simply to seek his care. as an infectious diseases doctor, i see many hiv / aids patients. to me they are not merely a subset of patients that must be constantly observed for cd4s and viral loads. we have to extend our care to their anxiety, the lack of understanding by their family members, the fear of discrimination, cultural conflicts, concurrent problems such as the use of illicit drugs, or the problems of unsafe sexual practices. compassion, dedication, comprehensiveness, continuity, and many other virtues described by rakel are a must in hiv / aids care. there need be no distinction between a classic generalist and specialist in one s approach to hiv / aids care. in fact, our patients desperately need the watchful eyes of both generalists and specialists. meza and passerman propose the integration of narrative medicine and evidence - based medicine.16 they do not consider these to be antagonistic concepts. a good narrative guides physicians and patients to ask appropriate questions to resolve the patients narrative dilemma. this is the first step towards entering into evidence - based medicine. unless you ask clinically appropriate questions that closely relate to the patient, a high quality search for evidence and detailed evaluation of articles will not result in the benefit or happiness of the patient. integrating all dichotomies will harmonize separate concepts as one, which will benefit all of the parties concerned. i would like to propose the same notion here with the concept of the genecialist to preemptively resolve any potential conflicts between generalists and specialists. one is interested in harmonization and integration, and the other is interested in specialization and disintegration.17 either view is possible and arbitrary, as explained in structuralism. more than likely, it is integration, rather than disintegration that has a greater affinity with the field of medicine. karl marx tried to overcome the dichotomy and class struggle between the bourgeoisie and proletariats, rather unsuccessfully. he foresaw the latter would overcome and destroy the former in the class struggle, but that prediction turned out to be untrue. i would like to propose a better way to overcome our dichotomy in the field of medicine : the coalescence of both entities and aufheben to nurture a new concept of the genecialist, for better patient care. | generalists and specialists are often considered two completely distinct species, which culminates in the establishment of a concept of dichotomy. however, these dichotomy can at times fuel tension and even erupt into open conflict. in order to resolve this issue, the author herein proposes the concept of a genecialist. the genecialist refers to a hybrid comprising elements inherent to both generalists and specialists. this potentially overcomes the multitude of issues associated with both generalists and specialists in the practical aspects of medicine. the coalescence of these two contrarieties may hold the key to improving the future of health care. mediating and integrating both categories into one consolidated entity carries the potential to stem the tide of class warfare between generalists and specialists. |
abundant epidemiological studies in populations and preclinical studies in animal models demonstrate that high density lipoprotein (hdl) protects against the development of atherosclerosis. the scavenger receptor class b type 1 (sr - b1) is a multiligand receptor that has high affinity for hdl and can mediate the bi - directional exchange of lipids between bound hdl and cells. sr - b1 expressed in hepatocytes mediates cellular uptake of cholesterol from hdl, driving a process called reverse cholesterol transport : the net movement of cholesterol by hdl from peripheral tissues including macrophages in the artery wall, to the liver for disposal via the bile or for recycling back into the bloodstream complexed with new lipoproteins. in addition to this well accepted role for sr - b1 in cholesterol transport, more recent studies have pointed to a role in hdl - dependent signaling in cells including endothelial cells, macrophages, platelets and other cell types. we have recently demonstrated that hdl acts as a chemotactic factor for macrophages in vitro, and that this involves hdl dependent signaling via sr - b1, pdzk1 and sphingosine 1 phosphate (s1p) receptor 1 (s1pr1), leading to phosphorylation of akt. inactivation of the expression of sr - b1, pdzk1 or akt1 or antagonism of s1pr1 impairs the ability of macrophages to undergo chemotaxis towards hdl. similarly, others have demonstrated that hdl signals via sr - b1 in platelets and in hematopoietic stem / progenitor cells (hspc) leading to pi3k / akt activation, which dampens platelet activation and hspc proliferation. hdl signaling has been most well studied in endothelial cells, in which hdl signaling via sr - b1 leads to phosphorylation of pi3k / akt, and of endothelial no synthase (enos) leading to its activation (fig. 1). hdl signaling in endothelial cells also results in cell migration and proliferation, and suppresses activation of expression of the inflammatory adhesion molecules vascular cell adhesion molecule-1 (vcam-1) and intercellular adhesion molecule-1(icam-1). hdl signaling via sr - b1 involves pdzk1 (shown binding to the c - terminal tail of sr - b1 via one of the two possible pdz domains known to bind there), activation of akt1 and enos. germline whole body knockout of sr - b1, pdzk1, akt1 and enos (red boxes) on atherogenic apoe or ldlr ko backgrounds have all been shown to trigger either spontaneous (sr - b1/apoe) or diet induced coronary artery atherosclerosis and myocardial infarction (pdzk1, akt1 or enos on apoe ko ; sr - bi on apoe - hypo or ldlr ko backgrounds). more detailed descriptions of hdl signaling pathways mediated by sr - b1 can be found in references. vcam-1 and icam-1 play key roles in adhesion of monocytes to activated endothelial cells of arteries at sites prone to atherosclerosis development. endothelial cells can become activated to express high levels of vcam-1 and icam-1 through signaling induced by inflammatory cytokines such as tumor necrosis factor (tnf), or by non - laminar, turbulent blood flow ; the latter being a key determinant of the location of atherosclerosis development within the arterial tree. exposure of monocytes to hdl has been reported to suppress the vcam-1 binding activity of cd49d and the icam-1 binding activity of cd11b, thereby reducing monocyte recruitment by activated endothelial cells in vitro. these findings suggest that hdl induces atheroprotective cellular responses locally in the blood vessel / at the sites of lesions that are dependent on sr - b1 but may be independent of bulk cholesterol transport. to investigate the role of sr - b1 on atherosclerosis, sr - b1 deficient mice were generated and mated to apolipoprotein (apo) e deficient mice to generate sr - b1/apoe double knockout (dko) mice. these mice develop increased aortic sinus atherosclerosis compared with control apoe ko mice when fed a normal chow diet. surprisingly, these mice also develop occlusive atherosclerosis in a substantial proportion (approximately 30%) of coronary arteries, accompanied by extensive myocardial fibrosis, electrical conductance abnormalities including st segment deviations, cardiac dysfunction characterized by reduced left ventricular ejection fraction and early death by approximately 8 weeks of age when fed a normal chow diet. krieger and co - workers also generated a related strain of mice which lacked sr - b1 on a hypomorphic (hypo) apoe background. these sr - b1-ko / apoe - hypomice appear normal when fed a normal chow diet, low in fat and cholesterol ; however feeding the mice diets containing high fat, high cholesterol and sodium cholate induces the development of occlusive coronary artery atherosclerosis and greater aortic sinus atherosclerosis as compared to similarly fed sr - b1 expressing apoe - hypo mice. the induction of occlusive atherosclerosis in coronary arteries is rapid, requiring only 3 - 4 weeks, and, as in sr - b1/apoe dko mice, is accompanied by extensive myocardial fibrosis, cardiomegaly, st segment deviations in electrocardiograms, and left ventricular dysfunction. the timing of disease development in the sr - b1-ko / apoe - hypo mice, as measured by survival and cardiomegaly, is affected by the composition of the diet, such that increased dietary cholesterol and the presence of sodium cholate, a bile salt derivative which results in increased levels of plasma cholesterol, result in more rapid onset of cardiomegaly and reduced survival. husbandry conditions can also affect the survival of the atherogenic, high fat / high cholesterol diet - fed sr - b1-ko / apoe - hypo mice, such that survival is prolonged when mice are group - housed vs. housed singly. surprisingly, mice housed in groups were reported to exhibit lower levels of plasma cholesterol than those housed singly, although the reasons for this appear unclear. however, it was suggested that this might contribute to the prolonged survival of the group - housed atherogenic diet fed sr - b1/apoe - hypo mice. the effects of the different diets on the level of atherosclerosis in coronary arteries or the aortic sinus, however was not described. we have recently reported that sr - b1/ldlr dko mice also exhibit reduced survival when fed atherogenic diets, and, similar to effects observed in sr - b1-ko / apoe - hypo mice, the average lifespan of atherogenic diet - fed sr - b1/ldlr dko mice was reduced by increased cholesterol content, and presence of sodium cholate in the diet. when female sr - b1/ldlr dko mice were fed the paigen atherogenic diet (15% fat, 1.25% cholesterol, 0.5% sodium cholate) beginning at 12 weeks of age, they exhibited a 50% survival of only 3.5 weeks from the onset of feeding, similar to the median survival reported for the sr - b1-ko / apoe - hypo mice fed the same diet. removal of sodium cholate prolonged the 50% survival to 9.4 weeks of diet feeding, while mice fed a normal chow diet supplemented with 2% cholesterol exhibited a 50% survival of 11.4 weeks. in contrast sr - b1/ldlr dko mice fed a western type atherogenic diet (21% fat and 0.15% cholesterol) from 12 weeks of age, or a normal chow diet (6.2% fat, and no added cholesterol) from weaning did not exhibit any reduced survival up to 22 weeks of age. for each diet / feeding time, female sr - b1/ldlr dko mice exhibited statistically significantly increased (3.5 - 10 fold) atherosclerosis in their aortic sinuses when compared with similarly aged and fed female ldlr single ko mice. sr - b1/ldlr dko mice fed each of the atherogenic diets, but not mice fed the normal chow diet, developed significant coronary artery atherosclerosis (fig. 2), with approximately 20% (western diet) to 30% (paigen diet) of coronary arteries on average, being completely occluded by atherosclerotic plaques. in contrast, virtually none of the coronary arteries examined in any of the atherogenic diet fed ldlr single ko control mice contained atherosclerotic plaques (although some fatty streaks were detected in paigen diet fed ldlr ko mice). furthermore, mice fed the cholesterol - supplemented diet, the paigen diet and the paigen diet lacking sodium cholate all exhibited substantial levels of cardiac fibrosis and cardiomegaly compared to age, gender and diet matched control ldlr single ko 's. surprisingly, the sr - b1/ldlr dko mice fed the western type diet for 12 weeks did not develop substantial cardiac fibrosis, although they did develop cardiomegaly to a similar degree as mice fed the other atherogenic diets. interestingly, we detected platelet accumulation in coronary artery atherosclerotic plaques in the atherogenic diet fed sr - b1/ldlr dko mice, and the abundance of platelet - positive atherosclerotic coronary arteries was highest in mice fed the paigen atherosclerotic diet (fig. whether the accumulation of platelets in atherosclerotic coronary arteries in the paigen diet fed sr - b1/ldlr dko mice is the result of thrombosis secondary to plaque rupture remains to be determined. together these data demonstrate the diet dependence of coronary artery atherosclerosis and cardiac fibrosis in the sr - b1/ldlr dko mice. in an experiment described in, an sr - b1/ldlr double ko mouse was fed the paigen atherogenic diet for 3.5 weeks. shown is a series of cross sections through the same coronary artery (passing through the septum of the heart), just below (a) and (b) and at (c) a bifurcation. (a) oil red o (lipid - red)/hematoxylin (nuclei - blue) stained section. (b) immunofluorescence staining for cd41 (platelet - red) ; counterstained with dapi (nuclei - blue). interestingly, we observed that the levels of coronary artery atherosclerosis did not correlate with the levels of aortic sinus atherosclerosis in the sr - b1/ldlr dko and ldlr single ko mice fed the five different diets. in particular, we observed similar levels of aortic sinus atherosclerosis in 22 week old normal chow diet fed sr - b1/ldlr dko mice and 13.5 week old sr - b1/ldlr dko mice that had been fed the paigen atherogenic diet for 3.5 weeks. the levels of aortic sinus atherosclerosis in these mice were also similar to those in ldlr single ko mice fed the 2% cholesterol supplemented diet for 12 weeks or fed the paigen atherogenic diet lacking sodium cholate for 10 weeks. despite similar levels of aortic sinus atherosclerosis, the only group of these four to develop severe coronary artery atherosclerosis were the sr - b1/ldlr double ko mice fed the paigen diet for 3.5 weeks - no coronary artery atherosclerosis was detected in ldlr single ko mice fed either of the high cholesterol diets and very little was detected in the sr - b1/ldlr dko mice fed the normal chow diet for the times indicated. this observation clearly demonstrated that coronary artery atherosclerosis was not merely a manifestation of increased overall atherosclerosis burden, but rather suggests different factors determined the development of atherosclerosis in the aortic sinus and coronary arteries of these mice. we observed that the majority of the non - atherosclerotic coronary arteries (75%-100%) analyzed in the atherogenic diet fed sr - b1/ldlr dko mice exhibited high levels of vcam-1 and icam-1 staining, whereas substantially fewer coronary arteries from similarly fed ldlr single ko mice were positive for vcam-1 and icam-1. this suggests that one consequence of inactivating sr - b1 expression is increased activation of coronary artery endothelial cells to express these adhesion molecules, which are known to mediate monocyte adhesion and recruitment into the vessel wall, and influence the sites of atherosclerosis development. these results are consistent with the finding that sr - b1 in endothelial cells mediates hdl dependent suppression of vcam-1 and icam-1 induction by tnf-. whether or not the increased vcam-1 and icam-1 in coronary arteries of the sr - b1/ldlr dko mice is the direct effect of a lack of sr - b1 expression in endothelial cells, or is an indirect effect of, for example, increased levels of il-6 and/or tnf- in circulation, or altered properties / levels of circulating lipoproteins, remains unclear. although total plasma cholesterol levels were lower in the atherogenic diet fed sr - b1/ldlr dko mice (corresponding to reduced hepatic lipoprotein production), the lipoproteins present in plasma of the dko mice were characterized by abnormally high unesterified cholesterol (reaching 50%-80% of total cholesterol) in the sr - b1/ldlr dko mice as compared to the lower levels of unesterified cholesterol (25%-27% of total cholesterol) in ldlr single ko mice, consistent with what has been previously described for sr - b1/apoe dko and sr - b1 ko / apoe - hypo mice. the effects of altered lipoprotein composition and structure in sr - b1/ldlr dko mice on endothelial cell activation, however, has not been examined directly. others have reported that hdl treatment of monocytes can suppress monocyte binding to vcam-1and icam-1 and monocyte adhesion to endothelial cells. consistent with this, we have demonstrated that monocytes from sr - b1 ko / apoe - hypo mice exhibit greater binding to vcam-1 and icam-1 and that restoration of sr - b1 expression in bone marrow - derived cells in sr - b1 ko / apoe - hypo mice resulted in reduced monocyte recruitment into atherosclerotic plaques and reduced aortic sinus and coronary artery atherosclerosis in mice fed the paigen atherogenic diet. in contrast, bm - specific restoration of sr - b1 expression did not restore altered lipoprotein cholesterol levels, raising the possibility that the protective effects reflected the direct activity of sr - b1 in monocytes or other bm derived cells. hdl triggers the activation (phosphorylation) of akt and akt dependent phosphorylation and activation of enos in endothelial cells both in culture and in vivo. this signaling process is dependent on sr - b1 and the multi - subunit adaptor protein, pdzk1, that binds to the carboxy terminal cytosolic domain of sr - b1 (fig. pdzk1 is required for the stability of sr - b1 protein in the liver and inactivation of pdzk1 in mice results in dramatically reduced levels of sr - b1 protein in liver hepatocytes, but does not alter sr - b1 protein levels in endothelial cells or macrophages. instead, in endothelial cells and macrophages, pdzk1 appears to be necessary for sr - b1 mediated hdl signaling. curiously, global knockout of each of pdzk1, akt1 or enos on the apoe ko background all lead to diet - induced coronary artery atherosclerosis and myocardial fibrosis, resembling the phenotype of sr - b1/apoe dko mice and atherogenic diet fed sr - b1/apoe - hypo and sr - b1/ldlr dko mice (although the degree of coronary artery atherosclerosis, myocardial fibrosis or effects on survival do not appear to be as severe as those observed in mice lacking sr - b1) (fig. 1). thus, krieger, kocher and colleagues demonstrated that feeding pdzk1/apoe dko mice a high fat, low cholesterol western diet (21% fat, 0.15% cholesterol) increased aortic sinus but not coronary artery atherosclerosis, but feeding the mice the paigen diet for 3 months induced substantial coronary artery atherosclerosis (with up to 40% of coronary arteries examined containing plaques that occluded more than half of the lumen) and myocardial fibrosis. these mice exhibited increased lipoprotein total cholesterol compared with similarly fed apoe ko mice but did not exhibit the increased lipoprotein unesterified cholesterol content characteristic of sr - b1 ko mice, possibly because low residual levels of sr - b1 protein in liver may have been sufficient to clear unesterified cholesterol normally. hdl treatment of endothelial cells and macrophages in culture induces akt phosphorylation and pharmacological inhibition of akt phosphorylation or knockout of akt1 prevents hdl signaling in these cells. dko mice have been reported to develop increased aortic atherosclerosis compared to similarly fed apoe ko mice, and, reminiscent of sr - b1-deficient and pdzk1-deficient apoe ko mice, also develop high fat diet induced occlusive coronary artery atherosclerosis. furthermore, high fat diet feeding (16 weeks) was also reported to induce coronary artery atherosclerosis (in addition to increased aortic atherosclerosis), cardiac fibrosis and reduced cardiac function in enos / apoe double ko mice, but not in apoe ko mice fed the same diet for up to 20 weeks. it is important to point out that the phenotypes reported for sr - b1/apoe, pdzk1/apoe, akt1/apoe and enos / apoe double ko mice do not match exactly : sr - b1/apoe dko mice develop occlusive coronary artery atherosclerosis spontaneously and rapidly (within 2 - 5 weeks) on normal chow diet while pdzk1/apoe, akt1/apoe and enos / apoe dko mice all require feeding with atherogenic diets (with different concentrations of fat, cholesterol and cholate). furthermore, a subset of high fat diet fed enos / apoe dko mice also develop abdominal aortic aneurysms, which has not been reported in sr - b1-deficient (sr - b1/apoe dko, or in sr - b1ko / apoe - hypo or sr - b1/ldlr dko mice fed atherogenic diet diets), or pdzk1/apoe or akt1/apoe dko mice fed atherogenic diets. to be sure, coronary artery atherosclerosis in mice does not solely arise from inactivation of components of the hdl / sr - b1/pdzk1/akt1/enos signaling pathway. substantial coronary artery atherosclerosis accompanied by myocardial infarction has also been observed in atherogenic diet fed apoe ko mice with macrophage overexpression of urokinase type plasminogen activator and in long term atherogenic diet fed ldl receptor / apoe double knockout mice. nevertheless, the finding that sr - b1, pdzk1, akt1 and enos are linked by hdl signaling and the observation that atherogenic strains that lack these factors develop either spontaneous or diet - induced coronary artery atherosclerosis, suggests the possibility that impaired hdl signaling may contribute to the development of coronary artery atherosclerosis in these mice. it is tempting to speculate that the observed spontaneous or diet - induced coronary artery atherosclerosis in apoe and/or ldlr ko mice also lacking sr - b1, pdzk1, akt1 or enos may reflect impaired hdl signaling in endothelial cells, resulting in increased endothelial cell activation in normally resistant coronary arteries. this is consistent with our observation of only very low levels of vcam-1 and icam-1 protein in coronary arteries of high fat diet fed ldlr ko mice, and substantially increased levels in coronary arteries of similarly fed sr - b1/ldlr dko mice. whether this is in fact due to impaired hdl signaling in endothelial cells (as predicted from in vitro studies cited earlier) or due other factors resulting from impaired global sr - b1 expression (e.g. altered lipoprotein structure, composition or levels ; altered systemic inflammation ; altered stress response etc) is currently unclear. resolution of this question awaits the analysis of mice with tissue selective ko or re - expression of sr - bi. hdl signaling via sr - b1, pdzk1 and akt1 appears to play an important role in homeostatic and/or atheroprotective responses in a variety of cell types including endothelial cells, macrophages and monocytes, hspc 's, smooth muscle cells and others. in endothelial cells, this signaling pathway leads to activation of enos and a variety of atheroprotective outcomes including proliferation, recovery after injury, increased barrier function and suppression of endothelial cell vcam-1 and icam-1 expression. global inactivation of sr - b1, pdzk1, akt1 and enos in mice predisposed to atherosclerosis due to inactivation of apoe and/or ldlr, all lead to spontaneous or diet - induced coronary artery atherosclerosis and myocardial infarction. these data suggest that hdl signaling via an sr - b1/pdzk1/akt1/enos axis plays an important role, at least in mice, in the normal resistance of coronary arteries to development of atherosclerosis. hdl signaling may explain, at least in part, the protective role that hdl exerts on human coronary artery disease. a better understanding of hdl signaling pathways, including the key cell types responsible for hdl signaling - mediated protection against coronary artery atherosclerosis in mice may provide important insights into strategies for therapeutic interventions in human coronary artery disease. | abstractatherosclerosis is a leading underlying factor in cardiovascular disease and stroke, important causes of morbidity and mortality across the globe. abundant epidemiological studies demonstrate that high levels of high density lipoprotein (hdl) are associated with reduced risk of atherosclerosis and preclinical, animal model studies demonstrate that this association is causative. understanding the molecular mechanisms underlying the protective effects of hdl will allow more strategic approaches to development of hdl based therapeutics. recent evidence suggests that an important aspect of the ability of hdl to protect against atherosclerosis is its ability to trigger signaling responses in a variety of target cells including endothelial cells and macrophages in the vessel wall. these signaling responses require the hdl receptor, scavenger receptor class b type 1 (sr - b1), an adaptor protein (pdzk1) that binds to the cytosolic c terminus of sr - b1, akt1 activation and (at least in endothelial cells) activation of endothelial no synthase (enos). mouse models of atherosclerosis, exemplified by apolipoprotein e or low density lipoprotein receptor gene inactivated mice (apoe or ldlr ko) develop atherosclerosis in their aortas but appear generally resistant to coronary artery atherosclerosis. on the other hand, inactivation of each of the components of hdl signaling (above) in either apoe or ldlr ko mice renders them susceptible to extensive coronary artery atherosclerosis suggesting that hdl signaling may play an important role in protection against coronary artery disease. |
for many mothers, giving birth to a child means a clear break from the community to which they had previously belonged. report that changes in mothers ' social situations from the puerperal period to the child - rearing period are related to depression through lack of a sense of belonging. similarly, hagerty and williams report that lack of a sense of belonging, loneliness, and social support are powerful predictive factors of depressive symptoms ; redeveloping positive interpersonal relationships is an imperative issue for postpartum mothers. immediately after giving birth to her child, a mother must build new relationships with people she meets through child - rearing and rebuild already - established relationships with friends and family as a mother. in modern japan, however, there is a continuing trend toward nuclear families combined with a declining birthrate, couples ' working patterns and lifestyles are diversifying, and economic disparity is becoming a problem. in current communities, the opportunities for child - rearing mothers to meet people with similar backgrounds in their neighborhoods are decreasing. furthermore, the spread of individualism makes socializing even more difficult. according to ogihara and uchida, while european - american individualism is an active individualism, japanese individualism has a tendency to preserve a steady sense of distance from other people or decide to end relationships. uchida. suggest that the spread of individualism in japan is decreasing happiness from interpersonal relationships. in this societal background, mothers are placed in situations in which they must intentionally build child - rearing communities to obtain the social support necessary for their personal mental health and child - rearing. according to a 2013 report, the yearly number of cases at child counseling centers related to child abuse exceeded 73,000. child - rearing isolated from regional communities is thought to be one of the factors. therefore, in previous research, the authors first clarified the people in the community mothers met through child - rearing. from this study, we found that mothers ' child - rearing communities must be intentionally made by mothers themselves and require a high degree of interpersonal relationship - building skills during opportunities for interaction. from exchanges among mothers in a child - rearing support program in england, peters and skirton also report on the difficulty of finding close friends who can be mutually supportive during child - rearing. the known skills for building positive interpersonal relationships are emotional and social intelligence [1113 ] and personal competence [1315 ]. at the core of these interpersonal relationship skills are empathy [16, 17 ] and trust. they are composed of skills including cognition and prosocial behavior [19, 20 ]. while referencing findings from these previous studies, this study clarified the skills mothers need to build positive relationships with the various people they meet during the initial stages of the child - rearing process. to clarify the skills mothers need to build positive relationships with the various people they meet during the initial stages of the child - rearing process, we used the qualitative descriptive study. the participants of this study were mothers who visited child - rearing support centers and salons. participant inclusion criterion was to be the mother of a child aged between 1 and 4 years. it did not matter whether they had spouse or not, and the number of children and whether the mothers felt anxious regarding their children 's health or not also did not matter. the total of participants was 24 mothers. when we began this study, we explained the study purpose and ethical considerations regarding the participants to the head of each facility orally and in writing. the study participants were chosen through staff mediation among the mothers who were attending the child - rearing salon that day. however, since social mothers tended to be selected through staff mediation, the author also recruited. at that time, five participants declined participation ; two mothers refused the request and three mothers were forced to decline because of their intensely crying children. ultimately, the author recruited four people and 20 people were recruited by staff at the child - rearing salon venue. since regional influences were expected to influence mothers ' exchanges with others, we performed sampling from various local municipalities. when selecting the study area, we recruited 13 people at four child - rearing salon and childcare centers located in a city with population of around 1.9 million, three people from a municipality with population of around 90,000, two people from municipalities with population of around 20,000, and six people at three facilities located in a municipality with population of under 10,000. we created an interview guide for asking about the mothers ' interaction experiences from the time they gave birth to their babies ' present ages. a small room adjacent to the salon was prepared for conducting interviews and space was secured to maintain privacy. in addition, baby - sitting support staff was arranged so the mothers would be able to focus on the interview. efforts were made to build an environment in which mothers could speak easily based on a relationship of trust between participant and researcher. interviews were recorded on an ic recorder and data was created with verbatim transcription. the length of the interviews ranged between 25 and 58 minutes depending on the participant, with an average of 36 minutes. in this study, the scope of theory development was focused on mothers ' interpersonal relationship - building skills and we aimed at theoretical saturation. we then performed additional four interviews and finished by sampling all 24 interviews, ensuring the soundness of the generated theory. in addition, because most mothers ' narratives were narratives of successful child - rearing, we examined reverse examples to investigate the generated theory 's properties and dimensions. for the reverse examples, we conducted interviews with five experienced public health nurses engaged in child - rearing support and inquired about cases in which the mother could not sufficiently build relationships with others. the fieldwork consisted of 37 visits to 18 parenting salons for participant observation of mothers ' behavior and interactions with staff and other mothers. we read the narrative data repeatedly in order to become familiar with latent mean in the mothers ' story. next, we extracted and coded data as initial codes focused on interpersonal relationship - building skills from the mothers ' narratives on feelings and behaviors during episodes of interaction with people they met through child - rearing. the initial codes were examined for similarities and integrated into secondary and final codes to create subcategories to explain specific concepts. to vividly describe the mothers ' interactions with others, the subcategories attempted to represent the tone of the mothers ' narratives to the extent possible. furthermore, for in vivo codes such as if our children are the same age, we can share the same problemsa comment expressed by many of the mothers the context of this comment 's background and the experiences and shared awareness of participants who made this comment were carefully explored. in this way, the characteristics of the subcategories such as bravery, volition, sympathy, and assessing the situation were carefully explored. furthermore, we determined the main subcategory through exploring the relationships between subcategories and categories. these categories are the mothers ' skills and are the core concepts that explain the theory in this study. researchers reviewed and checked codes and data as necessary in the process of categorization while conducting a posteriori analysis. during the theoretical coding process, we asked five mothers who participated in the interviews to do member checks to ascertain the appropriateness of our data interpretation, and corrections were made if necessary. the participant observations conducted over multiple sessions were beneficial in verifying the appropriateness of the generated concepts and exploring new perspectives. in addition, throughout all parts of the research process, we obtained reviews by fellow researchers knowledgeable about public health nursing with a wealth of qualitative research experience. the study 's purpose and ethical considerations were explained orally and in writing to the mothers who agreed to cooperate ; consent was received in writing. a small room adjacent to the salon was prepared for conducting interviews and space was secured to maintain privacy. in addition, baby - sitting support staff was arranged so the mothers would be able to focus on the interview. efforts were made to build an environment in which mothers could speak easily based on a relationship of trust between participant and researcher. this study was reviewed and approved by the research ethics committee of the faculty of health sciences of hokkaido university. the impact of the mothers ' backgrounds on the results was nullified in the end through the process of increasing the abstraction level in the categories. the participants included mothers who had one or more children ; however, in the interviews, they mainly described episodes in raising their first child. commonalities by categories were found, although there were differences in community exchanges by region of residence, dwelling history, and proximity to relatives. the mothers ' skills used to build positive relationships with others are shown in table 2. the representative subcategory of this category was for my child 's sake, i think i can gather courage and participate. the mothers knew about the childcare support centers and child - rearing salons but were worried about getting along with the group of other mothers and found it difficult to participate by themselves. therefore, even if they were unable to initiate conversations with other mothers, with the help of staff members they challenged themselves to have new encounters. this category comprised subcategories related to cognitive aspects of sociability such as i can generally build good relationships with anyone and subcategories related to behavioral aspects such as i try to be first one to say hello. within this category, there was a range of dimensions from positive to negative, such as i would actually like to talk more with the other mothers, but i am not good at smoothly developing conversation. for the mothers, perceptions such as it is difficult for me to maintain relationships if there are differences in our personalities and values existed as premises for initiating interaction with other people. to verify this mothers estimated the benefits of establishing connections with another person, such as i want to create relationships in which i can casually receive information when something happens to my child. they are also consciously thinking of ways to sustain their social relationships, as explained by the code if i remember the name of the other person 's child, it is easy for me to say hello when i see them in the neighborhood. i want to be in mutually caring relationships with friends was a concern for maintaining good relationships with each other. mothers were choosing conversation topics while calculating the closeness / intimacy with the other person. at the same time, they were determining the fine sense of distance that was neither too close nor too distant and were preserving that distance as they developed interactions. although troubles among children such as little fights over toys were common, other mothers ' attention to this trouble was an important element in building positive relationships among mothers. in japan, relationship - building between the wife and the husband 's parents is especially important. by having children, mothers felt an even stronger connection to their husbands ' families. mothers sensed that their in - laws wanted to see their grandchild regularly so they intentionally asked for their support. however, they sometimes held back, concerned about the burden on their in - laws ; while calculating the subtleties, they sought a positive relationship and sense of distance for both sides. they handled conflicts in child - rearing methods by being respectful of their in - laws ' thinking while taking care not to wound their pride. the skills mothers used to build positive relationships with people they met during child - rearing were explained by six categories. these categories are abilities and skills that are required in every step of interpersonal relationship. ability to strive for new encounters, ability to try to interact socially with others, and ability to choose compatible people are required at the first step of making friends in opportunities such as child - rearing support centers. the next step in developing relationships is keeping and developing the connection. ability to continuously maintain good relationships and ability to take action suitable to a situation are important skills in this step. according to kooker. self - management includes controlling self - destructive or impulsive emotions, striving for sincerity and honor in contact with others, and a goal - oriented mindset. we understood mothers also required these competencies. the relationship with parents - in - law, it is often said that getting married means marrying one 's husband 's family. thus, most mothers are required to build a good relationship with their parents - in - law. in fact, their parents - in - law provide concrete support such as financial aid. thus, ability to build positive relationships with parents - in - law may be an important skill in their child - rearing. in addition, barone. describe social network orientation as individual beliefs or attitudes that people have when seeking new networks for gaining support and estimating and determining how helpful accessing those networks would be. as described above, this study 's results contributed to providing a more concrete understanding and were compatible with existing theories. for today 's mothers, the skills elucidated in this study are necessary skills for being able to independently raise children so that they can smoothly accept support from others and create better personal child - rearing support. in addition, the categories confirmed cognitive and behavioral skills related to japan - specific values cherishing peace and harmony and maintaining a delicate sense of distance with others. the reality of weakening child - rearing support in local communities and support activities for rebuilding that support have become important issues not only in japan but also in other countries. the interpersonal relationship skills discussed in this study are both teachable and learnable [24, 25 ]. rather than attributing the responsibility of child - rearing to mothers individually, child - rearing support specialists are calling for support that helps develop these skills while accumulating successful relationship - building skills one at a time through child - rearing. for public health nurses involved in child - rearing support, this study 's results contribute to the framework of understanding mothers ' interpersonal relationship - building skills. the japanese government began a new policy in 2014, including child - rearing support center projects. this outcome is useful to find mothers who need support and how public health nurses can help those mothers. lastly, we describe study limitations. this study 's participants had no anxiety about their children 's health conditions and were healthy and relatively sociable mothers who were able to attend child - rearing support centers and salons of their own volition. therefore, more specific data may be extracted from cases with mothers raising children who are ill or disabled, mothers who are ill or disabled, and single mothers. to build relationships with others, in addition, we could not explore the relationship between each element of mothers ' interpersonal abilities, priorities, and so forth. this study clarified the skills mothers used to build positive relations with people they met during child - rearing. the mothers ' skills elucidated in this study are supported by previous studies and provided a more concrete understanding of these theories. in addition, for mothers, the skills elucidated in this study are necessary skills for independently raising children so that they can smoothly accept support from others and create a better personal child - rearing support team in communities that have recently been experiencing weakening interpersonal connections. moreover, this study confirmed cognitive and behavioral skills that relate to japan - specific values cherishing peace and harmony. | this study elucidated the skills of mothers who used to build positive interpersonal relationships with people they met through child - rearing. the research method was qualitative descriptive study. the participants were 24 mothers who had children between ages of one and four years. these participants were recruited at child - rearing salons and childcare centers located in various municipalities in japan. the survey period was from september 2013 to july 2014. the mothers ' interpersonal relationship - building skills were described by six categories : ability to strive for new encounters, ability to try to interact socially with others, ability to choose compatible people, ability to continuously maintain good relationships, ability to take action suitable to a situation, and ability to build positive relationships with parents - in - law. cognitive aspects of assessing and understanding the interactions that occur during exchanges and behavioral aspects guided by these cognitions were identified within these skills. this study contributed to providing a framework to understand mothers ' interpersonal relationship - building skills for public health nurses involved in child - rearing support. |
in the past 30 years, the global prevalence of obesity has increased among all age groups. obesity leads not only to an increase in adipose tissue mass but also to the infiltration of proinflammatory cells and secretion of inflammatory cytokines [1, 2 ]. therefore, obesity is characterized by low - grade inflammation in local and systemic sites as demonstrated by robust secretion of proinflammatory cytokines, including il-6, as well as active recruitment of leukocytes. substantial evidence supports the hypothesis indicating that inflammation may contribute to insulin resistance, which further induces a series of diseases such as diabetes, hypertension, fatty liver disease, and coronary heart disease, thereby threatening human health [4, 5 ]. autophagy includes three basic forms, namely, macroautophagy, microautophagy, and chaperone - mediated autophagy (cma). macroautophagy (henceforth termed autophagy) is a lysosomal degradation pathway, which can degrade the organelles, longevity protein, and lipid drops and thus provide energy for the body [7, 8 ]. when the body faces various pressures caused by acute stress, autophagy plays a key role in maintaining the stability of the internal environment, particularly in regulating apoptosis and resisting the invasion of pathogenic microorganisms. self - renew, repair, and differentiation of cells are important for metabolism and maintenance of energy balance. studies have shown that autophagic dysfunction is closely related to metabolic disorders, such as insulin resistance, diabetes, obesity, and osteoporosis. high mobility group box 1 protein (hmgb1) is a nonhistone nuclear factor and a highly conserved protein. hmgb1 can bind to chromosomal dna to adjust the refactoring of chromatin [11, 12 ]. hmgb1 is abundant in the vast majority of mammalian cells and plays a key role as a signal molecule extracellularly. hmgb1 can be passively released from necrotic cells or actively secreted from inflammatory cells [15, 16 ]. aseptic injury to cells increases the level of hmgb1 in serum and tissues. as such, hmgb1 is associated with low - grade inflammation diseases, such as obesity and type 2 diabetes. some research found that hmgb1 interacted with autophagy through its different receptors, outside the cells by receptor of advanced glycation end products (rage), within the nucleus through heat shock protein beta-1 (hspb1), and within the cytoplasm through becn1. however, little is known about how hmgb1, autophagy, and adipocytes interact to regulate adipocyte development and differentiation. the present research mainly focused on the effects of hmgb1 on autophagy and cell differentiation in adipocytes. antibodies were obtained from the following sources : hmgb1 and gapdh from abcam, lc3 from cell signaling, and p62 from proteintech group. the following reagents were purchased from sigma : 1-methyl-3-isobutylxanthine, dexamethasone, insulin, oil - red - o dye, and hematoxylin and eosin. rearing environment indoor temperature was controlled at 20c to 25c, relative humidity within 40% to 60%, lights 12 h every day, along with free drinking water in the cage. six - week - old male mice were randomly divided into two groups, namely, the normal - diet (nd) group and high - fat diet (hfd) group, with 10 mice in each group. mice were fed correspondingly with standard chow (10% kcal in fat) or hfd (45% kcal in fat) for 16 weeks. mouse epididymal adipose tissue samples were fixed in 4% paraformaldehyde and paraffin - embedded, then cut into 4 m thick sections, and deparaffinized in xylene and rehydrated in a descending ethanol series. the sections were stained with hematoxylin and eosin using standard pathologic procedures. finally, photomicrographs of all sections were taken by leica dm500 microscope. 3t3-l1 preadipocytes were maintained and cultured in dulbecco 's modified eagle 's medium (dmem) with existing 10% heat inactivated fetal bovine serum and 1% penicillin / streptomycin at 37c in 5% co2. 3t3-l1 preadipocytes were first cultured in a medium supplemented with 10 g / ml insulin, 1 m dexamethasone, and 0.5 mm 1-methyl-3-isobutylxanthine for 2 days. differentiated adipocytes were cultured in dmem containing 10% fetal bovine serum [1, 20 ]. to invest the effects of hmgb1 protein on autophagy in various differentiation periods of 3t3-l1 adipocytes, the adipocytes were treated with hmgb1 protein 24 h before differentiation. in detail, the 3t3-l1 preadipocytes were induced at 0, 2, 4, 6, 8, and 10 days, and the 0.2 g / ml recombinant hmgb1 proteins were added at days 1, 1, 3, 5, 7, and 9 and removed at days 0, 2, 4, 6, 8, and 10. 3t3-l1 preadipocytes were grown to approximately 50% to 70% confluence in a six - well plate and then transfected with 75 pmol hmgb1 sirna and control sirna by using lipofectamine 2000 in accordance with the manufacturer 's instructions. after transfection for 6 h, the medium was replaced with a normal medium. differentiated adipocytes were stained by oil - red - o, as follows : 0.5 g oil - red - o powder was dissolved in 100 ml of isopropanol overnight. the solution was filtered and stored in a brown bottle at 4c. after the medium was discarded, cells were then washed with pbs and stained with the oil - red - o dye solution for 1 h. subsequently, cells were washed with pbs and treated with 60% isopropanol for 5 min. finally, cells were washed and photographed under a microscope. 3t3-l1 cells were collected and lysed in ice - cold lysis buffer (5.0 mm tris buffer, ph 7.4, 150 mm nacl, 1% np-40, 0.1% sds, 0.5% deoxycholic acid, 1 mm edta, 1 mm pmsf, 2 g/l aprotinin, 2 g/l leupeptin, and 1 mm naf) for 40 min and then subsequently boiled and quantified. after the samples were resolved by sds - page for 2 h, proteins were transferred to nitrocellulose membranes at 200 v for 90 min. membranes were blocked in 5% nonfat milk for 2 h and then incubated with specific primary antibodies for 2 h or overnight at 4c. after being washed with tris - buffered saline with tween (tbst) thrice, membranes were incubated with peroxidase - conjugated secondary antibodies for 1 h. finally, membranes were washed with tbst and developed by enhanced chemiluminescence. blots were quantified using amersham imager 600 and analyzed with image j. total rna was purified using trizol reagent and converted into cdna by superscript iii reverse transcriptase. the sequences of primers for pcr amplification were as follows : gapdh : forward, 5-aggtcggtgtgaacggatttg-3 ; reverse, 5-tgtagaccatgtagttgaggtca-3 ; ppar- : forward, 5-ttttcaagggtgccagtttc-3 ; reverse, 5-tctgtgacgatctgcctgag-3 ; fabp4 : forward, 5-tccagtgaaaactttgatgattat-3 ; reverse, 5-acgcattccaccaccagtttatca-3. quantitative pcr analysis was performed using sybr select master mix in 7500 fast real - time pcr system. the thermal profile for real - time pcr was 50c for 2 min, 95c for 10 min, followed by 40 cycles of 95c for 15 s and 60c for 1 min, and then 95c for 30 s and 60c for 15 s. relative expression was calculated using the 2 method. the level of peripheral blood hmgb1 was detected in mice at 16 weeks in accordance with the elisa kit 's protocol. the spectrophotometry (od) value of the microplate reader was set at 450 nm wave length. one - way analysis of variance with bonferroni 's corrections was used for multiple comparisons. to determine the changes in hmgb1 and autophagy protein lc3 in epididymal adipose tissues in obese mice, male c57bl/6 mice were fed with hfd for 16 weeks. the mean body weight for each group is shown in figure 1(a). with the extension of time, after 16 weeks, the mean body weight of the hfd group was about 1.5 times that of the nd group. moreover, the epididymal adipose tissue of the hfd group increased notably (figures 1(b) and 1(c)). we also found that the adipocyte sizes in the hfd group were significantly larger than those in the nd group (figure 1(d)). hmgb1 was assessed in epididymal adipose tissue and serum by western blot and elisa, respectively. compared with the nd group, the hfd group showed that hmgb1 expression increased in both epididymal adipose tissues and peripheral blood of obese mice (figures 1(e), 1(f), and 1(h)). western blot was also used to detect the expression of autophagic protein lc3 in epididymal adipose tissues. the lc3ii protein markedly increased, reaching 3.29 times that of the normal - diet groups (figures 1(f) and 1(g)). these data suggested that hfd promoted hmgb1 production and activated autophagy in the epididymal adipose tissues in mice. to determine the functional role of exogenous hmgb1 in autophagy, 3t3-l1 preadipocytes were treated with 0.2 g / ml recombinant hmgb1 protein for 24 h, which did not affect the normal cell proliferation and hmgb1 protein expression in cells (figures 2(a) and 2(d)). however, compared with the control group, the treatment group showed that the expression of autophagic protein lc3ii increased (figures 2(a) and 2(b)), whereas the p62 protein expression decreased significantly (figures 2(a) and 2(c)). western blot analysis revealed that the knockdown of hmgb1 significantly reduced hmgb1 protein expression (figures 3(a) and 3(b)). furthermore, the lc3ii protein decreased obviously, whereas the p62 protein increased markedly compared with the control group (figures 3(c) and 3(d)). to further explore the effects of hmgb1 on autophagy in various differentiation periods of 3t3-l1 adipocytes, we added 0.2 g / ml hmgb1 protein in the differentiation stages of adipocytes for 24 h. oil - red - o staining was performed to detect intracellular lipid droplets. as shown in figures 4(a) and 4(b), 3t3-l1 preadipocytes were successfully induced into the adipocytes in the control group. however, after adding exogenous hmgb1 for 24 h, the lc3ii expression increased in the differentiation on the second and fourth days but decreased in the differentiation on the eighth and tenth days compared with the control group (figures 4(c) and 4(e)). on the contrary, the p62 protein expression decreased in the differentiation on the second and fourth days (figures 4(c) and 4(f)). finally, the expression of adipocyte differentiation related genes peroxisome proliferator - activated receptor- (ppar) and fabp (fatty acid - binding protein) 4 was measured. no significant difference in ppar- was found between the two groups (figure 4(g)), but the fabp4 expression significantly decreased in 4 days and increased in 8 days (figure 4(h)). these data indicated that the hmgb1 protein exerted different effects on autophagy and lipid metabolism in the early and late stages of adipocyte differentiation. white adipose tissue is primarily responsible for energy storage of the body, whereas brown adipose tissue is mainly responsible for energy degradation through uncoupling protein 1 (ucp1). obesity is characterized by an excess of white adipose tissue and leads to adipocyte dysfunction, thereby increasing the risk for insulin resistance, type 2 diabetes mellitus, and cardiovascular diseases [26, 27 ]. hmgb1 is produced as a damage - associated molecular pattern molecule (damp) by necrotic cells or activated immune cells. our data also confirmed the increased expression of hmgb1 in the adipose tissues and peripheral blood of mice induced by hfd. autophagy is a process which passes intracytoplasmic ingredients to lysosomes for degradation and provides energy to the body. autophagy not only is very important in the maintenance of cellular homeostasis but also participates in the metabolism of lipid droplets and lipogenesis. research showed that, in both in vivo and in vitro experiments, the inhibition of autophagy increases lipid storage. in addition, knockdown of autophagy genes atg5 and atg7 in adipocytes can improve insulin sensitivity and relieve obesity. the suppression of autophagy also reduced the expression of adipogenesis - related genes, such as ppar- and c / ebps. previous studies proved that the knockdown of atg7 decreased tg accumulation in 3t3-l1 preadipocytes, generated obese mice with decreased weight and white adipose mass, and then enhanced insulin sensitivity. some research found that hmgb1 activated an autophagic response to oxidative stress, and the loss of hmgb1 inhibited autophagy. we observed the change of autophagy in 3t3-l1 preadipocytes when hmgb1 was disturbed by specific small interfering rna. our results showed that autophagy was weakened when the expression of hmgb1 was suppressed in 3t3-l1 preadipocytes. hmgb1 also played a vital role in regulating autophagy in response to metabolic stress and oxidative damage. however, little is known about the connections between the hmgb1 protein and the change of autophagy in adipose tissues of mice. our results displayed that hmgb1 increased along with autophagy activated in adipose tissue of obese mice induced by hfd. this finding implied that activated autophagy may be a protective mechanism under cell stress state induced by hfd. to gain insights into the effects of hmgb1 on autophagy in adipocytes, in vitro studies were performed in adipocytes. 3t3-l1 preadipocytes were first stimulated with the recombinant hmgb1 protein to observe the expression of autophagy - related protein lc3 (microtubule - associated protein 1a/1b - light chain 3) and p62. a cytosolic form of lc3 (lc3i) is conjugated to phosphatidylethanolamine to form lc3-phosphatidylethanolamine conjugate (lc3ii), which is recruited to autophagosomal membranes [34, 35 ]. thus, the formation of lc3ii appears to be correlated with the induction of autophagy. p62 serves as a link between lc3 and ubiquitinated substrates, and the inhibition of autophagy correlates with the increased levels of p62. our results showed that lc3ii increased and p62 decreased when cells were treated with recombinant hmgb1 protein. this finding suggests that exogenous hmgb1 induced autophagy in 3t3-l1 preadipocytes. in addition, as small interfering rna knocked down the expression of hmgb1, autophagy appeared to be weakened. these data indicated that hmgb1 played an important role in regulating the autophagy in 3t3-l1 preadipocytes. we observed hmgb1 activated autophagy in the early differentiation within 2 and 4 days but weakened autophagy in differentiation after 8 and 10 days. presumably, the activation of autophagy resisted external stress in the early differentiation stage but was unable to resist this constant pressure in the late stage of differentiation. these results demonstrated that hmgb1 played a different role in the differentiation of 3t3-l1 adipocytes. several transcription factors, such as ppar-, are involved in the differentiation of preadipocytes into mature adipocytes. fabp4, a lipid chaperone, is expressed in adipocytes and plays an important role in the regulation of insulin sensitivity. this finding was consistent with the expression of lc3, which suggested that hmgb1 regulated autophagy and further altered lipid metabolism in adipocytes. these results not only provided a new theoretical basis for hmgb1 and autophagy in adipocytes but also presented a new target for the treatment of obesity and its related metabolic diseases. | high mobility group box 1 protein (hmgb1) is a molecule related to the development of inflammation. autophagy is vital to maintain cellular homeostasis and protect against inflammation of adipocyte injury. our recent work focused on the relationship of hmgb1 and autophagy in 3t3-l1 cells. in vivo experimental results showed that, compared with the normal - diet group, the high - fat diet mice displayed an increase in adipocyte size in the epididymal adipose tissues. the expression levels of hmgb1 and lc3ii also increased in epididymal adipose tissues in high - fat diet group compared to the normal - diet mice. the in vitro results indicated that hmgb1 protein treatment increased lc3ii formation in 3t3-l1 preadipocytes in contrast to that in the control group. furthermore, lc3ii formation was inhibited through hmgb1 knockdown by sirna. treatment with the hmgb1 protein enhanced lc3ii expression after 2 and 4 days but decreased the expression after 8 and 10 days among various differentiation stages of adipocytes. by contrast, fabp4 expression decreased on the fourth day and increased on the eighth day. hence, the hmgb1 protein modulated autophagy - related proteins and lipid - metabolism - related genes in adipocytes and could be a new target for treatment of obesity and related metabolic diseases. |
nursing is a professional, which because of its stressful nature, being faced with unforeseen situations, shifting, organizational and personal factors, effect on mental and physical health of these practitioners. different studies has reported that in comparison with the mental and physical health of people of our country, the mental and physical health of this group of health care workers is a lower level. the current study sought to determine the relationship between anxiety disorders and specific types of sleep quality complaints in a large epidemiological sample and among the various anxiety disorders assessed in the current study, most had a moderately strong relationship with sleep problems. among the other variables of work environment thermal stability also depends on different factors such as physical activity, garment and environmental factors (such as dry bulb temperature, radiant temperature, air movement speed and air moisture). thermal comfort based on ansi / ashrae -ss 1981 standard is described as a condition which in, a human being is mentally satisfied with thermal condition. mental satisfaction of environmental conditions of the work place is one of the issues that should be considered, because care of patient needed to especial air condition so indoor air rarely circulate without door fresh air, this poor ventilation can be unpleasant for nurses and other health care workers. hence, they should be noticed seriously. among the problems of night shift nurses,, nursing has been in the spot light, because of its work injuries. in research, which was done on 2372 nurse in baltimore, usa, it was shown that more than one - fourth of nurses work about 12 hour that this more work hours continually lead to mental and physical fatigue and finally this factor is the negative effect on the sleep quality. legar shows that people with sleeping problems receive 7 times more injuries related to people who have enough sleep. the one important factor that can influence comfort level in buildings can be environmental factors. nurses in comparison with others, experience disorders such as disturbance in sleep quality, physical and mental tension, depression, job stress and other problem. in a study, effects of work - sleep cycle on physical health among shift nurses, the results determined that the night shift nurses contract physical illnesses more than nurses who work in day light. in another research, results showed that, there is a magnificent difference between the sleep quality of the night shift and fixed shift nurses. hence, this study was done with the purpose of determining the effects of state anxiety and thermal conditions on sleep quality and eye fatigue in shift work nurses. subjects were chosen voluntarily and simple randomly, which their average (standard deviation) of age, weight, height, body mass index and work record were 30.75 (6.42) years, 74.04 (13.15) kg, 174.65 (8.38) cm, 24.17 (3.27) kg / m and 9.7 (3.5) years respectively. the inclusion criteria were having at least 1 year work history, lacking of cardio vascular diseases, respiratory diseases and not receiving any sedatives drug. exclusion criteria was not willing to corporate or not filling out the questionnaires completely (two of the participants were removed). this cross - sectional research was done on the night shift nurses in isfahan al - zahra hospitalin 18 nursing work stations in 2012. in the present study, state anxiety measured by state - trait anxiety inventory that has the likert scale and withgrade4.this questionnaire had high intra - class correlation coefficients and the average reliability ration different groups. visual fatigue level measured by eye fatigue questionnaire omprising 15 questions that they has been designed in (0 - 10) likert range. sleep quality measured by pittsburg sleep quality index (psqi) that sensitivity and reliability of psqi were 89.6%and 86.5%respectively. this questionnaire yields 7 scores for the individual 's over all description of sleep quality, the period of sleeping, the period of effective sleep, effective sleep, sleep disorders, dose of receiving opiate, daily performance disorders scales and one overall score. the score of each questionnaire has been considered from 0 to 3 in which, 0, 1, 2 and 3, imply normal condition, presence of a little mean and intense problem respectively. data measured between 9 p.m. and 1 a.m. data analyzing was performed by descriptive statistics, pearson correlation analysis and regression analysis. subjects were chosen voluntarily and simple randomly, which their average (standard deviation) of age, weight, height, body mass index and work record were 30.75 (6.42) years, 74.04 (13.15) kg, 174.65 (8.38) cm, 24.17 (3.27) kg / m and 9.7 (3.5) years respectively. the inclusion criteria were having at least 1 year work history, lacking of cardio vascular diseases, respiratory diseases and not receiving any sedatives drug. exclusion criteria was not willing to corporate or not filling out the questionnaires completely (two of the participants were removed). this cross - sectional research was done on the night shift nurses in isfahan al - zahra hospitalin 18 nursing work stations in 2012. in the present study, state anxiety measured by state - trait anxiety inventory that has the likert scale and withgrade4.this questionnaire had high intra - class correlation coefficients and the average reliability ration different groups. visual fatigue level measured by eye fatigue questionnaire omprising 15 questions that they has been designed in (0 - 10) likert range. sleep quality measured by pittsburg sleep quality index (psqi) that sensitivity and reliability of psqi were 89.6%and 86.5%respectively. this questionnaire yields 7 scores for the individual 's over all description of sleep quality, the period of sleeping, the period of effective sleep, effective sleep, sleep disorders, dose of receiving opiate, daily performance disorders scales and one overall score. the score of each questionnaire has been considered from 0 to 3 in which, 0, 1, 2 and 3, imply normal condition, presence of a little mean and intense problem respectively. data measured between 9 p.m. and 1 a.m. data analyzing was performed by descriptive statistics, pearson correlation analysis and regression analysis. the mean score (standard deviation) of sa was 40.98 (18.56) with range 5.00 - 96.67. the mean score (standard deviation) of sleep quality was 63.64 (19.35) with range 2.38 - 95.24. the mean score (standard deviation) of thermal comfort was 53.47 (17.15) with range 16.67 - 83.3. the mean (standard deviation) of eye fatigue among these people was 29.06 (24.28) with range 3.65 - 78.00. pearson correlation between state anxiety and the other variables represent, areas follow : pearson correlation between state anxiety and eye fatigue was 0.57(p < 0001) [figure 1 ]. pearson correlation between state anxiety and sleep quality was 0.664(p < 0001) [figure 2 ] and correlation between state anxiety and thermal comfort was 0.276(p = 0.016) [figure 3 ]. pearson correlation between thermal comfort and eye fatigue shows reverse relation 0.38(p = 0.002) [figure 4 ]. pearson correlation between thermal comfort and sleep quality was 0.24(p = 0.033) [figure 5 ], pearson correlation between eye fatigue and sleep quality, which indicates positive and moderate relation was 0.66(p < 0.001) [figure 6 ]. scatter plot between state anxiety and eye fatigue in shift work nurses scatter plot between state anxiety and eye fatigues in shift work nurses scatter plot between state anxiety and thermal comfort in shift work nurses scatter plot between thermal comfort and eye fatigues in shift work nurses scatter plot between thermal comfort and sleep quality in shift work nurses scatter plot between eye fatigues and sleep quality in shift work nurses the relationship between psychological problems such as state anxiety and mental health workers is a very complex so that studies to examine these relationships are essential. however, the results of this study showed that between shift work nurses sleep quality and state anxiety, as a psychological problem, was negatively correlated. this means that with an increase in anxiety levels, quality of sleep declines that these results are consistent with ramsawh. negative adverse effect of anxiety can be observed on the thermal comfort of shift work nurses. this relationship can be theoretically confirmed. because anxiety as a state of fear, distress and confusion often associated with physiological reactions such as increased heart rate, increased respiration and evaporation, which ultimately causes the thermal discomfort in anxious individuals (garfingel). and another results of the study can be noted that a direct connection between increased levels of eye fatigue and increase anxiety. this association may be due to a lack of good sleep in shift work nurses, which in turn has enhances eye fatigue. sleep disorders is one of the most important issues among shift nurses, especially night shift nurses. high stress and disarranged shifts are important effective factors on sleep disorders and sleep quality. the results of the current research like the others (escrib.), which have conducted before, indicate that night shift nurses (participant in this research) suffer from sleep disorders, is one of the researches with the same results, which describes the poor sleep quality of shift nurses very clearly. one of its reasons could be lacking an adequate sleep model and this in adequate and disordered sleep model may have originated in personal and environmental factors. among the important and interesting results of this study is the pearson correlation relation between eye fatigue and sleep quality, which is a reverse and significant relation. this relation indicates that the less level of eye fatigue decreases, them or level of their sleep quality increases. atmospheric condition is one of the other environmental factors, influencing physical and mental health conditions of people. pearson correlation results between dependent variables of this study (including eye fatigue and sleep quality) and nurses thermal comfort indicates that increase in thermal comfort contributes to decrease in eye fatigue of nurses and improvement in their sleep quality ; therefore, it can be inferred that we can decrease eye fatigue of shift nurse sand improve their sleep quality by improving thermal condition of the workplace. finally, it is presumed that the relationship between eye tiredness and sleep quality can be a mutual relationship. based on these results, it can be concluded that improvement of thermal conditions and reduce state anxiety level can be reduce eye fatigue and increase the sleep quality of shift work nurses. | psychological problems as state anxiety (sa) in the work environment has negative effect on the employees life especially shift work nurses, i.e. negative effect on mental and physical health (sleep quality, eye fatigue and comfort thermal). the purpose of this study was determination of effects of state anxiety and thermal comfort on sleep quality and eye fatigue in shift work nurses.methods:this cross - sectional research conducted on 82 shift - work personnel of 18 nursing workstations of isfahan hospitals in 2012. to measure the sa, sleep quality, visual fatigue and thermal comfort, spielberger state - trait anxiety inventory, pittsburg sleep quality index, eye fatigue questionnaire and thermal comfort questionnaire were used respectively. the data were analyzed with descriptive statistics, student test and correlation analysis.results:correlation between sa and sleep quality was 0.664(p < 0001), pearson correlation between sa and thermal comfort was 0.276(p = 0.016) and between sa and eye fatigue was 0.57 (p < 0001).conclusion : based on these results, it can be concluded that improvement of thermal conditions and reduce state anxiety level can be reduce eye fatigue and increase the sleep quality in shift work nurses. |
despite all preventive and therapeutical efforts, melanoma is still the most aggressive and deadliest skin cancer especially in persons of fair complexion. to a certain extent, primary prevention campaigns already achieved an earlier diagnosis of thinner tumors with a better prognosis. incidence rates are nonetheless increasing worldwide mainly due to unreasonable sun exposure habits, especially in young adults. once diagnosed, prognosis and therapy is stratified so far by several clinicopathological risk factors such as tumor thickness, sentinel lymph node status, ulceration, and the recently added mitotic rate. in view of an often unpredictable rather heterogeneous biological behavior mainly in > 4 mm thick (stage iic) or locally advanced melanoma (stage iii), the ajcc classification remains of limited clinical relevance in particular for these high risk patients. moreover, we currently do not have reliable tissue biomarkers that mark the disease of the individual patient for progression or complete remission. at the same time, an enormous amount of basic research within the last decade has dramatically changed the molecular understanding of melanoma. proof of several specific genomic key mutations such that braf could not only be causally linked to disease progression but also gave rise to new, highly effective therapies targeted specifically at those mutated molecules. while the multistep carcinogenesis of melanoma is still too little understood in its complexity in order to foresee when, how, and what kind of mutation develops in an invasive or metastatic tumor, genome - wide genetic analysis of primary or metastatic tumors will undoubtedly change future classifications and subsequent treatment algorithms. but are standard clinical prognostic parameters such as age, location, and metastasis already outdated ? could dermatopathology, the current cost - efficient diagnostic gold standard, consequently be redundant ? will we possibly be able to correlate certain histomorphologic features to specific genetic aberrations and their consecutive pathological or compensatory molecular cascades in order to recognize, treat, or even prevent the systemic metastasic impact of this tumor in our patients ? these important questions arise and may contribute to a better classification of melanoma patients. with the focus on their metastatic potential, our review summarizes the current knowledge of genetic, as well as molecular features of malignant melanoma and examines their possible correlation. moreover, we discuss the clinical implications as well as current therapies that may target these new hallmarks of melanoma. a growing body of evidence has already addressed melanoma as an umbrella term for several biological distinct subtypes as a result of multiple causative genetic aberrations, impaired pathways, or epigenetic changes. epidemiology, in contrast, strongly indicates that uv - induced dna damage is the primary cause of melanoma development, even though certain regions in which melanoma subtypes occur, such as mucosal or acral tumours, are not typically exposed to ultraviolet light. numerous studies about phenotypic risks such as age, gender, and skin type favour sun exposure as the major cause for thinner tumors of less incidence in young patients (4 mm [40, 41 ]. despite its inclusion in the ajcc classification already in 2001, the knowledge about why, when, and for what reason ulceration occurs and how it favours tumor progression is at best theoretical. studies concentrated on width, depth, and proportion of ulceration, its association with mitotic rate or vascular involvement, and tumor vascularity. the most plausible hypothesis that considered ulceration as a consequence of tumor proliferation, and therefore secondary epidermal thinning and contact ulceration, has been reevaluated. hence, ulceration challenges the control functions of keratinocytes, melanoma cells are enabled to transform more easily, therefore favoring tumor progression. more common in melanoma than in any other neoplasia, regression is defined as a partial or complete disappearance of the tumor without treatment. due to the loss in pigmentation in terms of a blue or grey - whitish discoloration, it is clinically highly apparent in this particular tumor entity. with an incidence of approximately 1035% of patients with primary malignant melanoma, associated with variable degrees of inflammatory and stromal changes, this particular phenomenon proceeds from an early dense lichenoid infiltrate of lymphocytes and dermal edema to a late fibrosis and a usual melanosis within a thickened papillary dermis. especially when the tumor is pigmented, melanophages as the histopathological telltale sign are often present. although the current understanding of regression is clearly that of an immune - mediated, cancer - autonomous process, neither its biological significance nor the underlying molecular or genomic aberrations are so far recognized. possible explanations vary from an increased t - cell response, an inhibited angiogenesis, to a forced apoptosis of tumor cells [53, 55 ]. while a positive host immune response may supersede wider excision margins or sentinel lymph node biopsy [56, 57 ], regression may, however, on the other side indicate a formerly deeper infiltrating tumor and thus a lower threshold for sentinel lymph node biopsy. especially in thin melanomas < 1 mm, regression as a left - over of a presumably thicker tumor therefore still leads to wider surgical margins and a lower threshold for sln biopsy. the most convincing, although unproven, hypotheses for a regression - driven tumor progression so far are the hammon 's effect, which postulates a natural selection of aggressive residual tumor clones as a result of regression [59, 60 ] and bastian 's telomere crisis, which argues that a massive senescence and cellular apoptosis equally favor the selection of genomic aberrations and therefore progression. future epidemiologic studies investigating the impact of regression of the primary tumor for the prognosis of melanoma are certainly required to further investigate those intriguing details. tumor proliferation as defined by mitotic rate has been confirmed as an independent adverse prognostic parameter in many solid neoplasia including melanoma [6164 ]. due to the fact that its increase is significantly correlated with reduced survival rates primarily within melanoma of less than 1 mm tumor thickness, it has recently replaced clark 's level as the primary criteria for defining the subcategory of t1b in ajcc classification 2009 [3, 65 ]. the lack of a universally agreed approach of how to document mitotic figures led to many studies that did not include mitotic rate in their analyses up to now. as recently detailed by the ajcc manual, starting with dermal areas that contain most mitoses (so - called hot spots), and extending the approach later to adjacent fields up to 1 mm, now allows for the first time a reproducible assessment although this approach is time consuming to the dermatopathologist. so far, only two sorts of genes and their pathways are identified to be overrepresented in melanoma with higher mitotic activity. replication origins firing (rof) genes such as mcm4 and mcm6 as well as the oncogene securin are strongly correlated with metastases and therefore poorer prognosis even after considering other prognostic parameters such as sex, age, location of the primary, thickness, and ulceration [29, 67 ]. as much effort has been made in defining the biological relevance of these dermatohistopathological parameters, they can not reliably distinguish the metastatic behaviour of certain subgroups such as stage iic melanoma. moreover, the exact diagnosis in some cases of melanoma might be problematic altogether as the individual assessment of these criteria differs among pathologists. in addition, benign melanocytic proliferations such as atypical nevi can also display a number of those features, given that routinely performed immunohistochemical markers, for example, s100b and hmb-45 are of little help in distinguishing nevi from melanoma. taken these reflections into account, a more molecular understanding of melanoma might therefore be desirable. inevitably, the understanding of the molecular basis of malignant melanoma has to be further improved to identify the critical drivers and passengers during oncogenesis of melanoma [70, 71 ]. the core issue obscuring the best possible treatment of malignant melanoma is still its unpredictable pattern of progression and metastasis. well - established prognostic parameters alone or in combination are so far not effective enough to accurately predict the outcome for every individual patient. biologically distinct as malignant melanoma is, the greatest therapeutical potential lies without doubt in the understanding of what key indicators influence the course of the disease most, regardless whether they may be genetic, possibly molecular, least likely clinical, or even combined, and therefore predispose for the risk of systemic disease. the multistep process of carcinogenesis in malignant melanoma is, however, complex and at best only in part understood. a number of excellent reviews have summarized the exciting developments in the understanding of this tumor in depth. to date, four pivotal, nonlinear, and rather netlike interwoven defective signaling pathways have been implicated. the following scheme gives a simplified overview of these pathways with their most common aberration and the percentage of mutations detected within these signaling pathways. certain rare subtypes such as uveal melanoma also have been found to have mutations in gnaq or gna11 that also lead to constitutive activation of these signaling pathways (figure 1). proven to be one of the most frequently mutated cascades in melanoma, the mitogen - activated protein (map) kinase pathway shows several pathologically activated mutations that may contribute to malignant transformation. the most common mutations or cytogenetic amplifications occur in the braf, the kit, the nras, or the cdkn2a genes. in 812% of familiar malignant melanoma alone, mutations of cdkn2a gene that are linked to chromosome 9p21 arise [75, 76 ]. unlike regular sites of cutaneous melanoma, uncommon subsets of melanocytic neoplasia such as uveal melanoma or malignant blue nevus lack frequent oncogenetic mutations in ckit, nras, or braf [7779 ]. notwithstanding other oncogenes such as the alpha subunit of a class of heterotrimeric gtp - binding proteins (gq), namely gnaq and gna11, are activated. hypermorphic mutations in those genes were found to contribute to skin darkening and therefore melanocyte biology in mice. proven to occur early in progression, they seem, however, not to be related to clinical outcome so far [81, 82 ]. when active, gnaq and gna11 alternatively upregulate the map kinase pathway. operating downstream of several g - protein coupled receptors, gna11 has presumably a more potent adverse effect than gnaq in locally advanced or metastasized tumors although overall survival did not differ. gnaq mutations are, however, considered to be more sensitive to the upcoming therapeutical mek inhibition. cross - linked via nras, the map kinase cascade also initiates the pi3k and thereby the pi3k signaling pathway, another defective cascade found in a large percentage of melanomas. apart from nras, either deletion of pten or overexpression of akt mainly lead to the stimulation of mtor, a central regulator of cell growth and proliferation that has raised substantial interest in this signaling pathway in melanoma. of central importance for benign as well as malignant melanocytes, mitf and its cascade were found to represent a central transcription factor that regulates differentiation in the pigment cell system. in addition to -msh and acth that activate mitf via the mc1r, it is also physiologically regulated by map kinase and pi3k signaling pathway [85, 86 ]. in the development of melanoma, however, an optimized level of mitf as an oncogene for proliferation and survival of tumor cells needs to be maintained by braf. insufficiently high or low expression of mitf results in tumor - protective differentiation, cell cycle arrest, and subsequent apoptosis. mitf amplification, single based mitf substitution and even mutation of its regulator sox10 have all been proven lately to be causative for altered mitf function in both primary and metastatic melanoma [89, 90 ] underscoring the involvement of mitf in melanomagenesis. although mutations of the -catenin gene and apc have already been detected, the wnt signaling pathway has not been extensively implicated in melanoma development this far, due to the fact that defective -catenin is rarely identified although it clearly acts as a melanoma - specific antigen [91, 92 ]. under physiological conditions, wnt - signaling proteins bind to frizzeled receptors, thereby stabilizing -catenin with subsequent release from a multiprotein complex. it then accumulates in the nucleus and initiates as a coactivator the transcription of a multitude of target genes. in case of genetic mutations of -catenin, such as in malignant melanoma, it forms a complex with lef-1 (lymphoid enhancer - binding protein), which in turn leads to malignant transformation of the cell [93, 94 ]. in particular wnt-2, a survival factor in human carcinogenesis, has lately become focus of intensified research as a biomarker and a potential target to subclassify and treat malignant melanoma [96, 97 ]. besides the main canonical wnt signaling pathway, a variation of the so - called noncanonical pathway with altered receptors and enzymes, and even a signal regulated in a paracrine manner (the so - called notch cascade), specific inhibitors in terms of small molecular antagonists or rna aptamers have nonetheless been developed to potentially target this pathway, an intruiging possibility given the important role of this pathway in the so - called tumor - initiating cells in other tumor entities. wnt2 has also been found to be overexpressed in malignant melanoma. of therapeutic interest, a specific anti - wnt-2 monoclonal antibody has been proven to inhibit wnt signaling and subsequently induce apoptosis. the complexity of these crosstalking circuits is increased even more by the fact that one genetic alteration is not enough to make a melanoma. cumulative genetic instability gradually induces arbitrary genomic aberrations that lead to uncontrolled replication and growth, inhibition of apoptosis, and finally the ability to invade and metastasize due to a darwinian - like selection process of the tumor cells [101103 ]. considering further stem cell - determined, epigenetic, tumor - environmental, or immunologic changes, the variety of possible influencing factors on the classic hallmarks of cancer is multiplied beyond measure, and the knowledge of critical constitutional and somatic genetic parameters is not yet complete. recent progress in the understanding of genetic aberrations in malignant melanoma has likewise prompted significant efforts in defining so called epigenetic changes that accompany the malignant transformation in melanocytes. defined as any changes in gene expression that are not achieved through alterations in the primary sequence of the genomic dna, epigenetics influence a wide range of alternative gene functions such as cell cycle regulation, cell signaling, differentiation, dna repair, apoptosis, invasion, metastasis, angiogenesis, and immune recognition. although their precise contribution to tumor progression is still unknown, they were proven to efficiently restore the expression of aberrantly silenced genes and thereby to reestablish silenced signaling pathway function. the most clearly identified epigenetic mediators so far are the methylation of dna in the context of cpg dinucleotides, the posttranslational changes of histone proteins and, though less characterized, the influence of micrornas (mirnas). the reactivation of sleeper genes and the maintenance of these epigenetics aberrations requires functioning enzymes such as dna methyltransferases (dnmt) or histone deacetylases (hdac), and histone methyl transferases (hmt), respectively. in case of dna methylation, three different dnmts are implicated in new methylation patterns with gene - specific hypermethylation on the one hand as well as genome - wide hypomethylation on the other. in addition to genetic alterations, epigenetic dna hypermethylation is, therefore, a complementary, frequent, and important mechanism to inactivate tumor suppressor genes such as cdkn2a. while hypermethylation silences tumor suppressor genes, global hypomethylation might this could lead to a diversified and significantly impaired methylation disbalance of multiple genes that eventually initiates genomic instability, tumorigenesis, and cancer progression [106, 107 ]. as common as this phenomenon of hypomethylation is in many tumors, little is known so far about target genes regulated by this event in melanoma. similar to the discussion of driver and passenger mutations in genetic aberrations, the biological significance of several identified aberrantly hypomethylated epigenetic genes, for example, cancer - testis antigen (ctas), prame, and mage continue to be poorly understood. nonetheless, given the broad relevance of these pathways in almost every tumor entity, substances have already been developed for therapeutical approaches, and the epigenetic status of certain genes may potentially predict the biological function and could serve as a biomarker [110, 111 ]. along with dna methylation patterns, initial studies about histone acetylation have addressed a possible role in melanoma development and progression. in particular hypoacetylation - mediated downregulation of cdkn1a and, similarly, proapoptotic proteins such as bax, bak, bid, and bim may profoundly influence cell cycle and apoptosis of the cell and thereby lead to tumor progression or therapeutic resistance [113, 114 ]. in the demanding packing and outpacking machinery of genomic dna into nucleosomes and chromatine, respectively, at least three groups of histone acetyltransferases (hat) and 18 identified histone deacetylases (hdac) are involved thus far. complicating this picture, histone methyl transferases (hmts) modulate the chromatin compaction grade of the dna that finally determines the transcriptional status of target genes. in contrast to dna methylation, the knowledge of the posttranslational aberration of histones is altogether scarce and mainly gathered indirectly by treatment results of hdacs thus far. promising results of multiple hdac inhibitors concerning vascular endothelial growth factor (vegf), generation of reactive oxygen species (ros), cell death, senescence, and especially intrinsic as well as extrinsic apoptosis in the transformed cells have already been described in various solid tumor entities [117119 ]. proapoptotic stimuli are, however, known to be less effective in human melanoma cell lines. recently discovered key mediators such as the cleavage of poly - adp ribose protein (parp) and hdac inhibitors like the short fatty acid vpa have led already to promising results with antitumor activity in combination therapy with anthracyclines in melanoma. the level of understanding of the molecular mechanism in histone posttranslational modifications has yet to become more refined to predict the outcome of such promising therapies in subgroups or individual melanoma patients. the most recently discovered players in epigenetic regulation have been noncoding microrna (mirna). once transcribed in the nucleus and further processed by several intermediate stages, they are finally incorporated into a rna - induced silencing complex that recognizes their target mirna. in addition to more than a hundred currently confirmed mirnas, more than 1000 mirna have been predicted by bioinformatics. despite the limited data available so far, mirnas are proven to play pivotal roles in the epigenetic pathogenesis of human cancer. as proof of principle, several key mirnas especially the lack of an inhibition by mir-137 and mir-182 was found to result in an overexpression of mitf, a master regulator in benign melanocytes as well as melanoma. on the other hand, overexpression of mir-182 mir-34b, mir34c, mir199a, and mirnas involved in the expression of the oncogene met modify target gene expression in accordance with the stage of cancer development. considering the fact that mirnas themselves are also targets of epigenetic regulations as, for example, mir-34a, which is proven to be silenced by a cpg - mediated methylation in up to 60% of primary melanomas, further studies are mandatory to define their role in melanoma biology more precisely. as one of the most devastating forms of cancer in terms of life expectancy and outcome, metastatic melanoma was until recently an almost intractable disease. this was largely explained by the fact that mono- or polychemotherapy, the standard of care for over 30 years, only benefits a very small subset of patients. with the discovery of an activating mutation of braf in 5060% of all melanoma, with 90% of these tumors carrying a substitution at v600, sorafenib, a multikinase inhibitor and one of the first targeted therapies in clinical testing, has unfortunately shown little efficacy in patients with activated map kinase pathway (and therefore braf positive) patients. consequently, more selective braf inhibitors were subsequently tested in clinical trials, which in case of vemurafenib (also known as plx 4032) and gsk2118436 have demonstrated unprecedented clinical results in metastatic malignant melanoma harboring braf mutation [7, 128, 129 ]. within two weeks, the majority of patients stated a symptomatic improvement, and approximately 60% showed an objective response according to response evalutation criteria in solid tumors (recists). in the subsequent extension phase of the trial, 81% patients demonstrated tumor regression, and the progression - free survival was at an average of 7 months. dose - dependant adverse events like rash, photosensitivity, fatigue, and arthralgia were well managed by either dose reduction or by the termination of the treatment if necessary. apart from pyrexia, rash, fatigue, headache, nausea, and vomiting, severe adverse events such as squamous cell carcinoma and keratoacanthoma were reported. a series of publications, however, quickly discovered novel mechanisms that paradoxically activate the map kinase pathway in the presence of braf inhibitors [130, 131 ]. due to three isoenzymes of raf (a - raf, b - raf, and c - raf), the inhibition of one of them such as b - raf can induce a compensatory transactivation of c - raf, which in turn activates downstream mek and the subsequent pathway [130, 132 ]. as a consequence of gatekeeper mutations that sterically prevent the inhibitor binding to the active side in raf, the crossactivation of c - raf is not always initiated and even to a certain extent inhibited by the given drug. atp competitive inhibitors for instance are supposed to stabilize the interaction between b - raf and c - raf. besides c - raf as a paradoxical bypass of b - raf, other erk - dependent mechanisms such as n - ras mutation, cox overexpression, or mek1 mutations contribute to an acquired resistance to b - raf. complicating the picture, even erk - independent alterations like pdgfr overexpression, igf1r activation and pten loss have been identified to reactivate erk signaling in b - raf mutant tumors [134, 135 ]. although the benefit of b - raf inhibition as monotherapy has been sufficiently confirmed, rapidly occuring secondary resistance mechanisms in tumors will most likely favor combination therapies targeting other genetic hot spots in melanoma such as mek, ras, and kit. ras, in particular n - ras mutations, occur in approximately 1525% of malignant melanoma. they inhibit the gtpase - mediated activity of ras and thus keep it in an continuously active state. demanding as task to develop an agent is that would rival gtp, several interacting pathways such as map kinase or pi3 kinase seem to play an important role in the n - ras mutant subset of melanoma [137, 138 ]. mutually exclusive to b - raf v600e mutation, nras mutations have been shown to be sensitive to mek - targeted therapies particularly in combination with pi3k, akt, or mtor inhibitors. kit mutations have so far been found in a small subgroup of melanomas, in particular acral or mucosal tumors that are not related to sun exposure. according to the results in gastrointestinal stroma tumors (gists), kit inhibitors such as imatinib and sunitinib, and newer inhibitors such as nilotinib or dasatinib have been described, however, to be less responsive [139, 140 ]. encouraging to this subgroup of patients, anecdotal reports have shown complete remission lasting up to one year. despite several promising new agents (table 1), there are, however, still no therapeutic strategies that would reliably conquer the complexity of pathways resulting in a highly aggressive malignancy in melanoma. considering several multimarker assays using in vivo samples and cell culture of primary melanomas and metastasis together, melanoma development itemizes to several hundreds of involved genes that seem too plentiful to be individualized for a targeted therapy in a single patient, even though new, potentially essential, marker genes have been identified and are currently tested. the very view of resistance, unwanted side effects, and rapid progression after initial responsiveness clearly emphasize the importance of a thorough, genotypical stratification, and a driver - focused synergistic therapy. the development of an oncogene hierarchy with differentiation into important drivers and bystanding passengers seems therefore necessary. the recently gained knowledge about the functional importance of muted genes in a high proportion of malignant melanoma has fundamentally changed the diagnostic and therapeutic approach. in view of the focus on braf, nras, kit, and pten, four key genomic defective alterations and their corresponding pathways are identified that without any doubt refine and extend the understanding of its bewildering biological complexity. although an improved classification [4, 18, 22 ] and corresponding risk stratifications and target - oriented therapies (table 1) are within reach, or in case of the latter even under effective investigation, a restriction to some precious few control factors seems to be a too easy answer. the serious question remains, how do the highly relevant histopathological parameters translate in a benefit for distinct subsets of the melanoma patients ? the answer probably lies in the identification of the biological achilles heel of individual tumors. as convincingly shown, molecular analysis of subsets of melanoma has at first revealed mutations in ckit. other positive examples are the more recent successful translation of the knowledge of the braf mutational status (e.g., v600e) into elegant mutation specific, and at least short - term successful therapy in these patients. however it is not surprising that in a large number of melanoma patients such single mutations do not precisely delineate the biological behaviour of the tumor at the time of primary melanoma diagnosis. thus, it is likely that this straightforward approach is too narrow, given that in a considerable fraction of melanomas so far unknown oncogenes or tumor suppressors, or combinations thereof may control tumor cell fate. most likely unbiased approaches to melanoma using 21st century technology of genetic profiling will yield intriguing results. as much as the classic hallmarks of cancer withstood the test of time : recently discovered characteristics such as antiapoptotic parameters, the role of tumor stem cells, telomerases, or circulating tumor cells, as well as other tumor - environmental and epigenetic phenomena [106, 115 ] have also to be taken into account and may translate into successful therapy. but hopefully, as hanahan and weinberg lately stated, this phenotypic myriad in melanoma [19, 149 ] may portray just a few of the causal principles of distinct tumor cell types that need to be clarified in order to improve the treatment and outcome in our melanoma patients. so, in the era of molecular profiling, the gist of the matter what 's really risky in melanoma | due to intensified research in recent years, the understanding of the molecular mechanisms involved in the development of melanoma has dramatically improved. the discovery of specific, causal mutations such as braf or kit oncogenes not only renders a targeted and thus more effective therapeutic approach possible, but also gives rise to a new genetic - based classification. targeting just a few out of several potential mutations, braf - inhibitors such as plx 4032 achieved already tremendous results in the therapy of metastatic melanoma. up to now, the correlation of clinical, histomorphologic, and genetic features is, however, not understood. even more, is it not well known precisely what kind of molecular changes predispose the primary melanoma for metastasis. the identification of morphological surrogates and prognostic parameters in tumors with such genetic alteration seems therefore crucial when differentiating and classifying this heterogeneous tumor entity in more detail and thus facilitates the stratification of prognosis as well as therapy. this review summarizes the current understanding of carcinogenesis and gives a detailed overview of known morphologic and potentially future genetic prognostic parameters in malignant melanoma. |
we analyzed serum samples that were collected in may 2009 from 125 dairy cows in durazno county (33230s, 56310w), durazno state, uruguay (figure 2). the cattle herds had no clinical sings of disease at the time of serum collection. to determine the presence of neutralizing antibodies in the serum samples, we used an orthopoxvirus plaque - reduction neutralization test as previously described (9). the serum titer was defined as the highest dilution that inhibited > 70% of virus plaques compared with negative controls (4). chronologic detection of vaccinia virus in south america. zoomed - in map shows location of durazno county, uruguay, where serum samples were collected from dairy cattle in 2009 to test for the presence of vaccinia virus. brazil states : ac, acre ; am, amazonas ; al, alagoas ; ap, amap ; ba, bahia ; ce, cear ; es, esprito santo ; go, gois ; ma : maranho ; mg, minas gerais ; ms, mato grosso do sul ; mt, mato grosso ; pa, para ; pb, paraba ; pe, pernambuco ; pi, piau ; pr, paran ; rj, rio de janeiro ; rn, rio grande do norte ; ro, rondnia ; rr, roraima ; rs, rio grande do sul ; sc, santa catarina ; se, sergipe ; sp, so paulo ; to, tocantins. because previous studies have detected viral dna in the serum of animals and humans with and without clinical manifestations (4,10,11), we performed a molecular investigation to identify orthopoxvirus. we used quantitative pcr (qpcr) to amplify vacv growth factor gene (c11r) dna. this qpcr tool has high sensitivity and specificity and, thus, has been routinely used as an orthopoxvirus diagnostic tool by our group (12). for molecular characterization the pcr a56r products obtained from c11r pcr positive samples were sequenced in both orientations and subjected to capillary electrophoresis (3130 genetic analyzer, bigdye terminator cycle sequencing kit v3.1 ; applied biosystems, foster city, ca, usa). we used the clustalw (http://www.clustal.org/) method to align sequences with previously published orthopoxvirus sequences from genbank ; alignments were manually checked with mega6 (http://www.megasoftware.net/). we constructed phylogenetic trees using the neighbor - joining method with 1,000 bootstrap replicates and the tamura 3-parameter model in mega6. we detected neutralizing antibodies against orthopoxvirus in 28 (22.4%) of 125 serum samples from cattle in uruguay ; titers were 100 neutralizing units (nu)/ml in 10 (35.7%) samples, 200 nu / ml in 11 (39.3%) samples, 400 nu / ml in 5 (17.9%) samples, and 800 nu / ml in 2 (7.1%) samples (technical appendix figure 1, panel a). to confirm that the orthopoxvirus seropositivity represented seropositivity to vacv, we used the c11r gene to molecularly test dna in the serum samples ; 2 (1.6%) samples were positive by qpcr, and 1 of those was also positive by the plaque - reduction neutralization test. one of the 2 c11r pcr positive isolates was also positive for the a56r gene ; we sequenced the gene and named the strain vacv uruguay. alignment of the a56r nucleotide sequence showed the presence of an 18-nt signature deletion, which is also present in sequences of brazilian - vacv group i, but not group ii, viruses. unlike sequences for other vacvs, the sequence for vacv uruguay had an at polymorphism (technical appendix figure 2, panel a). vacv uruguay exhibited higher identity with group i (98.6% identity) viruses from brazil and argentina than to group ii (97.3% identity) viruses from brazil (technical appendix figure 1, panel b). furthermore, in the phylogenetic tree based on a56r nucleotide sequences, the vacvs from uruguay clustered with group i vacvs that had been detected during outbreaks in brazil and with viruses from argentina (technical appendix figure 2, panel b). since 1999, vacv has been isolated from symptomatic and asymptomatic cattle, humans, and wildlife from the north to the extreme south of brazil (5,6,8), and in 2014, vacv was described in bovine serum samples from argentina (4). although no exanthematous vacv outbreaks have been reported among cattle in uruguay, we detected orthopoxvirus antibodies and vacv dna in serum samples from dairy cattle in the country, indicating they have been exposed to vacv. uruguay shares a border with brazil, and its western border is shared with entre rios province in argentina, where vacv dna has been detected. in addition, uruguay shares its northern and eastern borders with rio grande do sul state, brazil, where pelotas vacv has been isolated from horses (4,5). our finding of orthopoxvirus antibodies and vacv dna indicates a possible undetected or silent circulation of vacv in uruguay. considering the importance of the livestock sector in all countries of south america, concern exists about the possible spread of vacv beyond brazil, argentina, and uruguay (46,14). despite surveillance by veterinarians, efforts to stop the spread of vacv at borders may be hampered by the movement of infected rural workers, the marketing of asymptomatic live animals, and the misdiagnosis of vacv infection. furthermore, vacv has been shown to circulate in wild environments, and it has been hypothesized that rodents may serve as vacv hosts, as they do for other orthopoxviruses, and facilitate the spread of vacv in border areas (15). bovine vaccinia outbreaks in south america were first reported in brazil, but we can not rule out the possibility of autochthonous circulation of vacv in uruguay and other countries in south america. additional studies are needed to elucidate vacv seroprevalence in other countries in south america, and further research is needed to clarify the transmission pathways related to the spread of vacv in south america. orthopoxvirus antibody titers in serum samples from 125 cattle in durazno, uruguay, and molecular characterization of a newly isolated virus, vacv uruguay. | we detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in uruguay. two samples were pcr - positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in brazil. autochthonous circulation of vaccinia virus in uruguay and other south american countries can not be ruled out. |
the study included 225 boys aged 716 years, recruited from two centers of a football academy in the area of tunis (latitude, 35n). children with liver, renal, or bone disease ; intestinal malabsorption ; or cancer ; and those taking vitamin d supplements, anticonvulsant drugs, or systemic corticosteroids were not included. the study was carried out from january to march 2014, a period during which temperatures varied between 10c and 22c and the humidity ranged from 70 to 75%. besides 2 h per week of outdoor physical activity as part of the school program, each child attends three weekly outdoor sessions in the football academy. the training sessions are scheduled from 5 pm to 6 pm on friday and saturday, and from 9 am to 10 am on sunday. skin color of each participant was determined by two investigators (ib and mf), together with one parent of the child, and classified as fair, corresponding to fitzpatrick skin types 1 and 2, or dark, corresponding to fitzpatrick skin types 3 and 4 (19). written parental permission was obtained for each participant. weight, height, and sitting height were measured with the subjects barefooted and lightly clothed. body mass index (bmi) was calculated as weight per height squared (kg m). participants were divided according to the world health organization (who) child growth standards for bmi in three groups ; normal - weight group (bmi85th percentile), overweight group (85th percentile 97th percentile) (20). triceps and subscapular skinfolds thickness was measured with harpenden 's skinfold calipers (baty international, west sussex, england). biologic maturity was assessed by incorporating anthropometric variables (weight, standing height, and sitting height) and was calculated using the equation of mirwald (22) ; maturity offset=9.236+(0.0002708leg lengthsitting height)+(0.001663ageleg length)+(0.007216agesitting height)+(0.02292weight by height ratio). this assessment is a non - invasive and practical approved method of predicting age in years from the peak height velocity (phv) as a measure of maturity offset. for the purpose of data analysis, children were divided into three groups : pre - phv (3 to 1 year from phv), around phv (1 to + 1 year from phv), and post - phv (+ 1 to + 3 years from phv). the daily vitamin d intake in children 's diet was assessed using a 3-day food record (including 2 weekdays and 1 day over the weekend), combined with a food frequency questionnaire (ffq) that the parents mostly completed. a 35-items ffq to quantify the consumption of nutrients naturally rich in vitamin d, such as fatty fish, meat, eggs, milk, and other dairy products. the ffq was modified to estimate vitamin d intake and consumption frequency across nine categories (never, 1 time / month, 12 times / week, 23 times / week, 34 times / week, 1 time / day, 2 times / day, 3 times / day, and 4 times / day). ease of administration of this ffq was enhanced by the use of a food photograph album of tunisian food products that emphasized portion sizes. four food groups were selected as follows : fish (50 g / serving), eggs (50 g / serving), red meat (50 g / serving), and milk (200 ml / serving). consumption frequencies of milk and egg were standardized into servings per day and into servings per week for fish and red meat. the data about the mean daily intake of nutrients were processed using the professional nutri pro 7 program (nutri pro 7 software, cerden, brussels, belgium). blood was centrifuged at 2000g for 20 min and the plasma was frozen at 40c until analysis (within 3 months). plasma glucose, calcium, phosphorus, and c - reactive protein (crp) were assessed on architect c8000 analyzer (abbott laboratories, abbott park, il), using the respective reagents kits. plasma 25-hydroxyvitamin d (25-ohd) and insulin concentrations were measured by chemiluminescence immunoassay methods using the liaison analyzer (diasorin inc., vitamin d status was evaluated according to the standards of the institute of medicine (iom). vitamin d deficiency, insufficiency, and sufficiency were defined as plasma 25-ohd concentrations below 12 g / l, 12 to 20 g / l, and over 20 g / l, respectively (24). insulin sensitivity / resistance was assessed using two indexes ; the homeostasis model assessment of insulin resistance (homa - ir) and the quantitative insulin sensitivity check index (quicki), according to the following equations (25, 26) : homa - ir=[(fasting insulin in u / ml) (fasting glucose in mg / dl)/405 ] ; quicki=1/[log(fasting insulin in u / ml)+log(fasting glucose in mg / dl) ]. data were analyzed using spss for windows (version 18.0 ; spss inc., chicago, il). values are expressed as mean (sd) or median (inter quartile range, iqr) for continuous variables and as a percent for categorical variables. comparisons between groups were performed using analysis of variance or the mann whitney test for continuous variables and pearson chi - square test or fisher 's exact test for categorical variables as appropriate. unadjusted and multi - adjusted odd - ratios with 95% confidence intervals were calculated as an estimate of the risk of vitamin d deficiency / insufficiency for several potential risk factors. a binary logistic regression model was used to identify predictors for vitamin d deficiency, while adjusting for possible confounding factors. adjustment was performed on maturation status (pre - phv / around and post - phv) ; body mass (normal - weight / overweight and obese) ; skin color (fair / dark) ; and dichotomous variables for fat mass (97th percentile) (20). triceps and subscapular skinfolds thickness was measured with harpenden 's skinfold calipers (baty international, west sussex, england). biologic maturity was assessed by incorporating anthropometric variables (weight, standing height, and sitting height) and was calculated using the equation of mirwald (22) ; maturity offset=9.236+(0.0002708leg lengthsitting height)+(0.001663ageleg length)+(0.007216agesitting height)+(0.02292weight by height ratio). this assessment is a non - invasive and practical approved method of predicting age in years from the peak height velocity (phv) as a measure of maturity offset. for the purpose of data analysis, children were divided into three groups : pre - phv (3 to 1 year from phv), around phv (1 to + 1 year from phv), and post - phv (+ 1 to + 3 years from phv). weight, height, and sitting height were measured with the subjects barefooted and lightly clothed. body mass index (bmi) was calculated as weight per height squared (kg m). participants were divided according to the world health organization (who) child growth standards for bmi in three groups ; normal - weight group (bmi85th percentile), overweight group (85th percentile 97th percentile) (20). triceps and subscapular skinfolds thickness was measured with harpenden 's skinfold calipers (baty international, west sussex, england). biologic maturity was assessed by incorporating anthropometric variables (weight, standing height, and sitting height) and was calculated using the equation of mirwald (22) ; maturity offset=9.236+(0.0002708leg lengthsitting height)+(0.001663ageleg length)+(0.007216agesitting height)+(0.02292weight by height ratio). this assessment is a non - invasive and practical approved method of predicting age in years from the peak height velocity (phv) as a measure of maturity offset. for the purpose of data analysis, children were divided into three groups : pre - phv (3 to 1 year from phv), around phv (1 to + 1 year from phv), and post - phv (+ 1 to + 3 years from phv). the daily vitamin d intake in children 's diet was assessed using a 3-day food record (including 2 weekdays and 1 day over the weekend), combined with a food frequency questionnaire (ffq) that the parents mostly completed. a 35-items ffq to quantify the consumption of nutrients naturally rich in vitamin d, such as fatty fish, meat, eggs, milk, and other dairy products. the ffq was modified to estimate vitamin d intake and consumption frequency across nine categories (never, 1 time / month, 12 times / week, 23 times / week, 34 times / week, 1 time / day, 2 times / day, 3 times / day, and 4 times / day). ease of administration of this ffq was enhanced by the use of a food photograph album of tunisian food products that emphasized portion sizes. four food groups were selected as follows : fish (50 g / serving), eggs (50 g / serving), red meat (50 g / serving), and milk (200 ml / serving). consumption frequencies of milk and egg were standardized into servings per day and into servings per week for fish and red meat. the data about the mean daily intake of nutrients were processed using the professional nutri pro 7 program (nutri pro 7 software, cerden, brussels, belgium). blood was centrifuged at 2000g for 20 min and the plasma was frozen at 40c until analysis (within 3 months). plasma glucose, calcium, phosphorus, and c - reactive protein (crp) were assessed on architect c8000 analyzer (abbott laboratories, abbott park, il), using the respective reagents kits. plasma 25-hydroxyvitamin d (25-ohd) and insulin concentrations were measured by chemiluminescence immunoassay methods using the liaison analyzer (diasorin inc., vitamin d status was evaluated according to the standards of the institute of medicine (iom). vitamin d deficiency, insufficiency, and sufficiency were defined as plasma 25-ohd concentrations below 12 g / l, 12 to 20 g / l, and over 20 g / l, respectively (24). insulin sensitivity / resistance was assessed using two indexes ; the homeostasis model assessment of insulin resistance (homa - ir) and the quantitative insulin sensitivity check index (quicki), according to the following equations (25, 26) : homa - ir=[(fasting insulin in u / ml) (fasting glucose in mg / dl)/405 ] ; quicki=1/[log(fasting insulin in u / ml)+log(fasting glucose in mg / dl) ]. data were analyzed using spss for windows (version 18.0 ; spss inc., chicago, il). values are expressed as mean (sd) or median (inter quartile range, iqr) for continuous variables and as a percent for categorical variables. whitney test for continuous variables and pearson chi - square test or fisher 's exact test for categorical variables as appropriate. unadjusted and multi - adjusted odd - ratios with 95% confidence intervals were calculated as an estimate of the risk of vitamin d deficiency / insufficiency for several potential risk factors. a binary logistic regression model was used to identify predictors for vitamin d deficiency, while adjusting for possible confounding factors. adjustment was performed on maturation status (pre - phv / around and post - phv) ; body mass (normal - weight / overweight and obese) ; skin color (fair / dark) ; and dichotomous variables for fat mass (< 20%/20%), homa - ir (< 1.5/1.5) ; and the daily intake of vitamin d (< 8.5/8.5 g), milk (< 500/500 ml), red meat (< 100/100 g), fish (< 100/100 g), and eggs (< 50/50 g), defined as the respective continuous variables split at the median. serum crp and insulin concentrations and homa - ir and total energy intake were significantly higher and quicki lower in obese and overweight children compared to normal - weight children. plasma 25-ohd concentrations ranged between 3.80 and 31 g / l, and did not differ according to body mass (fig. vitamin d inadequacy was noted in about 85% of the children, with 40.9% having deficiency and 44% having insufficiency. plasma 25-ohd was positively correlated with dietary intake of proteins (r=0.407, p<0.001), milk (r=0.542, p<0.001), red meat (r=0.282, p<0.001), fish (r=0.502, p<0.001), and eggs (r=0.512, p<0.001) (fig. 2). however, no significant correlation was observed with phv, bmi, fat mass, homa - ir, or quicki. compared with vitamin - d - sufficient children, those with vitamin d deficiency or insufficiency showed lower intakes of proteins, milk, red meat, fish, and eggs (fig. vitamin d deficiency and insufficiency were associated with lower dietary intakes of vitamin d, proteins, milk, red meat, fish, and eggs. however, no association was observed with phv, bmi, fat mass, homa - ir, or skin color (table 2).. correlations of plasma 25-hydroxyvitamin d with daily intake of red meat, fish, eggs, and milk in tunisian active children (n=174). daily intake of proteins, red meat, fish, eggs, and milk according to the vitamin d status in tunisian active children (n=174). clinical, nutritional, and biochemical characteristics of children according to body mass (n=225) values are expressed as meansd or median (inter quartile range) ; homa - ir, homeostasis model assessment of insulin resistance ; quicki, quantitative insulin sensitivity check index. plasma 25-hydroxyvitamin d and multi - adjusted odd - ratios for vitamin d deficiency / insufficiency in children according to confounding variables phv, pick of high velocity ; or, odd - ratio ; 95% ci, 95% confidence interval ; 25-ohd, 25-hydroxyvitamin d adjusted for maturation status, bmi, skin color, fat mass, homa - ir, and intake of vitamin d, milk, red meat, fish, and eggs this study showed that vitamin d inadequacy is common among active tunisian children and is associated with a low intake of vitamin d food sources, but not with maturity status, adiposity, or insulin resistance. the high prevalence of vitamin d inadequacy in tunisian children is consistent with similar findings in other parts of the world (410). this finding is somewhat surprising since these are active children who live in a sunny environment and engage in a reasonable level of outdoor activities. our study had been conducted during the winter when plasma 25-ohd levels are usually at their nadir (3). because of cool environmental temperatures, children were wearing clothes that covered the trunk and limbs, minimizing the skin area exposed to the sun 's rays and preventing vitamin d synthesis. also, the training sessions were held in the early mornings or late afternoons when the sun 's rays are least efficient for vitamin d synthesis (7, 27). although dark skin color is a risk factor for hypovitaminosis d, obviously, that distinction is of little importance when most of the skin areas are covered with the clothing. however, the most important factor behind the observed vitamin d inadequacy in most children is probably the inadequate intake of dietary vitamin d sources. our data demonstrate that active children living in sunny environments may still have vitamin d inadequacy, which underlines the importance of monitoring plasma 25-ohd concentrations in children. in all the children participating in the present study, the daily oral vitamin d intake was well below the us institute of medicine recommended daily allowance (rda) of 15 mcg (24). there is no dietary recommendation for vitamin d for children and adolescents in tunisia, as it is assumed that sun exposure will ensure an adequate vitamin d status. our finding of low plasma 25-ohd levels in 85% of the participants highlights the need for evidence - based dietary recommendations for tunisian children. our study also showed a clear relationship between vitamin d deficiency / insufficiency and low intake of vitamin d food sources (i.e. fish, meat, milk, and eggs). (10) showing a positive correlation of serum 25-ohd with the consumption of vitamin d food sources in korean adolescents. in sunny areas, although sun exposure is the major source of vitamin d in the body, vitamin d inadequacy may occur. this suggests that concomitant appropriate dietary intake is required. the assumption that, in sunny environment, sun exposure alone may provide adequate plasma 25-ohd levels is often false. when sun exposure is limited as a consequence of low - sunshine seasons, pollution, dark skin, or clothing, the dietary intake of vitamin d may be the more significant contributor to vitamin d status. in these conditions, the present study showed no association between plasma 25-ohd and adiposity as assessed either by bmi percentile or fat mass. some previous studies reported an inverse relationship between plasma 25-ohd and adiposity (7, 9, 11, 28), while others found no association (8, 13, 14, 29). in a sample of quebec youth, bmi was negatively associated with 25-ohd levels in girls but not in boys (30). hypothetic mechanisms of low plasma 25-ohd levels in obesity include sequestration of vitamin d in the fat depot, impaired mobilization from the fat depot, and reduced skin and dilution throughout the body. other important factors in the obese include limited sun exposure due to few outdoor activities and reduced exposed skin area because of clothing (31, 32). this may explain the lack of an association between vitamin d and adiposity in our series. indeed, the overweight or obese and normal - weight children included in this study spend as much time outdoors and wear similar clothes, and thus receive the same dose of sunshine. the relationship between adiposity and the low vitamin d status described in some studies may be related to short exposure to the sun, rather than an excess of body fat per se. in line with the lack of an association with adiposity, our study showed no association between plasma 25-ohd and insulin resistance, a condition usually associated with obesity. in fact, the relationship between vitamin d and insulin resistance is still a subject of debate (1720, 33). our study has focused on a broad sample of children and adolescents, and its findings arose from multivariate analyses adjusting on several potential confounders for vitamin d status. the study has controlled for the time spent outdoors and thus on the amount of sun exposure, which is an important predictor of vitamin d status. although sun exposure was not measured with precision, all participants have comparable outdoor activities and equivalent sun exposure. this group of children is probably typical of urban and suburban children from the mediterranean region. the trans - sectional design prevents the evaluation of vitamin d status year - round, rendering the findings only suitable for the winter season. however, based on parents occupation, most participants have average to high socioeconomic status. nutritional assessment was achieved in only 77% of participants and vitamin d intake was estimated using a non - tunisian food database. because the vitamin d content of foods is not given in the food composition database for tunisia, the presumed concentrations of vitamin d were obtained from european food composition tables, which make estimations less precise. to overcome this issue, we considered the consumption of vitamin d food sources in tunisian diet. the development and validation of a vitamin d database for tunisian food is necessary to allow future estimations of vitamin d intake. our study did not look for health problems that may be related to vitamin d deficiency, an aspect that is beyond the scope of the study. further studies should address the health consequences of hypovitaminosis d in exposed populations. in conclusion, vitamin d intake is also low in most children, due to little consumption of vitamin d food sources. finally, circulating 25-ohd concentrations are related to vitamin d food sources intake, suggesting that dietary intake is a key contributor in vitamin d status when sun exposure is limited. given the key role of vitamin d in growth and health in general, every measure should be undertaken to achieve sufficient vitamin d status in these children. these measures include education in order to ensure adequate and safe sun exposure and appropriate consumption of vitamin d - rich / fortified foods. further research is needed to establish an optimal combination of sun exposure and food intake / supplementation ensuring year - round sufficient circulating 25-ohd in children and adolescents living in a sunny climate. the study was supported by funds of research unit 05/ur08 - 08 and research laboratory lr99es11, ministry of higher education and scientific research of tunisia. | backgroundvitamin d inadequacy is widespread in children and adolescents worldwide. the present study was undertaken to assess the vitamin d status in active children living in a sunny climate and to identify the main determinants of the serum concentration of 25-hydroxyvitamin d (25-ohd).methodsthis cross - sectional study included 225 children aged 715 years practicing sports in a football academy. anthropometric measures were performed to calculate body mass index (bmi), fat mass, and maturity status. a nutritional enquiry was performed including 3-day food records and food frequency questionnaire. plasma 25-ohd and insulin were assessed by immunoenzymatic methods ensuring categorization of vitamin d status and calculation of insulin sensitivity / resistance indexes. a logistic regression model was applied to identify predictors for vitamin d inadequacy.resultsvitamin d deficiency (25-ohd<12 g / l) was observed in 40.9% of children and insufficiency (12<25-ohd<20 g / l) was observed in 44% of children. in a multivariate analysis, vitamin d deficiency and insufficiency were associated with a lower dietary intake of vitamin d, proteins, milk, red meat, fish, and eggs. however, no significant relationship was observed with maturation status, adiposity, or insulin resistance.conclusionstunisian children and adolescents are exposed to a high risk of vitamin d inadequacy despite living in a sunny climate. circulating 25-ohd concentrations are related to the intake of vitamin d food sources but not to maturation status or body composition. ensuring sufficient and safe sun exposure and adequate vitamin d intake may prevent vitamin d inadequacy in children from sunny environments. |
stanford type a aortic dissection (taad) is a catastrophic clinical condition, occurring mainly in middle and old age groups, which requires emergency open surgical intervention. although surgical results have improved with improvements in cardiac surgery techniques over the last two decades, overall in - hospital mortality remains as high as 30%.1 the older people usually have poor physical condition, and they are often associated with other multiple basic diseases. therefore, owing to the significant mortality and morbidity of open surgery, patients of high risks are not considered for this procedure.2 previous reports have described that, in a small selected subset of patients, stent graft placement in the ascending aorta for taad is feasible and may represent a novel effective and safe procedure.37 computed tomography (ct) scan imaging studies in european and american population showed that endovascular repair was anatomically feasible in ~30% of taad patients with a proximal entry tear (et) in the ascending aorta. this percentage of patients may increase up to 39% with the use of extra - anatomic bypass, such as a left - to - right carotid carotid bypass, in order to obtain an adequate distal landing zone.8,9 recently, our previous anatomical study suggested that 38% of chinese patients with taad could potentially be treated by stent grafting.10 current endovascular treatment for taad, as with type b aortic dissection, involves sealing the proximal et and remodeling the dissected aorta but can be more challenging because of complex features of this aortic segment.1114 endografts should be precisely deployed between the sinotubular junction (stj) and the innominate artery (ia). however, ~36% of taad patients were not suitable for endovascular treatment due to the absence of available endografts, and endografts specifically designed for ascending aorta are not currently available.9 off - license techniques such as using endografts designed for the descending aorta or aortic cuff are unlikely to offer a robust solution but to compromise outcome.3 traditional thoracic endograft had to be shortened intraoperatively in order not to cover the supra - aortic trunks.13 the ideal endograft configurations of taad should be determined and developed before endovascular treatment can be implemented in the daily practice. the objective of this study is to perform a ct - based characterization of taad with a primary et in ascending aorta and make a preliminary exploration to the endograft configurations specific for ascending aorta. the delivery system and mechanism of deployment compatible with ascending aortic pathology were also described. this is a retrospective study of ct scan data of all patients treated for taad at nanjing drum tower hospital between 2008 and 2014. penetrating aortic ulcer (pau) affecting the ascending aorta was considered a classic dissection, and ct scans of pau were also included in the study. the exclusion criterion was the absence of a workable (maximum slice thickness of 1.25 mm and high - quality arterial phase) preoperative ct scan with digital imaging and communications in medicine (dicom) data and patients with retrograde taad who had primary et distal to the ia. ct angiography was performed in patients with high - resolution ct operating at 120 kv/250 ma with a 512512 matrix. the ct scanning called for injection of 100 ml of iodinated contrast agent automatically injected with 4 ml / s. demographic data were recorded, including patient age, sex, previous major conditions, and the continence of the aortic valve. this study was approved by the internal review board of nanjing drum tower hospital. written informed consent was obtained from all the participants. all ct scans with dicom data were uploaded to a picture archiving and communication system (pacs) workstation and reconstructed using an endosize workstation (therenva, nanjing, france). a centerline of flow (clf) was generated using the well - proven centerline algorithms. the calculated centerline was confirmed manually by scrolling through the images in the sagittal, axial, and coronal planes, ensuring that it accurately reflected the center point of the arterial lumen. a two - dimensional format was reconstructed perpendicular to the clf.15 important anatomic landmarks, including primary et and stj, were identified. et was accurately determined by the evidence of a clear region of intimal disruption, and contrast existed above or below the tears within the aortic wall (figure 1). the aorta was divided into the greater curvature, lesser curvature, and anterior and posterior segments, and the location of the et was classified into one of these four areas.8 the stj was defined as the place where the sinus of valsalva became the tubular ascending aorta. stj was selected as the zero point for the aorta, and et location was defined in terms of distance from proximal et to the zero point (figure 2). the distances between stj and et, stj and the closest coronary artery, stj and the tip of aortic valve, stj and ia, and et and ia, as well as the length of et were measured. diameter measurements were obtained from intima to intima (inner wall to inner wall) for a circular aortic true lumen. if the perpendicular cross - sectional shape of the true lumen was elliptical or crescentic rather than circular, additional mathematical modeling (maximum + minimum diameter)/2 was required to obtain correct diameter (figure 3). then, the diameters at stj level, et level, proximal to ia, proximal to left common carotid artery, and proximal to left subclavian artery were obtained from the reconstructions perpendicular to the clf. descending thoracic aorta maximal diameter, abdominal aorta maximal diameter, and external iliac minimal diameter were measured. the potential candidacy for endovascular therapy was defined in our previous report : proximal landing length (distance between stj and et) 20 mm, distal landing zone length (distance between et and ia) 20 mm, presence of available access iliofemoral vessel (diameter > 7 mm), absence of severe aortic regurgitation (grade 3 or 4), and coronary bypass originating from the ascending aorta.10 anatomical data of the suitable patients were used to determine the endograft configurations for ascending aorta. proximal diameter and distal diameter of the endograft were selected based on the diameter measured at et and ia levels, with 10% oversizing with respect to the true lumen, but not exceeding the original aortic diameter. the maximum length of the endograft was the distance from stj to ia, in order not to cover the supra - aortic trunk or potentially damage normal structure of aortic root. considering the difficulty in accurate deployment in the ascending aorta, in our opinion, the length of the endograft might be slightly shorter than the distance from stj to ia but should not be less than the sum of the minimum landing zone and the length of et. statistical analysis was performed using sas software (version 9.1 ; sas institute inc., cary, nc). quantitative data were represented as the mean standard deviation, whereas categorical and ordinal data were reported as frequencies and percentages. differences between the mean values of the group were assessed by student s t - test. the analysis of measurement method comparison data was performed to compare intraobserver variability according to bland and altman.16 from january 2008 to june 2014, 182 patients diagnosed with taad had visited our hospital. of them, 56 patients were excluded from the study due to suboptimal ct scans and retrograde taad. the remaining 126 patients were adequate to allow assessment of anatomic characteristics, with a mean age of 52.17.6 years. of these patients, 89 patients (70.6%) were male, and 103 (81.7%) and 39 (30.9%) patients had hypertension and diabetes, respectively. two reviewers measurements were compared to assess variability, and there were only minor differences in the measured data due to the standardized clf techniques used, and an excellent interobserver repeatability coefficient was obtained (kappa 0.79), indicating no significant differences between the observers (p>0.05). ets were located 19.122.6 mm distal to the stj and 38.225.3 mm proximal to ia, and the detailed distribution of ets is clearly indicated in figure 4. it was also found that most ets were commonly located along the greater curvature of the ascending aorta (88 patients, 69.8%), with the minority occurring along the lesser curvature (15 patients, 11.9%), the posterior aorta (13 patients, 10.3%), and the anterior segments (ten patients, 8.0%). when tears were located distally to stj, the average distance from the stj to the highest coronary artery orifice was 8.92.3 mm (range, 5.013.8 mm), and that from the stj to the aortic valve in the center of the aorta was 17.44.1 mm (range, 10.229.3 mm). according to the assumed criteria, 48 (38.1%) patients were deemed to be suitable for endovascular treatment. no proximal landing zone (33 patients), no distal landing zone (12 patients), disturbed aortic valve (19 patients), coronary bypass originating from the ascending aorta (ten patients), and absence of available access vessels (four patients) for these 12 patients with et close to the origin of ia (length < 20 mm), the distal landing zones could be extended via extra - anatomic carotid carotid bypass, in situ fenestration, or using an arch - branched stent graft, then the percentage of patients suitable for endovascular repair increased up to 47.6%. besides that, six pau patients with et located in the ascending aorta were included in this study. ets of these patients were located 40.827.8 mm distal to the stj. among these six patients, four (66.7%) were anatomical candidates for endovascular repair, and the rest two were unsuitable patients due to absence of the distal landing zone or coronary bypass originating from the ascending aorta, respectively. however, the results were not significantly different between type a pau and taad due to the small sample size (66.7% versus 37.5%, p=0.159). for 126 taad patients, a downward trend of the true lumen diameters from the stj level to the ia level is observed in figure 5, indicating that conical endograft might have advantages in the ascending pathology. taking into consideration 48 patients eligible for endovascular treatment, endografts with a proximal diameter of 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50 mm were required in one (2.1%), two (4.2%), two (4.2%), three (6.2%), four (8.3%), seven (14.6%), nine (18.7%), seven (14.6%), six (12.5%), three (6.2%), two (4.2%), and two (4.2%) patients, respectively. moreover, endografts with a distal diameter of 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46 mm were required in one (2.1%), two (4.2%), seven (14.6%), nine (18.7%), 12 (25.0%), seven (14.6%), four (8.3%), three (6.2%), two (4.2%), and one (2.1%) patients, respectively. proximal diameters differed significantly from distal diameters of the endografts (39.9 versus 36.2 mm, p<0.01), which also implied that conical endograft might be compatible with the ascending pathology. the lengths of the endograft required in the ascending aorta were 50, 60, 70, 80, and 90 mm in five (10.4%), 22 (45.9%), 13 (27.1%), six (12.5%), and two (4.2%) patients, respectively. successful endovascular treatment for the taad with et in ascending aorta is related to the selection of suitable patients with appropriate anatomy and the interest in the production of device for such applications. the information needed for such an analysis is typically obtainable from high - resolution ct in conjunction with clf analysis.17,18 sobocinski confirmed the feasibility of endograft placement in a subset of type a dissections based on a ct study. ronchey took advantage of high - resolution ct to tailor an aortic stent graft for four patients with taad, and their good outcomes demonstrated that a preoperative ct study was efficient and adequate to characterize this disease. moon used an electrocardiogram - gated ct scan reconstructed at 55% or 70% of rr interval to minimize motion artifact in the ascending aorta dissection, and this four - dimensional ct with temporal resolution rendered their study more accurate. unfortunately, electrocardiogram - gated ct was not typically performed in our emergency setting ; however, this method would get more accurate anatomical characteristics and should be applied routinely in future endovascular treatment for taad.8 in this study, the et location, proximal and distal landing zones, true and false lumen, patency of the false lumen, and the branch vessels as well as the extent of the dissection were evaluated with high - resolution ct and clf analyses based on endosize workstation. our intimal tears were located 19.122.6 mm distal to the stj, and most of the intimal tears were located on the greater curvature. the mean length of the ascending aorta was 69.4 mm, which is slightly shorter but without significant difference on comparison of the mean length of 70.5 mm reported in another study.8 thus, there was not much difference in morphological characteristics between the chinese population and other races,8,9 indicating that future devices could be designed as a whole. the anatomical characteristics were then assessed to determine the suitability of endovascular treatment for such situations according to our criteria. the results showed that, in 126 taads with primary et in ascending aorta, 48 (38.1%) patients were anatomically suitable for endovascular treatment. previous studies have proven that endovascular suitability was dependent mainly on an appropriate landing zone and good aortic valve function.610 as no fenestrated or branched endografts were commercially available to allow flow into coronary arteries, we considered that suitable length of the proximal landing zone were essential, and 20 mm as the minimum proximal length was enough for endograft to seal et. patients with inadequate proximal landing zones lead to a considerable risk of obstruction of the coronary, hemodynamic forces in this arterial segment, and the potential for fatal retrograde dissection.3,5 patients with severe aortic regurgitation (grade 3 or 4) were considered unfit for endovascular repair. although transcatheter aortic valve implantation has become a feasible therapeutic option for the management of severe aortic regurgitation,19 no study has combined this technology with endograft to treat taad associated with severe aortic regurgitation. so grade 3 or 4 aortic regurgitation was considered a contraindication for endovascular treatment. in the study, we thought four (12.7%) patients were difficult and at high risk for endovascular repair because of the absence of adequate femoral vessels (24 french). patients without adequate femoral vessel could use the left carotid artery as an access,20 alternatively, a small thoracotomy could be used to deploy the endograft through the apex of the heart.21 for those 12 patients with et located near the innominate trunk and absence of distal sealing zone (length < 20 mm), a branched endograft, hybrid, or telescope technique was efficient to maintain perfusion to the branch vessels while covering et.22 finally, the percentage of patients suitable for thoracic endovascular aortic repair would increase up to 50.1% (64/126). however, the risk of performing endovascular repair would increase if access to femoral artery and distal landing zone was not available.9 it is worth mentioning that pau has been acknowledged as a pathologic variant of false - lumen aortic dissection, and six type a pau patients (4.8%) with et located in the ascending aorta were included in this study. of these six patients, four (66.7%) were anatomical candidates for endovascular repair. the higher suitable rate in pau patients indicated that endovascular repair might have advantages in this pathology, but with a small sample size, the result was not statistically significant on comparison of taad patients. endograft placement in the ascending aorta may be difficult because the proximal and distal landing zones are very close to stj and ia. no commercially available endovascular treatment modalities designed for taad increase additional challenge to the procedure. it is apparent that the ascending aortic pathologies need specific endograft solutions.5 thus, the development of novel devices for taad is fundamental before these techniques can be widely adopted. in this study, we first defined the endograft configurations for the ascending aorta, which could not be replaced by off - license techniques using the aortic cuffs.12 the anatomical characteristics of taad patients suitable for endovascular treatment were used for the analysis of the endograft configurations. proximal and distal diameters of the endograft were based on diameters measured at et and ia levels with oversizing of 10%. ronchey and nordon adopted a maximum oversizing of 10% relative to the native true lumen at the site of the proximal seal zone to minimize aortic trauma. their experiences show that this technique is feasible in select patient categories and seems to be associated with favorable results.6,12 significant oversizing was avoided to minimize risk of inadvertent retrograde dissection while placing the endograft in an aorta with a highly fragile intima, and ; aortic dilatation due to excessive oversizing might be associated with aortic valve insufficiency.23 more importantly, we found that conical devices were needed in the ascending pathology, where the true lumen at the ia level was usually smaller than that at stj and et levels. the average length of endograft for our taad was 67.211.1 mm, which was shorter than the available devices for the descending aorta. when tears were located distal to stj, the average distance from the stj to the highest coronary artery orifice was 8.92.3 mm, and the average distance from the stj to the aortic valve in the center of the aorta was 17.44.1 mm. these measurements had implications for proximal device design, meaning the bare springs frequently used in descending aortic endograft must be questioned given that the potential to compromise aortic valve closure coupled with the likely failure of apposition of such stents to the wall of nontubular aortic root.11 therefore, in our opinion, shorter, larger, and bare spring - free conical endograft was preferred to seal the et, while presenting no harm to the fundamentally weak aorta. in some cases, a mismatch may exist between the length of the inner and outer curvature of the ascending aorta, which might easily result in a kink of the graft. instead of using one graft, two- or three - stacked grafts 5 cm long could solve this problem.11 developments of the delivery system and mechanism of deployment compatible with ascending pathology were fundamental. the delivery system should have a long shaft due to the distance from the femoral artery (726.170.2 mm) and needs to be flexible to negotiate the arch and maintain torque control. the tip of the device must cross the aortic valve and sit in the left ventricle at deployment, and short tip could limit cardiac trauma.20 easier - use deployment mechanism allows more guided placement and reduces windsock effect. a constraining loop around the body of the graft enables repositioning of the stent graft, and then the constraining wire and loop are removed. to permit exact graft deployment and prevent accidental covering of the coronary arteries, the temporary rapid ventricular pacing (heart rate 180 beats / min) was used to decrease blood pressure and cardiac output this technique has been described and used by many authors as a means of ensuring accurate deployment of the endograft.6 while some advocated partial right atrial inflow occlusion for controlled systemic hypotension during thoracic endovascular aortic repair.24 interestingly, lu found that delivery system nose cone contact with the left ventricular wall induced ventricular tachycardia or fibrillation, resulting in hypotension and decreased cardiac output that allowed the endograft to be deployed accurately. the use of intraoperative transesophageal echography has been recommended to assist with precise endograft deployment, especially when the proximal deployment location is very close to the coronary sinus.25 limitations of this study should be acknowledged. diameters of the endograft for taad were based on the measurements on gated ct scans, because it has been shown that diameters of the thoracic aorta can change by up to 17.8% through the cardiac cycle.26 endografts designed for taad should consider the mobile diameters of the pulsating ascending aorta. above all, this study was just a purely theoretical study exploring the endograft configurations for taad, and future studies are mandatory. endovascular development offered a therapeutic modality for cases of surgically untreatable taad, especially in anatomically suitable patients and the older patients. we suggested that shorter, larger, and bare spring - free conical endografts were preferred in the ascending aortic pathology. developments of the delivery system and mechanism of deployment compatible with ascending pathology were also fundamental. however, this study was just a purely theoretical study exploring the endograft configurations for taad, and further studies were mandatory. | backgroundendovascular option has been proposed for a very limited and selected number of stanford type a aortic dissection (taad) patients. we have performed a computed tomography (ct)-based taad study to explore appropriate endograft configurations for the ascending aortic pathology.methodstaad patients treated with optimal ct scans were retrospectively reviewed, and their entry tears (ets) were identified using three - dimensional and multiplanar reconstructions in an endosize workstation. after generating a centerline of flow, measurements, including numerous morphologic characteristics of anatomy, were evaluated and a selected subset of patients were determined to be suitable for endovascular treatments. proximal diameter and distal diameter of endograft were selected based on diameters measured at the et level and at the innominate artery (ia) level, with 10% oversizing with respect to the true lumen, but not exceeding the original aortic diameter. the length of the endograft was determined by the distance from the sinotubular junction to ia.resultsthis study covered 126 taad patients with primary et in ascending aorta, among which, according to the assumed criteria, 48 (38.1%) patients were deemed to be suitable for endovascular treatment. the diameters of ascending aorta from the sinotubular junction to the ia level presented a downward trend, and the proximal diameters differed significantly from distal diameters of the endograft for taad (39.9 versus 36.2 mm, p<0.01), implying that the conical endograft might be compatible with the ascending pathology. in the ascending aorta, lengths of the endograft should be 50, 60, 70, 80, and 90 mm in five (10.4%), 22 (45.9%), 13 (27.1%), six (12.5%), and two (4.2%) patients, respectively.conclusionin this selected number of chinese patients, the suitability of endovascular repair has been demonstrated based on the ct imaging. shorter, larger, and bare spring - free conical endografts were preferred in the ascending aortic pathology. |
delivery pain does not result from tissue trauma or damage ; rather, it is a part of a unique physiological process. severe delivery pain leads to the mother 's emotional turmoil and disturbs her mental health. it also has several negative effects on maternal and fetal physiological status as well as the delivery progress, including increase of oxygen consumption, increase of pulmonary ventilation, increase of cardiac output, delayed gastric emptying, inefficiency of uterine contractions, prolonged labor, decrease in uterine perfusion, fetal hypoxia, and metabolic acidosis, leading to obstetric interventions and their resultant complications. consequently, fear and anxiety resulting from delivery pain increase the mother 's pain and discomfort during the delivery process. in the study performed by lee. (2001), pregnant women mentioned severe pain as the most important factor in fear from delivery. thus, reduction of delivery pain is of great importance for decreasing the negative effects of the physiological processes which occur due to the mother 's pain and anxiety and lead to maternal and fetal damages. up to now, various pharmacological and nonpharmacological methods have been used for sedating delivery pain. in the recent years, physicians and researchers have come to the conclusion that they should employ safe and effective methods which do not disturb the delivery process, mother 's consciousness, and mother 's straining reflex and other physiological actions in order to reduce pain. it should be noted that these complications are usually detected following utilization of pharmacological methods. moreover, nonpharmacological methods do not have any side effects for the mother and the fetus, do not interfere with the labor progress, and are even desirable for both the mother and her fetus. one of these nonpharmacological methods is acupressure which is a kind of massage therapy developed in ancient china. some researchers believe that acupressure prevents transfer of pain stimulators, increases blood endorphin levels, and thereby reduces pain. chao (2007) and chung (2003) have also emphasized the effectiveness of acupressure in reduction of labor pain [2, 11 ]. one of these points is bl32 which is located in the second hole of sacral bone. doula 's supportive care includes emotional support (continuous presence, reassurance, and encouragement) and physical support (reduction of hunger, thirst, and pain, giving information about what is happening and how to deal with it, respecting the woman 's decision, and helping her to create relationships with the caregivers). supporting the mother has a considerable effect on reduction of labor pain. in this regard, hofmeyr. indicated that supporting the mother by the doula caused the mothers to report less pain. also, hodnett and osborn stated that continuously supporting the mother by the doula during the labor significantly reduced utilization of analgesics. doula calms the mother down during labor and suggests different positions to increase fetal descent. besides, doula 's support leads to less utilization of oxytocin for augmentation, lower rate of instrumental delivery, less utilization of epidural anesthesia and narcotics, and lower rate of cesarean delivery. considering the harmful effects of severe delivery pain on mother, fetus, and delivery outcomes, safe and effective control of pain nonetheless, a limited number of studies have been conducted on the effects of acupressure and supportive care on the delivery outcomes. thus, the present study aims to compare the effects of maternal supportive care and acupressure at bl32 acupoint on the pregnant women 's pain intensity and delivery outcomes. this randomized clinical trial was conducted in the delivery ward of the selected educational center of shiraz university of medical sciences (shoushtari hospital) in 2012. considering d = 5, = 0.05, 1 = 0.90, sd = 7, and the following formula, a 150-subject sample size (50 subjects in each group) was determined for the study : (1)n=2(z1/2+z1)2sd2d2. then, the subjects were selected through simple random sampling and were divided into supportive care, acupressure, and control groups using stratified block randomization. the inclusion criteria of the study were 1835 years of age, term pregnancy, singleton pregnancy, and healthy fetal membranes. also, the study participants had no history of medical, surgical, or mental problems and had faced no special problems during pregnancy. the participants ' uterine contractions started spontaneously and, at admission, the contractions occurred every 510 minutes and cervical dilatation was 3 - 4 cm. written informed consent was obtained from all the study participants. in the supportive care group, doula (the researcher) was constantly beside the mother from the beginning of the mother 's maternity ward admission (beginning of the active phase of labor at 3 - 4 cm cervical dilatation) to the end of the second stage of labor. supportive measures classified into psychological and emotional, educational, and physical categories were offered to the mother. psychological and emotional support included touching, empathy, compassion, encouraging the mother to continue cooperation in the labor process, reassurance, taking mother 's hands, maintaining eye contact, creating a sense of trust and confidence, continuous talking, and reduction of fear during labor. educational support included informing the mother about the natural process of childbirth and answering her questions. finally, physical support included cooling the mother, satisfying her hunger and thirst, and helping her change the positions in various stages of labor. these positions were as follows : the mothers followed activity positions, such as straddling a chair, leaning, tailor stretching, and lunging for 20 minutes at 38 cm dilatation. then, they were required to follow relaxing positions, such as semisitting and side - lying for 10 minutes. since 810 cm dilatation, the mothers followed fetal head descent positions, such as dangling, squatting, and hands and knees. in this study, 20 minutes were considered for changing maternal positions during labor in order to avoid boredom and monotonous conditions for the mother (figure 1). in the acupressure group, this point lies approximately one index finger length above the top of the buttock crease, approximately one thumb width either side of the spine (figure 2). pressure was applied by the beginning of contractions (3 - 4 cm cervical dilatation) and continued during the transitional phase of labor (7 - 8 cm cervical dilatation) up to the end of the first stage. in doing so, when the contractions began, the aforementioned point was pressed gently for 30 seconds. the amount of pressure was determined by an electrical engineer who was familiar with such interventions using a digital device. after the training, the pressure applied by the right and the left thumbs was measured as 1405 and 1277 mmhg, respectively. the pressure was applied by the beginning and stopped at the end of the contractions. to ensure that there was no difference in the amount of the pressure exerted by the right and left thumbs, the amount of pressure was computed using the related formula. the study data were collected using interview form (including demographic information, history of pregnancy, familial status, and pregnancy information), observation form (including evaluation of uterine contractions, fetal heart rate, labor progress, and delivery outcome), and visual analogue scale (vas). vas is a scale numerated from 0 to 10 with 0, 13, 46, 79, and 10 representing no, mild, moderate, severe, and the worst possible pain, respectively. the validity and reliability of this scale have been confirmed in the study conducted by molazem. in iran. in that study, the content validity of vas was determined using experts ' opinions and its cronbach 's alpha coefficient was computed as 0.80. after all, the data were entered into the spss statistical software (v. 16) and analyzed using wilcoxon nonparametric test, chi - square test, and one - way anova. in this study, the participants were similar regarding age (p = 0.496), number of pregnancies (1 or 2), cervical dilatation at admission (3 - 4 cm), gestational age (3741 weeks) (p = 0.158), educational level (p = 0.584), and occupation (p = 0.781). considering the intensity of pain, the results of wilcoxon nonparametric test showed no significant difference among the supportive care (6.14 0.926), acupressure (6.52 1.054), and control groups (6.20 1.088) before the intervention (p = 0.354). after the intervention, however, the intensity of pain reduced in the supportive care (3.54 1.328) and acupressure groups (3.44 0.907) compared to the control group (9.40 1.010) and the difference was statistically significant (p 0.05) (table 1). moreover, the results of chi - square test revealed a significant difference among the three groups regarding the mode of delivery (p < 0.001). accordingly, the rate of natural vaginal delivery in the supportive care group was 2% and 34% higher than that in the acupressure and control groups, respectively. the highest rate of natural vaginal delivery was observed in the supportive care group (94%) and the acupressure group (92%), while the highest rate of cesarean delivery was related to the control group (40%). overall, 18% of all the deliveries were carried out through cesarean section (table 2). in this study, all the participants ' pain intensity increased by increase in dilatation in the first stage of labor. yet, the intensity of pain was lower in the two intervention groups compared to the control group and the difference was statistically significant (p < 0.001). according to lowe (1996), the intensity of pain is extremely high in the first stage of labor, particularly in the transition phase (810 cm dilatation). based on western medicine, the intensity of uterine contractions is closely related to delivery pain. however, pharmacological interventions prevent effective uterine contractions. these medications increase uterine contractions, but they increase the delivery pain, as well. however, traditional chinese medicine (tcm) has shown that acupressure can maintain balance during labor, reduce labor pain, and improve the delivery process by increasing the uterine contractions. (2003) also reported that the participants of the acupressure group experienced less pain during labor compared to those of the touch and the control groups. reduction of pain by acupressure at bl32 acupoint can be justified by the gate control theory of pain. according to this theory, acupoints are the locations of sensory receptors with thin afferent fibers (a - delta and c - fibers) placed in the muscles. by stimulation of these points by needle, pressure, or transcutaneous electrical nerve stimulation (tens), the sensory receptors are activated and send the stimulations to the spinal cord. in this way, the spinal cord, midbrain, and hypothalamic - pituitary axis are activated and present their analgesic effects through releasing enkephalin and endorphin. in the present study, doula 's continuous support significantly reduced the intensity of labor pain compared to the control group, which is consistent with the results of many studies conducted on the issue. for instance, the study by mcgrath and kennell (2008) showed that continuous support during labor considerably decreased the need for analgesics. also, the results of the research by pascali - bonaro demonstrated that supporting the mother during labor facilitated delivery and decreased the intensity of delivery pain. this theory provides a novel concept of a widely distributed neural network in reception and perception of pain on one hand and individuals ' responding method and physical as well as psychological behavior on the other hand. according to this theory besides, based on the brain 's view towards the whole body, it not only responds to the sensory input, but it can also experience pain without any stimulation or input from the sensory neurons. hence, presence of the doula, her psychological support, and suggestion of various positions during labor led to a decrease in mother 's pain by changing her physical and psychological behavior. the findings of the current study revealed a significant difference among the three groups concerning the mode of delivery (p < 0.001). the highest rate of natural and cesarean delivery was observed in the supportive care and the control groups, respectively. doula can play a critical role in changing delivery into a desirable experience and reducing the rate of cesarean delivery by helping, supporting, consoling, and reassuring the women in labor and encouraging them to do physical activities. in the study conducted by zhang., the rate of vaginal delivery was 2-fold higher than that of cesarean delivery in the supportive group. similarly, scott indicated that the rate of cesarean section decreased by 47% in the continuous support group in comparison to the control group. in the studies by hodnett, also, the rate of cesarean delivery was higher in the control group compared to the supportive care group. all these findings are in agreement with those of the present study which showed the lowest rate of cesarean delivery in the supportive care group. of course, the current study had a major difference with the other ones. in this study, in addition to psychoemotionally supporting the women, doula suggested appropriate positions for opening the cervix at 38 cm dilatation and other positions for increasing fetal head descent at 810 cm dilatation. in the study by bloom, 4 and 6 deliveries were carried out through cesarean surgery in the active and resting groups, respectively, but the difference was not statistically significant. lawrence. also found no significant association between mother 's position and type of delivery. the difference between the results of the present study and those of other studies might be due to the difference in mother 's positions during labor. in this study, the effective positions in fetal head descent were recommended to the mother to reduce pressure on the inferior vena cava and improve oxygenation to the fetus. in bloom 's study, on the other hand, the women only walked during labor. in contrast, albers. indicated a significant relationship between mother 's activity in labor and type of delivery. in that study, the rate of cesarean delivery was 2-fold higher in the control group compared to the mothers who were active during labor (5.5% versus 2.7%). in the current study, the rate of natural vaginal delivery was significantly higher in the acupressure group in comparison to the control group and the highest rate of cesarean delivery was observed in the control group. the study findings showed that continuous support and position change during labor as well as acupressure reduced the intensity of pain and the rate of cesarean delivery. therefore, these two nonpharmacological methods can be used to improve the delivery outcomes and create a positive delivery experience. | delivery is considered as one of the most painful experiences of women 's life. the present study aimed to compare the effects of supportive care and acupressure on the pregnant women 's pain intensity and delivery outcome. in this experimental study, 150 pregnant women were randomly divided into supportive care, acupressure, and control groups. the intensity of pain was measured using visual analogue scale (vas). the supportive care group received both physical and emotional cares. in the acupressure group, on the other hand, bl32 acupoint was pressed during the contractions. then, the data were analyzed using descriptive and inferential statistics. the results revealed significant difference among the three groups regarding the intensity of pain after the intervention (p < 0.001). besides, the highest rate of natural vaginal delivery was observed in the supportive care group (94%) and the acupressure group (92%), while the highest rate of cesarean delivery was related to the control group (40%) and the difference was statistically significant (p < 0.001). the results showed that maternal supportive care and acupressure during labor reduced the intensity of pain and improved the delivery outcomes. therefore, these methods can be introduced to the medical team as effective strategies for decreasing delivery pain. this trial is registered with the iranian registry of clinical trial code irct2014011011706n5. |
an accurate implant - supported prosthetic framework is considered to have a simultaneous and circumferential contact between the implant abutment and the framework, without causing strains before functional loading.12 if the gap between the implant abutment and the framework is sufficient for bacterial adherence, inflammatory reactions will occur around the peri - implant soft tissues. moreover, the accuracy can minimize mechanical complications, decrease the development of strains, and provide long - term results regarding implant dentures.345 recently, some novel techniques are advocated to fabricate metal frameworks. compared with traditional cast technique, the new techniques are operated with machine and not by hand. these novel techniques are characterized as subtraction of raw material, as in milling technique ; or addition of raw material, as in selective laser melting (slm) technique.6 the marginal accuracy of prosthetic frameworks fabricated by milling, slm, and cast techniques has been extensively studied.78 however, the results are controversial.9 nesse compared the precision of the three methods in terms of internal and marginal fit and determined that slm restorations presented the poorest internal and marginal fit, whereas the milling method was the optimal technique.10 on the other hand, xu found that slm - fabricated cobalt chromium alloy (co - cr) crowns exhibited smaller marginal gap widths than those of cast crowns.11 furthermore, in most previous reports, the research target was the single crown and not the bridge. therefore, few studies have focused on the effect of span length on the adaptation of metal frameworks fabricated by different methods. the present investigation evaluated the adaptation of implant - supported co - cr frameworks fabricated by three techniques with different span lengths. apart from traditional impression replica technique, the frameworks were also evaluated by a new method to determine the overall extent of linear deformation. the null hypothesis was that the span length would not affect the adaptation of implant - supported co - cr frameworks fabricated by milling, slm, and cast techniques. six solid abutment analogs (straumann 048.5416, basel, switzerland) were fixed with plasticene in three standard female mandible molds at their corresponding positions (44 and 45, 43 and 45, 43 and 46). the spaces for pontics (44, 44 and 45) of the two of the molds were also filled with plasticene. the models were used to simulate different dentition defects restored by two implants with two-, three-, and four - unit fixed prostheses (figs. each model was fabricated with removable dies and scanned by 3shape d810 laboratory scanner (3shape, copenhagen, denmark) to create a 3d data file. these files were used to design the two-, three-, and fourunit fixed prosthetic frameworks in the virtual realm with the same parameters. six dots with a diameter of 0.2 mm each were designed randomly by a computer on the abutment surface of each framework for measurement. two dots were located at the buccogingival region (a1 and a2), two other dots were placed at the bucco - occlusal region (b1 and b2), and the last two dots were positioned at the linguogingival region (c1 and c2). with the same data, the frameworks of the two implants with two-, three-, and four - unit fixed prostheses were prepared by milling and slm techniques (n = 6 for each technique at each unit). the co - cr alloy blocks used to mill the framework (original multi and changer 20 rk, germany) contained 61.1% co, 32.0% cr, and 2.5% mo. the co - cr alloy powder used to fabricate the slm framework (bego eos - m280, germany) contained 63.3% co, 24.8% cr, and 5.1% mo. to cast the framework, the resin patterns of the prostheses (n = 6 for each unit) were first prepared by the milling system. these patterns were then subjected to phosphate - bonded investment (bego bellavest sh, germany) and casted with co - cr alloy (63.3% co, 24.8% cr, and 5.1% mo ; germany). the distances between six dots along the x, y, and z directions on each designed framework were measured by 3shape software. these distances were defined as standard distances and named as x1, x2, y1, y2, z1, and z2 (figs. all frameworks fabricated by the three techniques were scanned by 3shape d810 laboratory scanner and exported as stereolithography files with.stl as filename extension. the distances on each fabricated framework were also measured with 3shape software in the same way. the differences between the standard distances and the measured distances of each framework were calculated and expressed as absolute values. the marginal accuracy was also evaluated using impression replica technique.12 briefly, the framework was filled with a light body silicone (express xt, 3 m espe) and seated on the corresponding abutment with finger pressure for 2 - 3 minutes. the framework with light body silicone layer was then removed from the abutment and filled with medium - viscosity silicone (express xt, 3 m espe). each part was measured twice, and the width of the light body silicone layer represented the gap between the framework and abutment. the measurement was performed using a microscope (zeiss, stemi sv11) at 6.6 magnification (fig. the differences of the distances and gap widths for each framework are presented in fig. the differences of the distances in the frameworks made by milling technique were 0.020 0.017, 0.025 0.018, and 0.046 0.043 mm for the two-, three-, and four - unit fixed prostheses, respectively. the corresponding values in the frameworks made by slm technique were 0.052 0.045, 0.053 0.036, and 0.073 0.048 mm. the values on the cast frameworks were 0.143 0.052, 0.153 0.047, and 0.179 0.085 mm, correspondingly. in terms of span length, a significant difference was found between three- and four - unit frameworks, as well as between two- and four - unit frameworks made by milling technique (p <.05). a significant difference was also noted between two- and three - unit frameworks, as well as between two- and four - unit frameworks made by cast technique (p <.05). in terms of fabrication method, both milling and slm techniques presented smaller differences than cast technique at any unit prostheses (p <.05), but the milling technique exhibited smaller differences than the slm technique (p <.05). moreover, the impression replica method showed that the average marginal gap widths of the milled frameworks were 21.13 3.47, 24.18 5.45, and 44.44 8.61 m for the two-, three-, and four - unit fixed prostheses, respectively. the corresponding data for slm frameworks were 24.85 4.09, 35.89 4.84, and 57.31 7.97 m ; for cast frameworks, the data were 29.30 7.37, 39.72 6.99, and 63.76 7.79 m, respectively. a significant difference existed among the span lengths for any fabrication method (p <.05), as well as among the fabrication methods at any unit prostheses (p <.05). the results of this study rejected the null hypothesis as the framework span would affect the adaptation of implant - supported co - cr frameworks fabricated by milling, slm, and cast techniques. our results showed that, for all techniques, the differences of distances increased from two - unit to four - unit frameworks. when evaluated with the impression replica method, the average marginal gap widths also increased from two - unit to four - unit frameworks. for instance, lee evaluated the effect of span length on the fit of zirconia framework fabricated by cad / cam system and pointed out that the increase of span length might decrease the marginal and internal fit.13 anunmana compared the marginal and internal gaps of zirconia substructure of single crowns with those of three - unit fixed dental prostheses ; significant differences were found between the elements under study.14 tiossi also discovered that longer - span one - piece zirconia frameworks showed an increased microgap.15 although co - cr was different from zirconia, which demonstrated shrinkage compensation mechanism for the expected linear shrinkage, the co - cr frameworks exhibited the same trend as the span length increased. this finding implied that, in contrast to crowns or short - span frameworks, the more complicated shape and larger size affected the adaptation of long - span co - cr frameworks. with regard to the manufacturing method, the present study revealed that both milling and slm techniques displayed better adaptation than cast technique regardless of the span length of the frameworks. this result was different from that of nesse, who reported that slm restorations achieved the poorest internal and marginal fit.10 the reason might be attributed to the specimens in nesse 's experiment, which were delivered directly from production without internal airborne - particle abrasion, external polishing, or final adjustments. however, the intaglio surfaces of the specimens in our study were sandblasted with aluminum oxide prior to fit assessment so as to remove the obstacles influencing optimal seating. furthermore, our result was consistent with some other reports. for instance, witkowski compared the marginal accuracy of titanium copings fabricated by three different cad / cam systems and found that the marginal accuracy was significantly improved by the cad / cam system.16 moreover, sundar compared the marginal fit and microleakage of metal laser - sintered co - cr alloy copings and conventional cast ni - cr alloy copings by using a stereomicroscope and found that the copings fabricated by slm technique achieved better marginal fit and decreased microleakage in contrast to that of cast method.17 however, although both milling and slm techniques were operated by machine, the slm frameworks presented larger difference in distance and greater marginal gap than the milled frameworks. generally, the milling technique was characterized as subtraction of raw material and manufacturing under recrystallization temperature. therefore, the process slightly affected the deformation of the framework.1819 by contrast, the slm technique was characterized as addition of raw material, which selectively irradiated the metal powder material into a thin layer at high temperature.2021 hence, the slm frameworks might suffer from a greater deformation than the milled ones. finally, the adaptation evaluation showed that the results of both methods were not totally matched. this finding might be related to the larger standard deviations of the new method in contrast to those of impression replica method. given that the smallest diameter of the dots on the surfaces of the frameworks was 0.2 mm, the distances were difficult to measure with an accuracy of 0.01 mm. therefore, the large standard deviation of the measurements compromised the precision of the assessment. for more accurate analyses, more specimens are needed when using the new method. nevertheless, the two methods assessed the adaptation from different aspects. the adaptation was evaluated by analyzing the differences of corresponding distances on the designed and fabricated frameworks in the x, y, and z directions. however, the traditional impression replica method placed more emphasis on the accuracy of the prostheses individually. it measured the gap between the framework and the abutment. a smaller gap indicated better adaptation. therefore, for more effective and comprehensive analysis on the adaptation of the frameworks, a combination of the new evaluation method and the impression replica method should be considered. the differences of six distances on the designed and manufactured frameworks were not analyzed along the x, y, and z directions, but as only the sum of the values. as a result, some detailed information on each direction might be missing. to guarantee the comparability of the three techniques, the resin patterns of the cast technique were prepared by milling technique. hence, the adaptation of the cast technique should eliminate the error from the milling technique. finally, the number of specimens used in the experiment was limited, which might compromise the results of the new method. with the limitations of this study, both span length and fabrication method affected the adaptation of the implant - supported cobalt chromium frameworks. | purposethis study evaluated the effect of span length on the adaptation of implant - supported cobalt chromium frameworks fabricated by three techniques.materials and methodsmodels with two solid abutment analogs at different inter - abutment distances were digitized using a laboratory scanner. frameworks of two-, three-, and four - unit fixed prostheses were designed by a computer. six dots with a diameter of 0.2 mm were preset on the surface of each framework. a total of 54 implant - supported cobalt chromium frameworks were fabricated by milling, selective laser melting (slm), and cast techniques. the frameworks were scanned and exported as stereolithography files. distances between two dots in x, y, and z coordinates were measured in both the designed and fabricated frameworks. marginal gaps between the framework and the abutments were also evaluated by impression replica method.resultsin terms of distance measurement, significant differences were found between three- and four - unit frameworks, as well as between two- and four - unit frameworks prepared by milling technique (p<.05). significant differences were also noted between two- and three - unit frameworks, as well as between two- and four - unit frameworks prepared by cast technique (p<.05). the milling technique presented smaller differences than the slm technique, and the slm technique showed smaller differences than the cast technique at any unit prostheses (p<.05). evaluation with the impression replica method indicated significant differences among the span lengths for any fabrication method (p<.05), as well as among the fabrication methods at any unit prostheses (p<.05).conclusionthe adaptation of implant - supported cobalt chromium frameworks was affected by the span length and fabrication method. |
the brachial plexus is frequently involved by malignant tumors of the lung apex and breast, or as a result of radiotherapy. malignant bone tumors, such as osteosarcoma, arising from the cervical spine, clavicle or first rib may less commonly invade the plexus. however, primary tumors of the brachial plexus are rare. a review of the literature over the last decades demonstrates a scarcity of publications on this type of tumor. with the exception of the large series of 226 brachial plexus region tumors from the louisiana state university health sciences center (lsuhsc), most of the publications are case reports or small series of patients7,18,19). schwannomas and neurofibromas are the most common tumors of the brachial plexus region, and in most series in the literature neurofibroma is more prevalent6,18,21). peripheral non - neural sheath brachial plexus tumors are even rarer than tumors of neural origin and comprise a wide variety of benign and malignant tumors and tumor - like conditions14,18). with regard to those tumors, preoperative evaluation and planning of proper treatment this is a retrospective review of 22 surgically treated benign and malignant tumors of brachial plexus region to describe clinical presentation, the characteristics of brachial plexus tumor and clinical outcomes with a literature review. twenty - one patients with consecutive 22 surgeries for primary brachial plexus tumors were enrolled between february 2002 and november 2011 were included in this study. relevant medical history, presence of neurofibromatosis type 1 (nf1) and positive details of the neurological examination were documented. the location, size, mobility and tenderness on palpation of the tumor were assessed. plain x - ray films and computerized tomography (ct) scans of tumors close to the spine were examined to find evidence of intervertebral foraminal enlargement or vertebral erosion. magnetic resonance (mr) imaging was performed to document tumor location, margins, and relationship with adjacent structures. the histopathological diagnosis of all tumors based on light microscopy was reviewed. in some instances, the surgical exposure depended on the site of the lesion and was initially performed under loupe magnification. the lower lesions, involving cords and terminal nerves, required an anterior infraclavicular approach and when there was a more extensive involvement of the plexus, including its retroclavicular part, a combined anterior approach, with or without section of the clavicle, was employed. after gross identification of the tumor, the surgical microscope was brought into the field. in the beginning, special attention was devoted to the identification, isolation and mobilization of all adjacent plexus elements, which must be dissected together with non - neural tissues away from the tumor. under higher magnification, the proximal and distal elements directly involved and the margins of the tumor were defined. in the benign, solitary tumors, the thickened epineurium over the lesion was opened until the capsule and the mass were dissected away from adjacent nerve fascicles, in an extra capsular plane, usually as a single mass. fascicles entering the substance of the tumor or its capsule were isolated at the proximal and distal poles of the mass. intraoperative electrical stimulation and nerve action potential (nap) recording provided information about non - functioning fascicles entering and exiting the tumor. the extent of tumor removal varied from gross total resection to sub - total resection. a more aggressive resection was substituted by a less radical level of resection whenever a significant motor impairment could result. in these cases, twenty - one patients with consecutive 22 surgeries for primary brachial plexus tumors were enrolled between february 2002 and november 2011 were included in this study. relevant medical history, presence of neurofibromatosis type 1 (nf1) and positive details of the neurological examination were documented. the location, size, mobility and tenderness on palpation of the tumor were assessed. plain x - ray films and computerized tomography (ct) scans of tumors close to the spine were examined to find evidence of intervertebral foraminal enlargement or vertebral erosion. magnetic resonance (mr) imaging was performed to document tumor location, margins, and relationship with adjacent structures. the histopathological diagnosis of all tumors based on light microscopy was reviewed. in some instances, the surgical exposure depended on the site of the lesion and was initially performed under loupe magnification. the lower lesions, involving cords and terminal nerves, required an anterior infraclavicular approach and when there was a more extensive involvement of the plexus, including its retroclavicular part, a combined anterior approach, with or without section of the clavicle, was employed. after gross identification of the tumor, the surgical microscope was brought into the field. in the beginning, special attention was devoted to the identification, isolation and mobilization of all adjacent plexus elements, which must be dissected together with non - neural tissues away from the tumor. under higher magnification, the proximal and distal elements directly involved and the margins of the tumor were defined. in the benign, solitary tumors, the thickened epineurium over the lesion was opened until the capsule and the mass were dissected away from adjacent nerve fascicles, in an extra capsular plane, usually as a single mass. fascicles entering the substance of the tumor or its capsule were isolated at the proximal and distal poles of the mass. intraoperative electrical stimulation and nerve action potential (nap) recording provided information about non - functioning fascicles entering and exiting the tumor. the extent of tumor removal varied from gross total resection to sub - total resection. a more aggressive resection was substituted by a less radical level of resection whenever a significant motor impairment could result. in these cases, the duration of symptoms ranged from 6 months to 10 years prior to the surgical treatment. signs and symptoms included paresthesis and numbness (54.5%), radiating pain (22.7%), direct tenderness and pain (27.2%), palpable mass (77.3%). twelve patients presented preoperative sensory deficit (54.5%) and 9 patients among those patients accompanied with preoperative motor deficit (40.9%). on mri finding, almost all brachial plexus tumors showed well - defined mass with the long axis in line with the nerve of origin and homogeneous intermediate signal intensity (si) on t1-weighted sequences (fig. the tumors were hyperintense on t2-weighted images with inhomogeneous central low si (the target sign) (fig. 3). the tumors were hyperintense on t2-weighted images and showed strong enhancement following contrast administration. schwannomas showed globoid mass with well demarcation and surrounding nerved were suspended eccentrically from the tumor. grannular cell tumor showed multiple fusiform and lobulated enlargement of nerves, which were originated from several neural formamens. the 22 tumors comprised a heterogeneous group of lesions in terms of their size, location and pathological characteristics. the largest diameter of the smallest lesion and the largest lesion were 2.2 and 9.8 centimeters, respectively (average 6.98 cm). most of schwannomas appeared grossly as smooth globoid masses, which did not enlarge the nerve but are suspended eccentrically from it with a discrete attachment. complete resection of tumor could be achieved in all cases of schwannoma because of these gross findings. most of neurofibromas produced fusiform enlargement of the involved nerve, which made it impossible to distinguish between tumor and neural tissue. granular cell tumor appeared fusiform and multiple lobulate enlargement of the involved nerves, which was similar to gross feature of neurofibroma. but granular cell tumor was much harder consistency with poor demarcation between neural tissue and tumor, which impeded complete tumor resection. twenty tumors (90.9%) were benign and 2 tumors (9.1%) were malignant. benign tumors included 15 schwannomas (68.2%), 4 neurofibromas (18.2%) and 1 gct (4.5%). there were 1 malignant peripheral nerve sheath tumor (mpnst) and 1 malignant granular cell tumor. gross total resection was achieved in 16 patients (72.7%), including all schwannomas, 1 neurofibroma. subtotal resection was performed with a small residual lesion in 6 tumors (27.3%), including 3 neurofibromatosis associated with brachial plexus neurofibromas, 1 mpnst and 2 granular cell tumor in one patient. in case of granular cell tumor, subtotal resection was performed for the first surgery, and then small residual remnant had grown three timed size big just before the revision surgery (fig. mean follow - up period was 13.7 months (range 2 - 41 months). radiating pain and localized direct tenderness were improved immediately after surgery in 10 out of 11 patients who complained of pain at presentation, 1 patient developed new pain after surgery. motor deficit as an initial symptom was improved in 7 out of 9 patients who complained of motor weakness at presentation. newly developed motor deficit was improved during the follow - up period in 1 patient. profound motor weakness was developed in the other patient with granular cell tumor after the first surgery and recovered to nearly intact status (g4 +) for postoperative 6 months. the patient presented severe left shoulder pain with weakness of arm (g2). even though pain was improved, complete monoplegia was developed after revision surgery with no improvement until final follow - up. the duration of symptoms ranged from 6 months to 10 years prior to the surgical treatment. signs and symptoms included paresthesis and numbness (54.5%), radiating pain (22.7%), direct tenderness and pain (27.2%), palpable mass (77.3%). twelve patients presented preoperative sensory deficit (54.5%) and 9 patients among those patients accompanied with preoperative motor deficit (40.9%). on mri finding, almost all brachial plexus tumors showed well - defined mass with the long axis in line with the nerve of origin and homogeneous intermediate signal intensity (si) on t1-weighted sequences (fig. the tumors were hyperintense on t2-weighted images with inhomogeneous central low si (the target sign) (fig. 3). the tumors were hyperintense on t2-weighted images and showed strong enhancement following contrast administration. schwannomas showed globoid mass with well demarcation and surrounding nerved were suspended eccentrically from the tumor. grannular cell tumor showed multiple fusiform and lobulated enlargement of nerves, which were originated from several neural formamens. the 22 tumors comprised a heterogeneous group of lesions in terms of their size, location and pathological characteristics. the largest diameter of the smallest lesion and the largest lesion were 2.2 and 9.8 centimeters, respectively (average 6.98 cm). most of schwannomas appeared grossly as smooth globoid masses, which did not enlarge the nerve but are suspended eccentrically from it with a discrete attachment. complete resection of tumor could be achieved in all cases of schwannoma because of these gross findings. most of neurofibromas produced fusiform enlargement of the involved nerve, which made it impossible to distinguish between tumor and neural tissue. granular cell tumor appeared fusiform and multiple lobulate enlargement of the involved nerves, which was similar to gross feature of neurofibroma. but granular cell tumor was much harder consistency with poor demarcation between neural tissue and tumor, which impeded complete tumor resection. twenty tumors (90.9%) were benign and 2 tumors (9.1%) were malignant. benign tumors included 15 schwannomas (68.2%), 4 neurofibromas (18.2%) and 1 gct (4.5%). there were 1 malignant peripheral nerve sheath tumor (mpnst) and 1 malignant granular cell tumor. gross total resection was achieved in 16 patients (72.7%), including all schwannomas, 1 neurofibroma. subtotal resection was performed with a small residual lesion in 6 tumors (27.3%), including 3 neurofibromatosis associated with brachial plexus neurofibromas, 1 mpnst and 2 granular cell tumor in one patient. in case of granular cell tumor, subtotal resection was performed for the first surgery, and then small residual remnant had grown three timed size big just before the revision surgery (fig. mean follow - up period was 13.7 months (range 2 - 41 months). radiating pain and localized direct tenderness were improved immediately after surgery in 10 out of 11 patients who complained of pain at presentation, 1 patient developed new pain after surgery. motor deficit as an initial symptom was improved in 7 out of 9 patients who complained of motor weakness at presentation. newly developed motor deficit was improved during the follow - up period in 1 patient. profound motor weakness was developed in the other patient with granular cell tumor after the first surgery and recovered to nearly intact status (g4 +) for postoperative 6 months. the patient presented severe left shoulder pain with weakness of arm (g2). even though pain was improved, complete monoplegia was developed after revision surgery with no improvement until final follow - up. many reports have been published about brachial plexus tumor since courvoisier reported the first description of the surgical excision of a brachial plexus tumor in 1886. in 1970 dart.6) reported 27 cases of plexus tumors, 22 of which were nerve sheath tumors. in that same year, fisher and tate10) reviewed the literature for all brachial plexus neoplasms reported earlier than 1970. otherwise, most other publications were confined to case reports or small series of nerve sheath tumors (nsts)2,5,7,10,11,13,14,20,21,26,27). clinical presentation of brachial plexus tumor is various according to location, extension, involved neural elements and pathology. symptoms are caused by direct nerve invasion, infiltration of surrounding tissues, or local mass effect22). the most common presenting symptom was pain (70%), followed by sensory loss (61%) and weakness (52%). about half of their patients harbored benign nsts : of these, 55% presented with a neurofibroma whereas 45% had a schwannoma. kehoe.15) reported on 104 patients who presented with a solitary benign nst, 15 of which involved the brachial plexus. the majority of their patients presented with the primary complaint of a palpable mass, less than half complained of local pain or paresthesia. more recently, siqueira.27) reported on a consecutive series of 18 patients who presented with brachial plexus tumors and tumor - like conditions. most common presenting symptom was mass (75%), pain (58%), followed by sensory change (41%). in our series, the most common presenting symptom was growing mass (95.4%), parenthesis and numbness (54.5%), sensory deficit (54.5%), motor deficit (40.9%), direct tenderness and pain (27.2%), followed by included radiating pain (22.7%). in cases of brachial plexus lesions, mr imaging is the study of choice to delineate the margins of the tumor from surrounding tissues with greatest contrast12,23). computerized tomography scanning is optimal at revealing osseous erosion around the spine or changes in neural foramina8). typical mr features of benign nsts include a well - defined oval mass with the long axis in line with the nerve of origin and homogeneous intermediate signal intensity (si) on t1-weighted sequences (fig. 1). the tumors are hyperintense on t2-weighted images with inhomogeneous central low si (the target sign) (fig. less commonly, schwannomas and neurofibromas may be mildly hyperintense or hypointense to muscle on t1-weighted images and show non - uniform enhancement3,29 - 31). features, including non - homogeneous contrast enhancement, an irregular, infiltrative margin and bone destruction, may be helpful to differentiate benign from malignant nsts. schwannomas and neurofibromas are the most common tumors of the brachial plexus region, and in most series in the literature neurofibroma is more prevalent14,21). huang.14) reported that about half of their patients harbored benign nsts : of these, 55% presented with a neurofibroma whereas 45% had a schwannoma. on the contrary, siqueira.27) reported that 9 presented with a schwannoma and 1 had a neurofibroma. in our series, the most common benign tumors of the brachial plexus region were schwannoma and neurofibroma as same as mentioned prior, but benign tumors included 15 schwannomas (68.2%), 4 neurofibromas (18.2%) and 1 granular cell tumor (4.5%). there were 1 malignant peripheral nerve sheath tumor (mpnst) and 1 malignant granular cell tumor. twenty tumors (90.9%) were benign and 2 tumors (9.1%) were malignant. artico reported on cases involving 119 peripheral nerve sheath tumors (pnsts), 11 of which involved the brachial plexus. in 93% of their patients with schwannomas and 81% of their patients with neurofibromas, motor function stabilized or improved postoperatively. at the 6-year follow - up, the best surgical results were observed in the patients with schwannoma, whereas the worst results were demonstrated in those with plexiform neurofibroma2). the interest in pre - therapeutic biopsy on benign lesions is limited because the sensitivity of this procedure is moderate and the procedure could damage intact fascicles or cause haemorrhage1,14,25). on the review of literature, postoperative outcomes were not influenced by surgical approach, but were related to grade of resection at surgery and pathological features of tumor. gross total resection (gtr) can be expected for relatively small and medium size tumors, benign schwannoma, some of neurofibroma and other benign tumors, including desmoids, ganglions, cysts, epidermoids, and lipomas, and relatively good postoperative outcomes and prognosis can be expected after gtr18,19). in case of large size schwannoma tumor capsule may be encountered after skin retraction in case of large size tumor, and then further procedure can be proceeded in side of the tumor capsule. on the contrary, manipulation of nerve tissues may be needed after skin retraction in case of small size tumor. whereas, gtr can not be expected in case of some neurofibromas, mpnst and other malignant tumors because those tumors have diffuse infiltration to surrounding nerves and connective tissue. subtotal resection (str) may be recommended for those tumors to preserve neurological function. even when benign tumors are carefully dissected from the nerve involved under magnification, transient incomplete nerve paralysis may occur27). hosoi calculated that mpnst develops in approximately 13% of patients with von recklinghausen disease27). based on longer - term observation of a large series of patients, brasfield and das gupta reported an overall incidence of 29%7). although a small number of cases of mpnsts have been reported in patients without nf, it has long been recognized that nf (especially nf type 1, von recklinghausen disease) is associated with a high incidence of mpnst. however, post - operative radiotherapy is currently recommended by oncology consensus as part of standard treatment guidelines for these tumors, despite clear surgical margins32). particularly in patients with small - sized sarcomas, surgical treatment may promote cure, but in bigger lesions (the majority of the cases), the patients should undergo adjuvant treatment after surgery. external radiation therapy prevents local recurrence more effectively than surgery alone. despite aggressive surgery with adjuvant therapies, one case of our series was granular cell tumor at the first surgery and was transformed into malignant granular cell tumor at 17 months later after surgery. granular cell tumor is benign or malignant depends histologically on evidence of increased cellularity, nuclear atypia and mitotic activity9). however, on rare occasions, a benign - appearing granular cell tumor may exhibit signs of malignancy, including invasion, local recurrence and distant metastasis before or after surgery. local recurrence rate is 69% from 3 months to 3 years or more after initial surgery, and distant metastasis rate is 80% of patients before or after surgery9,16,17). resection of tumor is the choice of treatment in the most of benign and malignant brachial plexus tumors. the appropriate surgical strategy is necessary in every brachial plexus tumor because postoperative outcome is influenced by grade of resection at surgery and pathological features of tumor. appropriate intraoperative microsurgical dissection technique and electrophysiologic monitoring are essential for favorable clinical outcome. in malignant tumors, surgical treatment should be planed and followed by adjuvant chemotherapy or radiation therapy to alleviate presenting symptom and reduction of tumor volume. | objectivethis is a retrospective review of 22 surgically treated benign and malignant tumors of brachial plexus region to describe clinical presentation, the characteristics of brachial plexus tumor and clinical outcomes with a literature review.methodstwenty-one patients with consecutive 22 surgeries for primary brachial plexus tumors were enrolled between february 2002 and november 2011 were included in this study. the medical records of all patients were reviewed.resultseleven male and 10 female patients were enrolled. mean age was 39 years. three patients had brachial plexus tumor associated with neurofibromatosis (13.6%). presenting signs and symptoms included parenthesis and numbness (54.5%), radiating pain (22.7%), direct tenderness and pain (27.2%), palpable mass (77.3%). twelve patients presented preoperative sensory deficit (54.5%) and 9 patients presented preoperative motor deficit (40.9%). twenty tumors (90.9%) were benign and 2 tumors (9.1%) were malignant. benign tumors included 15 schwannomas (68.2%), 4 neurofibromas (18.2%) and 1 granular cell tumor (4.5%). there were 1 malignant peripheral nerve sheath tumor (mpnst) and 1 malignant granular cell tumor. gross total resection was achieved in 16 patients (72.7%), including all schwannomas, 1 neurofibroma. subtotal resection was performed in 6 tumors (27.3%), including 3 neurofibromatosis associated with brachial plexus neurofibromas, 1 mpnst and 2 granular cell tumor in one patient.conclusionresection of tumor is the choice of tumor in the most of benign and malignant brachial plexus tumors. postoperative outcomes are related to grade of resection at surgery and pathological features of tumor. |
among the genes identified so far in drosophila melanogaster that play a role in dna damage repair, mus308 presents some unique properties, because its cdna sequence shows motifs characteristic of dna helicase and dna polymerases. the putative product of this gene was indeed isolated as a new dna polymerase, homologue to the escherichia coli dna polymerase i, carrying as well a dna helicase domain at the n terminus region. orthologues of this gene have been found in caenorhabditis elegans, arabidopsis thaliana [3, 4 ], and mammals [3, 59 ]. in humans, three genes encoding proteins with sequence similarities to mus308one similar to mus308 helicase, hel308, and two similar to the mus308 polymerase, polq [5, 7 ] and poln have been identified to date. polq, the most studied of these proteins, has also an atpasehelicase domain at the n terminus and is able to perform dna synthesis past an abasic site, following the a - rule ; however, there are contradictory results about its fidelity in a normal nondamaged template [10, 11 ]. the mus308 gene is involved in the repair of cross - linking adducts [12, 13 ] and also of monofunctional damage, probably persistent and difficult to repair by other systems, such as the o - ethylpyrimidine damage induced by n - ethyl - n - nitrosourea (enu) in postmeiotic male germ cells. in addition, at least parts of enu- and diethyl sulphate- (des-) induced damages were repaired by mus308 in female germ cells of drosophila. this protein works in a damage bypass mechanism [1, 13 ], which was originally related to homologous recombination, hr [14, 16 ]. nevertheless, the isolation of the dna polymerase encoded by this locus, its possible ability for dna synthesis through abasic sites [10, 11 ], and the requirement of a functional mus308 protein to prevent damage - induced dna strand breaks in vivo in somatic cells of drosophila, pointing to a translesion synthesis (tls) mechanism as the activity of this protein. in summary, along these years the work of our laboratory have demonstrated that mus308 works in the repair / processing of cross - links and oxygen ethylation damage [1315, 17 ] whereas n - ethylation damage is apparently not substrate of this system, because no effect of methyl methanesulphonate (mms) was detected either in germ cells or in somatic ones. additionally, its mechanism of action is poorly understood, because it could be related to hr [14, 16 ] or to tsl. because of this, we have proposed that the mus308 locus works in a bypass - mediated tolerance mechanism, btm [15, 17 ]. given the conservation of mus308 among higher eukaryotic organisms, this locus is likely a part of a repair system relevant to dna damage processing. therefore, it would be important to elucidate what types of dna damage, apart from cross - links and o - ethylpyrimidine adducts, are substrate of this system and to get information about which of the two possible mechanisms of action, hr or tls, is actually involved in the damage bypass process. to have more information about the role of mus308 in the processing of dna ethylation damage, we have studied here the effect of des in postmeiotic male germ cells, analysing maternal repair and using the vermilion system. this system combines the analysis of induced mutation frequencies, both at a single locus (vermilion, with a specific locus test) and at multiloci (700 loci in the x - chromosome, with the recessive lethal test), with the generation and analysis of mutation spectra. our data, together with other already published, indicate that mus308 protein is involved in the processing of all types of oxygen ethylation damage, that it is also involved in the processing of nitrogen ethylation damage, that it prevents cell death at least when the amount of dna damage is high, and that this protein could be working in a tls mechanism as well as in an alternative end - joining system (alt - ej). 6467 - 5), obtained from sigma qumica (spain), was dissolved in a solution of 3% ethanol-1% tween-80 in 33.1 mm phosphate buffer (16.5 mm na2hpo4, 16.6 mm kh2po4, ph 6.8), containing 5% sucrose. 1 - 2-days old brown (bw) males, in groups of 30 individuals, were placed in glass tubes, with eight layers of glass microfiber paper (whatmann, gf / a) at the bottom, soaked with 0.9 ml of different des concentrations. after 3 hours treatment, males were mated to in(1)sc^s1l sc^8r in(1)dl" " 49, y, scsc, v ; bw ; mus308 (i, v ; bw ; mus308) virgin females (for marker descriptions see). protocols for fractionation the progeny in mature sperm and spermatids for the recessive lethal (rl) test and for isolation of f1 and f2vermilion (v) mutants were described elsewhere. at least five different experiments were carried out for each concentration and, since there were no differences among them, data were pooled. statistical analysis of rl results was performed comparing mutant frequencies in treated flies with their respective negative controls, using the fisher exact test. the influence of mus308 in the repair or processing of ethylation - induced damage was measured through the mutability index (mi), and the statistical analysis of aguirrezabalaga. for each transmissible vermilion mutant, a homozygous strain was established to carry out the molecular analysis. all the isolated mutants were analysed. the isolation of dna and pcr amplifications were as described. sequencing reactions for the coding region were carried out using the dideoxy method, with a set of 10 internal primers. a fragment of about 1.8 kb, localized upstream of the coding region, was analyzed as described before in those mutants which did not show changes in the coding sequence. in order to exclude taq polymerase - introduced errors, at least two plaques or colonies from independent pcr reactions statistical analyses of differences between mutation spectra were carried out using the hypergeometric test for comparison of samples from mutational spectra [23, 24 ]. the rl and vermilion mutation frequencies, both spontaneous and induced by the different des concentrations under mus308 deficient (mus308) conditions, are presented in table 1. pooled data from mature sperm and spermatids are shown, because no differences between them were found in any case (not shown). all chemically induced rl mutation frequencies are statistically higher than the spontaneous one, although their values decrease as des concentration increases. comparisons with the results previously obtained in mus308 proficient (mus308) conditions reveal two relevant differences (table 1). first, the spontaneous rl frequency is statistically lower in mus308 than in mus308 conditions. second, in mus308 conditions, a decrease in rl frequencies is induced as des concentration increases, whereas the opposite, that is an increase was detected in efficient repair conditions. consequently, the value of the mutability index (mi) for 10 mm des is statistically higher than 1 whereas for 15 mm and 25 mm the mis are lower than 1 (table 1). to analyse the dose range between 10 and 15 mm des, and to compare both repair conditions in the same experiment, the obtained results confirmed that when the amount of dna damage is low or moderate, hypermutability is obtained (4.8% and 8.2% rl mutation frequencies in mus308 and in mus308, respectively, with a statistically significant mi of 1.7). it is noticeable that the very high rise in mutation frequency was detected between 1012 mm and 15 mm des in efficient repair conditions, but it is not unusual to find such a narrow window of increased activity in a chemical. the obtained results demonstrate that mus308 detects and processes des - induced dna damages. on one hand, low effectiveness des doses, such as 1012 mm (inducing low mutation frequencies), cause dna damage, mostly oxygen alkylations, that seems to be processed through an error - free pathway, as pointed by the observed hypermutability. on the other hand, with high effectiveness des doses (such as 15 and 25 mm), able to induce also considerable nitrogen alkylations, the obtained results indicate hypomutability ; this fact, together with a decreased induced fertility, suggest that a functional mus308 protein is necessary for the survival of the fertilized eggs. the analysis of vermilion mutation frequencies (table 1) show that, under mus308 conditions and considering all concentrations together, 2 mutants were isolated in f1 (0.11 10 mutation frequency) while most v mutants, 11, were isolated among the f2 offspring (1.46 10 mutation frequency). other 5 v mutants were isolated from mass cultures, but they are not included in the mutation frequency estimations. additionally, another mutant induced by des was identified by genetic analysis as a translocation between the x and y chromosomes that does not include the v locus. a comparison of these data with those obtained under mus308 proficient conditions reveals that the f1 and f2 induced mutation frequencies are much lower in mus308 than in mus308 conditions, indicating that the hypomutability observed with rl frequencies also extends to v mutation frequencies. these results are in agreement with those previously obtained with enu in the same cell type and under the same repair conditions. in that case, hypermutability was observed with a concentration that induced a moderate level of dna damage (1 mm enu), and similar results were found in rl and vermilion mutation frequencies analysis. results obtained here are consistent with a hr - mediated bypass of dna damage if at least part of this damage induces cell mortality. however, a bypass tolerance system mediated by tls could be also implicated in the processing of des induced damaged. thus, a dna polymerase could process error - free some dna damage, like oxygen alkylation, when the amount of dna damage is low, but the processing of other types of induced dna lesions, especially when they are present in high amounts (because other repair systems are saturated or inactive), like nitrogen alkylation, could be error - prone. moreover, there is another tolerance system, the alternative end - joining process (alt - ej), independent of ligase 4. mus308 was very recently discovered to be involved in this system, which processes dna double strand breaks generated by replication blockage. our results are compatible also with this system because nitrogen alkylations can be the source of dna strand breaks. details of des - induced mutants are shown in table 2. in d8 - 9 no mutation was found, and the same mutation was present in the independent mutants d8 - 21 and d8 - 30, as previously reported for other vermilion spectra [14, 18, 21, 31, 32 ]. in d8 - 26 no mutation was detected in the coding region nor in the proximal part of the promoter, but the distal part of the promoter could not be amplified, suggesting the presence of a mutation. additionally, d8 - 6 presented five different mutations none of which was found in any other mutant. no mutants were isolated either from the 24002 f1 and 15698 f2 flies analysed in the concurrent control experiments or in the historical control ; therefore, we consider that the observed mutations were induced by des. the v mutation spectrum, constituted by the 23 obtained mutations, is summarized in table 3 and includes two deletions (8.7%) and 21 base pair changes, distributed as follows : 11 gc - at (47.8%) and 4 at - gc (17.4%) transitions, and 4 at - ta (17.4%) and 2 gc - ta (8.7%) transversions. the pairing up of these mutations with the several adducts induced by des indicates that : (i) the gc - at and at - gc transitions in the des spectrum should be, respectively, the consequence of the o - ethylguanine and o - ethylthymine adducts [3336 ], induced by this chemical [3739 ] ; (ii) at - ta transversions are most probably due to n - ethylation, like the rest of transversions and the deletions [25, 27 ], because des does not ethylate o - thymine. the two found deletions occur between direct repeats and, as the translocation, they can be indirectly generated from n - ethylation, as described before. comparison of the relative mutation frequencies of this mus308 spectrum with those previously obtained under mus308 conditions (table 3) reveals clear differences (p =.07, with the hypergeometric test, and lower if the translocation is considered), including a strong decrease in the frequency of gc - at transitions, and increases in the frequencies of at - gc transitions, transversions and deletions under mus308 conditions. these results confirm that mus308 is processing o - ethylguanine and o - ethylthymine, as indicated before, and reveal that this protein is also processing n - ethylation damage. o - ethylguanine, like o - methylguanine, is a stable dna lesion that can mispair with t to produce gc - at transitions as indicated but can also pair correctly with c or can even block dna polymerases [41, 42 ]. o - ethylthymine is a dna damage with a long half - life, difficult to repair in mammals [4345 ]. although it is not considered a lethal lesion [33, 36 ], it is able to block dna replication in mammalian cells in a ner deficient background. ner is apparently implicated in its repair in drosophila [27, 32 ], although rather inefficiently, because at - gc transitions are one of the most frequently enu - induced damages in the repair - active premeiotic germ cells of this organism [21, 31 ]. therefore, this adduct fits with the proposed requirements for the substrates of mus308 [13, 14 ]. since at least part of the n - ethylation damage can be persistent and can block dna synthesis, its detection as substrate of mus308, especially when the level of dna damage is very high and repair is difficult, is not unexpected or strange. additionally, it can be considered that n - ethylation is a source of dna strand breaks, and this type of dna damage is substrate of mus308 in the alt - ej system. the sequence specificity of des- induced mutations was studied determining the base pairs 5 and 3 of the damaged nucleotide (table 2). the results of this analysis show that at pairs are present at 5 in 64% of gc - at mutations in mus308 conditions whereas 64%70% of this type of mutations is preceded by gc pairs at 5 in mus308 conditions and in ner deficient conditions, respectively. this means that the neighbouring sequences 5 to o - ethylguanine change depending on the mus308 status, which is in good accordance with the proposed polymerase function of mus308, specially considering that no influence of surrounding sequences was found before for this chemical in this locus [25, 27 ], nor were expected for an sn1/sn2 alkylating agent. in summary, the results presented in this paper demonstrate that mus308 processes oxygen and nitrogen alkylations, and they support its role in a tolerance mechanism that is especially relevant in case of high dna damage levels, because it prevents cell death. additionally, these results suggest that this protein could act through a tls pathway, because of (i) the detected neighbouring sequence influence, and (ii) its dna polymerase activity. finally, these results also agree with the mus308 role in the alt - ej system, for the processing of dna damage - inducing strand breaks, which can be compatible with the tls pathway. | the d. melanogaster mus308 gene, highly conserved among higher eukaryotes, is implicated in the repair of cross - links and of o - ethylpyrimidine dna damage, working in a dna damage tolerance mechanism. however, despite its relevance, its possible role on the processing of different dna ethylation damages is not clear. to obtain data on mutation frequency and on mutation spectra in mus308 deficient (mus308) conditions, the ethylating agent diethyl sulfate (des) was analysed in postmeiotic male germ cells. these data were compared with those corresponding to mus308 efficient conditions. our results indicate that mus308 is necessary for the processing of oxygen and n - ethylation damage, for the survival of fertilized eggs depending on the level of induced dna damage, and for an influence of the dna damage neighbouring sequence. these results support the role of mus308 in a tolerance mechanism linked to a translesion synthesis pathway and also to the alternative end - joinig system. |
participants for this project were identified from the diabetes research into adolescent transitions (drat) study. the drat project was a 3-year longitudinal project examining the self - management of young people with type 1 diabetes, living in rural and urban areas of new south wales and the australian capital territory. a total of 158 children and adolescents, 819 years of age, together with their families, were recruited into the drat study, independent of their source of care. as described elsewhere (1,2), the drat study involved three annual visits for which a researcher traveled to interview the young person and their family in their home. at each of these annual visits data relating to frequency of blood glucose monitoring were obtained from the memory of blood glucose meters for the preceding 2 weeks. a capillary blood sample was also collected, and hba1c values were determined by a single laboratory using high - performance liquid chromatography (bio - rad, hercules, ca ; upper limit of normal 6.0%). participants who had completed their first annual visit were posted a letter inviting them to be involved in this extension of the drat study, along with an information sheet, consent form, and personality data collection tool. to improve return rates, the package was followed up by a telephone call from the researcher who had met with the family at the first annual visit, and a reply paid envelope was included in the package to assist participants in returning completed forms. other participants were approached directly by researchers during their first annual visit, and personality data were completed at this time. a total of 142 of 158 drat participants agreed to take part in the personality study. personality data were gathered using the five - factor personality inventory for children (ffpi - c). the ffpi - c is a validated, untimed pen - and - paper questionnaire designed to measure personality dimensions of conscientiousness, agreeableness, emotional regulation, extraversion, and openness to experience in children and adolescents from 9 years and 0 months of age to 18 years and 11 months of age (13). the ffpi - c consists of 75 items (15 items per factor), with each item having two opposing anchor statements aimed at assessing trait variability on the personality dimensions. there are five circles between the anchor statements that allow the participant to choose the statement that best reflects their response (13). raw scores for the five - factor domains were summed and converted to t - scores using the manual provided (13). cronbach alphas ranged from 0.75 (openness) to 0.88 (conscientiousness). to assess for statistical significance of the relationships between personality traits and outcome measures over time, multivariate regressions using the five - factor domains as independent variables and hba1c or frequency of blood glucose monitoring as dependent variables were conducted for each year of data collection. pearson product correlations were computed between independent and dependent variables, and coefficients are reported in supplementary table 1. scores for emotional regulation suggested a curvilinear relationship with glycemic control (hba1c) ; therefore, a centered quadratic term for this trait was included in the regression analyses, allowing for linear and quadratic regression models to be compared. if the addition of a quadratic term improved the overall fit of a bivariate regression model, the relationship between the independent and dependent variables was considered to be of curvilinear nature (1416). control variables of age, sex, duration of diabetes, family responsibility, and interaction terms were entered into hierarchical regressions along with all five of the personality traits. after this, significant variables were re - entered into forced - entry regression models. the forced - entry models for the second and third years also included the initial outcome measure from the first year to control for its effects (i.e., hba1c from year 1 was included in year 2 and year 3 hba1c regressions, and smbg from year 1 was included in year 2 and year 3 smbg regressions). the contribution of personality was calculated as the difference in adjusted r between a model including all predictors of hba1c or smbg and a second model with personality variables excluded. to assess longitudinal change, mixed - design anovas were used using high and low baseline groups (upper and lower tertiles) for traits found to be significant predictors of outcomes. baseline personality traits were used based on the temporal stability of these factors (correlation coefficients r = 0.550.84). three groups (upper, middle, and lower quintiles) were created for analyses of emotional regulation and hba1c based on the nonlinear relationship that was found. the research protocol used in this project was approved by the university of western sydney and the university of sydney human ethics committees, as well as the relevant state area health units. personality data were gathered using the five - factor personality inventory for children (ffpi - c). the ffpi - c is a validated, untimed pen - and - paper questionnaire designed to measure personality dimensions of conscientiousness, agreeableness, emotional regulation, extraversion, and openness to experience in children and adolescents from 9 years and 0 months of age to 18 years and 11 months of age (13). the ffpi - c consists of 75 items (15 items per factor), with each item having two opposing anchor statements aimed at assessing trait variability on the personality dimensions. there are five circles between the anchor statements that allow the participant to choose the statement that best reflects their response (13). raw scores for the five - factor domains were summed and converted to t - scores using the manual provided (13). to assess for statistical significance of the relationships between personality traits and outcome measures over time, multivariate regressions using the five - factor domains as independent variables and hba1c or frequency of blood glucose monitoring as dependent variables were conducted for each year of data collection. pearson product correlations were computed between independent and dependent variables, and coefficients are reported in supplementary table 1. scores for emotional regulation suggested a curvilinear relationship with glycemic control (hba1c) ; therefore, a centered quadratic term for this trait was included in the regression analyses, allowing for linear and quadratic regression models to be compared. if the addition of a quadratic term improved the overall fit of a bivariate regression model, the relationship between the independent and dependent variables was considered to be of curvilinear nature (1416). control variables of age, sex, duration of diabetes, family responsibility, and interaction terms were entered into hierarchical regressions along with all five of the personality traits. after this, significant variables were re - entered into forced - entry regression models. the forced - entry models for the second and third years also included the initial outcome measure from the first year to control for its effects (i.e., hba1c from year 1 was included in year 2 and year 3 hba1c regressions, and smbg from year 1 was included in year 2 and year 3 smbg regressions). the contribution of personality was calculated as the difference in adjusted r between a model including all predictors of hba1c or smbg and a second model with personality variables excluded. to assess longitudinal change, mixed - design anovas were used using high and low baseline groups (upper and lower tertiles) for traits found to be significant predictors of outcomes. baseline personality traits were used based on the temporal stability of these factors (correlation coefficients r = 0.550.84). three groups (upper, middle, and lower quintiles) were created for analyses of emotional regulation and hba1c based on the nonlinear relationship that was found. the research protocol used in this project was approved by the university of western sydney and the university of sydney human ethics committees, as well as the relevant state area health units. overall, glycemic control remained stable over the 3-year study period, with a nonsignificant rise in hba1c from 8.6% (sd 1.4) in year 1 to 8.8% (sd 1.4) in year 2 and 8.7% (sd 1.3) in year 3. baseline characteristics of young people participating in the personality study and drat project correlations indicated that four of the five - factor domains conscientiousness, agreeableness, emotional regulation, and openness to experience were associated with hba1c levels at different points across the 3-year study period (supplementary table 1). hierarchical regressions indicated that conscientiousness, agreeableness, and the centered quadratic term for emotional regulation were significant independent predictors of hba1c levels. no significant interactions were identified between personality traits and demographic variables. despite a significant correlation between openness to experience and hba1c, a model including conscientiousness, agreeableness, and the quadratic term for emotional regulation significantly predicted 8% of the variance in hba1c scores at baseline [r = 0.08 ; f(3,115) = 3.21 ; p = 0.03 ]. the quadratic term for emotional regulation was the only significant independent predictor within this model (table 2). standardized coefficients and r in regression models predicting glycemic control (hba1c) and frequency of smbg over the 3-year study period a regression model that included the participants hba1c at baseline together with conscientiousness, agreeableness, and the quadratic term for emotional regulation significantly predicted their second - year hba1c [r = 0.55 ; f(4,120) = 37.28 ; p < 0.001 ]. for this year, conscientiousness and agreeableness were significant independent predictors of glycemic control, whereas the quadratic term for emotional regulation was nonsignificant. the contribution of these personality traits to the overall model was calculated at 9% (table 2). similar results were obtained for the third year of data collection. as shown in table 2, the full model, including baseline hba1c, significantly predicted third - year hba1c [r = 0.39 ; f(4, 126) = 20.49 ; p < 0.01 ]. once again, conscientiousness and agreeableness were the only significant personality trait predictors of glycemic control within this model. in year 3 while glycemic control did not change over the study period for the group as a whole [f(2, 278) = 1.34 ; p = 0.26 ], there were differences in hba1c between individuals with high and low levels of conscientiousness [f(1, 100) = 5.09 ; p = 0.03 ] and individuals with high and low levels of agreeableness [f(1, 96) = 9.79 ; p < 0.01 ]. 1a, while hba1c was similar at entry to the study (year 1), there was an interaction between conscientiousness groups and time [f(2, 200) = 4.48 ; p = 0.01 ]. glycemic control deteriorated over the 3-year study period in individuals in the lowest tertile of conscientiousness [f(2, 98) = 6.10 ; p < 0.01 ]. in contrast, no significant changes in hba1c values were found for the high - conscientiousness group [f(2, 102) = 0.37 ; p = 0.69 ]. a similar interaction was found between agreeableness group and hba1c over time [f(2, 192) = 4.91 ; p < 0.01 ]. individuals in the lowest tertile of agreeableness had worsening glycemic control [f(2, 88) = 5.00 ; p < 0.01 ], whereas no significant main effect of time on hba1c values was found for the high - agreeableness group [f(2, 104) = 0.64 ; p = 0.53 ]. since there was a statistically significant curvilinear relationship between emotional regulation and hba1c, the upper, middle, and lower quintile groups for baseline emotional regulation were compared to examine the role of this trait in long - term glycemic control. there was a significant interaction between emotional regulation and hba1c scores over time [f(4, 190) = 3.01 ; p = 0.02 ]. between - group differences also approached significance [f(2, 95) = 2.88 ; p = 0.06 ]. within - group analyses indicated no significant changes in glycemic control within the low emotional regulation group [f(2, 56) = 1.80 ; p = 0.18 ] or the high emotional regulation group [f(2, 74) = 2.18 ; p = 0.12 ]. however, there was significant decline in glycemic control within the moderate emotional regulation group [f(2, 60) = 5.44 ; p < 0.01 ]. 1c, individuals with low emotional regulation had consistently worse glycemic control across the study period compared with those with moderate emotional regulation, while hba1c trended down steeply in the final year for those high in emotional regulation. correlations indicated that conscientiousness, emotional regulation, and agreeableness were associated with blood glucose monitoring at different points across the 3-year study period (supplementary table 1). hierarchical regressions indicated that age and conscientiousness were the only significant independent predictors of blood glucose monitoring. a model including conscientiousness and age significantly predicted 7% of the variance in blood glucose monitoring at baseline [r= 0.07 ; f(2, 118) = 4.10 ; p = 0.02 ]. a model including baseline number of blood glucose estimations, age, and conscientiousness significantly predicted blood glucose monitoring behavior in the second year ; however, the trait of conscientiousness only contributed to 3% of the variance (table 2). in the third year, a similar model predicted 30% variance in smbg behavior [r= 0.30 ; f(3, 127) = 18.33 ; p < 0.00 ], with conscientiousness providing 5% of this contribution. older participants monitored less frequently than their younger counterparts, whereas participants who scored high in the domain of conscientiousness consistently performed a higher frequency of blood glucose monitoring (mean number of smbg in a fortnight : 81.0 [sd 43.7 ] vs. 66.8 [sd 24.7 ] in year 1 ; 79.0 [sd 33.7 ] vs. 62.4 [sd 22.3 ] in year 2 and 76.2 [sd 27.7 ] vs. 58.8 [sd 26.9 ] in year 3 ; f(1, 98) = 9.53 ; p < 0.01). overall, glycemic control remained stable over the 3-year study period, with a nonsignificant rise in hba1c from 8.6% (sd 1.4) in year 1 to 8.8% (sd 1.4) in year 2 and 8.7% (sd 1.3) in year 3. correlations indicated that four of the five - factor domains conscientiousness, agreeableness, emotional regulation, and openness to experience were associated with hba1c levels at different points across the 3-year study period (supplementary table 1). hierarchical regressions indicated that conscientiousness, agreeableness, and the centered quadratic term for emotional regulation were significant independent predictors of hba1c levels. no significant interactions were identified between personality traits and demographic variables. despite a significant correlation between openness to experience and hba1c, a model including conscientiousness, agreeableness, and the quadratic term for emotional regulation significantly predicted 8% of the variance in hba1c scores at baseline [r = 0.08 ; f(3,115) = 3.21 ; p = 0.03 ]. the quadratic term for emotional regulation was the only significant independent predictor within this model (table 2). standardized coefficients and r in regression models predicting glycemic control (hba1c) and frequency of smbg over the 3-year study period a regression model that included the participants hba1c at baseline together with conscientiousness, agreeableness, and the quadratic term for emotional regulation significantly predicted their second - year hba1c [r = 0.55 ; f(4,120) = 37.28 ; p < 0.001 ]. for this year, conscientiousness and agreeableness were significant independent predictors of glycemic control, whereas the quadratic term for emotional regulation was nonsignificant. the contribution of these personality traits to the overall model was calculated at 9% (table 2). similar results were obtained for the third year of data collection. as shown in table 2, the full model, including baseline hba1c, significantly predicted third - year hba1c [r = 0.39 ; f(4, 126) = 20.49 ; p < 0.01 ]. once again, conscientiousness and agreeableness were the only significant personality trait predictors of glycemic control within this model. in year 3, personality contributed 12% of the variance in hba1c results. correlations indicated that four of the five - factor domains conscientiousness, agreeableness, emotional regulation, and openness to experience were associated with hba1c levels at different points across the 3-year study period (supplementary table 1). hierarchical regressions indicated that conscientiousness, agreeableness, and the centered quadratic term for emotional regulation were significant independent predictors of hba1c levels. no significant interactions were identified between personality traits and demographic variables. despite a significant correlation between openness to experience and hba1c, a model including conscientiousness, agreeableness, and the quadratic term for emotional regulation significantly predicted 8% of the variance in hba1c scores at baseline [r = 0.08 ; f(3,115) = 3.21 ; p = 0.03 ]. the quadratic term for emotional regulation was the only significant independent predictor within this model (table 2). standardized coefficients and r in regression models predicting glycemic control (hba1c) and frequency of smbg over the 3-year study period a regression model that included the participants hba1c at baseline together with conscientiousness, agreeableness, and the quadratic term for emotional regulation significantly predicted their second - year hba1c [r = 0.55 ; f(4,120) = 37.28 ; p < 0.001 ]. for this year, conscientiousness and agreeableness were significant independent predictors of glycemic control, whereas the quadratic term for emotional regulation was nonsignificant. the contribution of these personality traits to the overall model was calculated at 9% (table 2). similar results were obtained for the third year of data collection. as shown in table 2, the full model, including baseline hba1c, significantly predicted third - year hba1c [r = 0.39 ; f(4, 126) = 20.49 ; p < 0.01 ]. once again, conscientiousness and agreeableness were the only significant personality trait predictors of glycemic control within this model. in year 3, personality contributed 12% of the variance in hba1c results. while glycemic control did not change over the study period for the group as a whole [f(2, 278) = 1.34 ; p = 0.26 ], there were differences in hba1c between individuals with high and low levels of conscientiousness [f(1, 100) = 5.09 ; p = 0.03 ] and individuals with high and low levels of agreeableness [f(1, 96) = 9.79 ; p < 0.01 ]. as shown in fig. 1a, while hba1c was similar at entry to the study (year 1), there was an interaction between conscientiousness groups and time [f(2, 200) = 4.48 ; p = 0.01 ]. glycemic control deteriorated over the 3-year study period in individuals in the lowest tertile of conscientiousness [f(2, 98) = 6.10 ; p < 0.01 ]. in contrast, no significant changes in hba1c values were found for the high - conscientiousness group [f(2, 102) = 0.37 ; p = 0.69 ]. a similar interaction was found between agreeableness group and hba1c over time [f(2, 192) = 4.91 ; p < 0.01 ]. individuals in the lowest tertile of agreeableness had worsening glycemic control [f(2, 88) = 5.00 ; p < 0.01 ], whereas no significant main effect of time on hba1c values was found for the high - agreeableness group [f(2, 104) = 0.64 ; p = 0.53 ]. since there was a statistically significant curvilinear relationship between emotional regulation and hba1c, the upper, middle, and lower quintile groups for baseline emotional regulation were compared to examine the role of this trait in long - term glycemic control. there was a significant interaction between emotional regulation and hba1c scores over time [f(4, 190) = 3.01 ; p = 0.02 ]. between - group differences also approached significance [f(2, 95) = 2.88 ; p = 0.06 ]. within - group analyses indicated no significant changes in glycemic control within the low emotional regulation group [f(2, 56) = 1.80 ; p = 0.18 ] or the high emotional regulation group [f(2, 74) = 2.18 ; p = 0.12 ]. however, there was significant decline in glycemic control within the moderate emotional regulation group [f(2, 60) = 5.44 ; p < 0.01 ]. as seen in fig. 1c, individuals with low emotional regulation had consistently worse glycemic control across the study period compared with those with moderate emotional regulation, while hba1c trended down steeply in the final year for those high in emotional regulation. correlations indicated that conscientiousness, emotional regulation, and agreeableness were associated with blood glucose monitoring at different points across the 3-year study period (supplementary table 1). hierarchical regressions indicated that age and conscientiousness were the only significant independent predictors of blood glucose monitoring. no significant interactions between demographics and personality traits were found. a model including conscientiousness and age significantly predicted 7% of the variance in blood glucose monitoring at baseline [r= 0.07 ; f(2, 118) = 4.10 ; p = 0.02 ]. a model including baseline number of blood glucose estimations, age, and conscientiousness significantly predicted blood glucose monitoring behavior in the second year ; however, the trait of conscientiousness only contributed to 3% of the variance (table 2). in the third year, a similar model predicted 30% variance in smbg behavior [r= 0.30 ; f(3, 127) = 18.33 ; p < 0.00 ], with conscientiousness providing 5% of this contribution. older participants monitored less frequently than their younger counterparts, whereas participants who scored high in the domain of conscientiousness consistently performed a higher frequency of blood glucose monitoring (mean number of smbg in a fortnight : 81.0 [sd 43.7 ] vs. 66.8 [sd 24.7 ] in year 1 ; 79.0 [sd 33.7 ] vs. 62.4 [sd 22.3 ] in year 2 and 76.2 [sd 27.7 ] vs. 58.8 [sd 26.9 ] in year 3 ; f(1, 98) = 9.53 ; p < 0.01). correlations indicated that conscientiousness, emotional regulation, and agreeableness were associated with blood glucose monitoring at different points across the 3-year study period (supplementary table 1). hierarchical regressions indicated that age and conscientiousness were the only significant independent predictors of blood glucose monitoring. a model including conscientiousness and age significantly predicted 7% of the variance in blood glucose monitoring at baseline [r= 0.07 ; f(2, 118) = 4.10 ; p = 0.02 ]. a model including baseline number of blood glucose estimations, age, and conscientiousness significantly predicted blood glucose monitoring behavior in the second year ; however, the trait of conscientiousness only contributed to 3% of the variance (table 2). in the third year, a similar model predicted 30% variance in smbg behavior [r= 0.30 ; f(3, 127) = 18.33 ; p < 0.00 ], with conscientiousness providing 5% of this contribution. older participants monitored less frequently than their younger counterparts, whereas participants who scored high in the domain of conscientiousness consistently performed a higher frequency of blood glucose monitoring (mean number of smbg in a fortnight : 81.0 [sd 43.7 ] vs. 66.8 [sd 24.7 ] in year 1 ; 79.0 [sd 33.7 ] vs. 62.4 [sd 22.3 ] in year 2 and 76.2 [sd 27.7 ] vs. 58.8 [sd 26.9 ] in year 3 ; f(1, 98) = 9.53 ; p < 0.01). the current study suggests that personality traits may be a predictor of how well a young person with type 1 diabetes does over time in terms of glycemic control and self - monitoring behavior. (12) showed that high agreeableness and high conscientiousness were associated with better glycemic control in the first 2 years of living with diabetes., the current study shows that these traits continue to play a role regardless of duration of diabetes. although the hba1c of the participants who were high on these two traits in our study was above target, it remained stable over the 3 years. of more concern, however, were those participants who were low on conscientiousness and/or agreeableness. in these individuals, therefore, low scores on these personality traits may be risk factors for poor or worsening glycemic control and confirm that baseline personality scores may have a continuous effect on diabetes management outcomes. it is intuitive that conscientiousness would also play a role in self - care behavior. indeed, skinner. (17) showed that conscientiousness, as well as emotional regulation, influenced type 1 diabetes self - management behavior through a mediating effect on a person s treatment beliefs. more broadly, conscientiousness has been found to be associated with better adoption of health - protective behaviors relating to issues such as tobacco use and physical activity (18). in this current study, those participants with high scores on conscientiousness performed a higher frequency of blood glucose estimations. importantly, this group continued to show high levels of self - care in relation to monitoring throughout the study period. conversely, participants with low scores on this trait, as well as older participants, monitored less frequently. although agreeableness was associated with glycemic control, it did not predict self - monitoring behaviors. of interest, the work by vollrath. (12) showed that children low on neuroticism (now known as emotional regulation) also had better glycemic control. emotional regulation refers to a person s capacity to control their emotional responses to their environment and others, and those scoring low on this factor are prone to be emotionally over reactive and more vulnerable to negative emotional states (19). (12), our study showed a curvilinear relationship between emotional regulation and glycemic control. individuals with either low or high scores on this trait had consistently higher hba1c values across the first 2 years of the study compared with those with moderate scores. there is some prior evidence of a curvilinear relationship between emotional regulation and progression of diabetes complications. a study by brickman. (20) demonstrated that, in adults with diabetes, extreme scores on emotional regulation (either low or high) were related to more rapid deterioration in renal function. (20) may be explained by the law of yerkes and dodson (21), which states that task performance increases with emotional arousal up to an optimum point, beyond which reductions in performance occur. therefore, people high in emotional regulation may be unconcerned about the long - term effects of poor glycemic control. those low in emotional regulation are likely to be distressed, and this may influence their ability to look after themselves. the findings of this study are significant, as they underline the importance of personality in determining self - management in young people with type 1 diabetes over time. personality traits show some continuity from childhood onwards ; however, traits are not generally considered to be fixed until 30 years of age (22). further research is needed to identify the extent to which a young person s personality can be modified. in the meantime, there may be benefits in clinicians taking personality into account and helping young people manage rather than change their personality. for example, knowing whether a young patient is not particularly conscientious would be helpful in understanding the possible reasons for their poor attention to regular blood glucose monitoring. focusing on simple strategies to raise behavior - awareness and to improve self - organization, time - management, and planning skills these strategies may include the use of telephone reminders or providing young people with blood glucose meters with alerts. in contrast, for those who are low on emotional regulation, strategies that focus on reducing stress and anxiety may be of higher priority than using reminders or fostering time - management skills. similarly, knowledge that a young person is particularly conscientious or emotionally well - regulated would free up precious clinic time to concentrate on other aspects of their care or equally provide a sound basis for engaging this particular attribute to ensure continued good self - management practices. future studies are needed to examine the potential of personality - based interventions in diabetes care. research trial interventions that develop, implement, and evaluate health care professionals skills in monitoring and fostering attributes of personality and their impact on improving self - management of this group of people are also needed. while this study was the largest of its kind, the results were based on a comparatively small sample of participants. relative to the sample size, a high number of correlations were performed, increasing the possibility of type i or type ii error. the general lack of significant relationships between personality traits and first - year outcomes is intriguing. however, the five - factor model variables showed high temporal stability within participants, and further detailed research is needed to understand these findings. despite these limitations, the results of this study indicate that personality may be one of the many factors that contribute to less than optimal self - care behaviors and suboptimal glycemic control in young people with type 1 diabetes. harnessing those personality traits associated with improved outcomes, as well as developing effective methods of working with those traits associated with worse outcomes, may serve to optimize diabetes care. | objectiveto determine whether personality traits (conscientiousness, agreeableness, emotional regulation, extraversion, and openness to experience) are associated with glycemic control and blood glucose monitoring behavior, and change or stability of these outcomes over time, in young people with type 1 diabetes.research design and methodsa 3-year longitudinal study was conducted using data from 142 individuals with type 1 diabetes, 819 years of age. personality was assessed at baseline using the five - factor personality inventory for children. data relating to glycemic control (hba1c) and frequency of blood glucose monitoring (based on meter memory) were collected annually. relationships between personality traits and hba1c and monitoring frequency were examined using regression models and mixed - design anova.resultsthree of the five - factor domains were independently associated with glycemic control. individuals high in conscientiousness and agreeableness had a lower and more stable hba1c across the 3-year study period. in contrast, the hba1c of individuals scoring low on these traits was either consistently worse or deteriorated over time. low or high emotional regulation scores were also associated with worse glycemic control. by the third year, these domains, together with initial hba1c, accounted for 39% of hba1c variance. conscientiousness was the only personality factor associated with blood glucose monitoring behavior.conclusionsresults of this study underline the importance of personality in contributing to diabetes outcomes. attention to a young person s personality, and appropriate tailoring of diabetes management to ensure an individualized approach, may help to optimize diabetes outcomes. |
the age - adjusted incidence of female thyroid cancer from 2005 to 2009 was 17.3 per 100,000, making it the 5th leading cancer diagnosis among women according to the national cancer institute 's surveillance, epidemiology, and end results (seer) program. recent reports estimate that thyroid cancer incidence has been increasing significantly at an average annual rate of 6.6% per year since 1998. from 2010 to 2012, 113,690 new cases of thyroid cancer are expected to be diagnosed among us women alone [35 ]. thyroid cancer is not only one of the leading types of cancer among women ; it is also one of the most survivable with current 5-year relative survival rates as high as 98%. the high incidence of thyroid cancer combined with its high survival makes it one of the most prevalent cancers in the country among women, contributing an estimated 6% (n = 376,969) to the total female cancer prevalence as of january 1, 2009. the gender, age, race / ethnic, and histological patterns among individuals diagnosed with thyroid cancer have been studied extensively [1, 615 ]. the association of geographic location and socioeconomic status (ses) with the incidence of thyroid cancer is not well studied, but evidence suggests that incidence rates vary by seer registry [7, 12, 13 ] and that individuals who live in areas with high ses or with a high proportion of insured individuals are more likely to have a higher incidence of thyroid cancer [9, 1618 ]. there is also a growing body of literature evaluating the association of gender, age, race / ethnicity, treatment, and geography (vis - - vis seer registry) on long - term thyroid cancer survival [6, 7, 11, 15, 1921 ]. although the literature is consistent with regard to the association of age and race / ethnicity with survival (i.e., younger women and non - hispanic whites are less likely to die from their disease [6, 7, 10, 11, 15, 2022 ]), treatment effects on survival are less consistent. some studies report significant survival advantages among patients who receive total thyroidectomy while others have found no survival benefits [22, 23 ] when compared to patients who do not receive cancer - directed therapies such as surgery and radioactive iodine (rai) treatment. the only study considering geographic variability in thyroid cancer survival found differential 5-year relative survival of anaplastic thyroid cancer by seer registry. however, authors attributed this to regional differences in diagnostic and surveillance practices and did not report systematic differences between registries for differentiated thyroid cancer (dtc), which is the most common type of thyroid cancer among young women. another long - term outcome among young women diagnosed with thyroid cancer is the development of subsequent primary cancers [14, 2431 ]. in the only study that evaluated the risks of subsequent primary cancers among women diagnosed under age 40, ronckers and colleagues reported that patients diagnosed under the age of 40 had a 39% increased risk for developing a second malignancy, whereas women diagnosed at age 40 or older had only a 6% increased risk for developing a second malignancy. to our knowledge, no studies to date have addressed competing risks of death and the association of geographic and socioeconomic factors with long - term survival among women diagnosed with dtc before the age of 40. this study describes long - term survival and expands previous research by exploring potential geographic and socioeconomic risk factors. in addition, we explore long - term survival outcomes in the context of competing risks. we used the national cancer institute 's seer database to obtain demographic, socioeconomic, geographic, tumor, treatment, and survival information necessary for analysis. the initial sample was restricted to a cohort of women diagnosed from 1975 to 2009 with histologically confirmed papillary (ptc) or follicular (ftc) thyroid cancers residing in one of 9 seer registries, including the states of connecticut, hawaii, iowa, new mexico, and utah and the metropolitan areas of san francisco, detroit, seattle, and atlanta. pre-1990 census county - level ses measures were not available in the seer database, and as a result when examining ses effects, we limited our analysis to women diagnosed from 1992 to 2009. this, however, allowed us to include three additional seer registries in the ses analysis : rural georgia and the metropolitan areas of san jose and los angeles (seer 12). following the methods of kosary and colleagues, we used the international classification of disease for oncology, 3rd edition (icd - o-3), primary site definition for thyroid cancer (c73.9) with the following ptc and ftc histology classifications : (a) ptc : m-8050, m-8260, m-8340, m-8350, m-8450 ; (b) ftc : m-8290, m-8330-m-8332. because our study includes cases diagnosed prior to january 1, 2001, when the icd - o-3 coding schema was adopted by registries, we excluded 1,482 cases with histologies which were not valid prior to icd - o-3 implementation (i.e., histologies which did not exist prior to 2001 based on icd - o-2). incident ptc and ftc were limited to primary cancers which were either the first and only cancer diagnosed or the first of multiple cancers diagnosed for each individual. subsequent primary cancers, which refer to new primary cancers that develop in a person with a history of cancer, were limited to cancers occurring after an initial primary diagnosis of thyroid cancer. women who were diagnosed with thyroid cancer after other primary cancers were also excluded (n = 3, 228). other exclusions were women with unknown age (n = 1) or unknown county of residence (n = 3) at diagnosis, other or unknown race / ethnicity (n = 557), and incident cases that were only reported via death certificates or were diagnosed at autopsy (n = 437). with the above inclusion and exclusion criteria, a total of 36,295 women were eligible for the study. however, women with missing or unknown surgical (n = 137) or radiation (n = 465) treatment or with missing or unknown stage (n = 641) were also excluded for ease in interpretation of outcomes or because of small numbers. the primary outcomes for overall survival (os) and disease - specific survival (dss) were deaths due to any cause and deaths due to thyroid cancer, respectively. cause of death was obtained using vital status and cause of death information from the seer database. all thyroid cancer patients were followed from the date of diagnosis (19752009) to december 31, 2009. deaths from thyroid cancer were defined using the seer cause - specific death classification system, which assumes that the cause - of - death coding systems are inherently prone to error, leading to misclassification and potential underreporting of causes of death due to cancer. the seer cause - specific death classification system addresses this potential bias by recoding causes of death, accounting for tumor sequence (i.e., only one tumor or the first of subsequent tumors), site of the original cancer diagnosis, and comorbidities (e.g., aids and/or site - related diseases). competing risk analysis was conducted using the same vital status and cause of death information to classify deaths from other (nonthyroid) cancers (ocs, other cancer survival) and deaths from all noncancer causes (ncs, noncancer survival). in addition to age at diagnosis and histological subtype, we evaluated survival outcomes by year of diagnosis, race / ethnicity, marital status, stage and tumor size at diagnosis, metropolitan / nonmetropolitan county designations, county - level ses, seer registry, and surgical and radiation treatment. these variables have been previously linked to cancer incidence and survival [79, 12, 13, 16, 3537 ]. race / ethnicity was defined using a combination of seer race and the hispanic identification algorithm (nhia) developed by the north american association of central cancer registries. seer race and nhia codes were used to classify women into four major groups : non - hispanic white (nh white), non - hispanic black (nh black), hispanic, and asian / pacific islander (api). marital status at the time of diagnosis was defined as married (married including common law), not married (single, divorced, widowed, or never married), and unknown. methodologies for classifying tumor stage and size at diagnosis follow those reported by aschebrook - kilfoy.. seer historic stage a was used to classify thyroid cancers by stage at diagnosis including local (limited to thyroid gland), regional (limited to thyroid gland and surrounding tissues), and distant (metastatic or systemic) stages. tumor size for thyroid cancers was not collected by seer registries until 1983 and therefore, was only included in the seer 12 ses analysis for the cohort diagnosed from 1992 to 2009. tumor size was grouped into five categories : < 1 cm, 1.12.0 cm, 2.14.0 cm, 4.0 + cm, and unknown size. geographic variables include seer registry and county - level metropolitan / nonmetropolitan designations based on 1974, 1983, 1993, and 2003 rural - urban continuum codes (ruccs) developed by the us department of agriculture. the rucc classification scheme delineates metropolitan counties based on the size of their metropolitan area population. nonmetropolitan counties are delineated based on their level of urbanization and adjacency to metropolitan areas. because rucc classification schemas changed over time, we assigned each cancer record a rucc designation based on the year of diagnosis : diagnosis year 1975 was assigned to rucc 1974, diagnosis years 19761985 were assigned to rucc 1983, diagnosis years 19861995 were assigned to rucc 1993, and diagnosis years 19962009 were assigned to rucc 2003. all rucc codes were consolidated and grouped into the two major groups : metropolitan (metro) and nonmetropolitan (nonmetro). women with unknown county were not assigned rucc designations and were, therefore, excluded from analyses (n = 3). socioeconomic status (ses) was derived using county - level data from the 1990 and 2000 decennial censuses and defined as the percent of persons whose income was below the federal poverty level (% pov). the ses analysis was limited by the availability of census data as % pov is only available in seerstat starting with the 1990 census. percent poverty was evenly distributed in tertile groups, with higher categories representing higher ses (or low - poverty areas). radiation treatment may include internal rai (i) ablation therapy or external beam radiation. women were grouped into two broad categories for surgical treatment with women who received lobectomies, partial / incomplete thyroidectomies, total thyroidectomies, and other nonspecific cancer - directed surgery classified as having surgical treatment and all others as not having surgical treatment. radiation and surgical treatment variables were based on seer coding guidelines which define treatment as cancer - directed first - course treatment administered as either part of the patient 's treatment plan or administered up to 12 months following diagnosis. we first evaluated demographic, geographic, tumor, treatment, and socioeconomic differences by comparing women residing in the seer 9 registries diagnosed with dtc from 1975 to 2009 who were under the age of 40 (< 40) to women diagnosed with dtc at age 40 or older (40 +) using chi - square tests of association for categorical variables and t - tests for continuous variables. to adjust for multiple comparisons and because we expected that small differences between groups would be statistically significant due to large sample sizes, we set significance at probability levels of p < 0.0038 based on a bonferroni correction with 13 bivariate associations (0.05/13 = p < 0.0038). we generated the kaplan - meier survival curves to evaluate long - term os and dss by age group (< 40 versus 40 +) and histological subtype (ptc versus ftc) and used multivariate cox proportional hazards (cox ph) regression to estimate the relative risk (hazard ratio, hr) of death from any cause (os) and from thyroid cancer (dss). all models included year of diagnosis, histological subtype, stage at diagnosis, race / ethnicity, marital status, metro / non - metro county designations, radiation and surgical treatment, and subsequent primary tumors. the second model was similar to the first but included seer registry to evaluate registry - specific differences in os and dss. the third model was created specifically to evaluate the association of ses on os and dss and, therefore, was limited to thyroid cancers diagnosed from 1992 to 2009 in seer 12. because tumor size for thyroid cancer was not collected until 1983, tumor size was only included in the third model. all women were followed from the time of diagnosis to the end of the study period (december 31, 2009), until death or loss to follow up. in the os models, events were considered as deaths from any cause (n = 4,007), and women who were lost to follow up or still alive on december 31, 2009, were censored (n = 31, 046). in the dss models, events were considered as deaths from thyroid cancer (n = 1, 025) as defined by seer cause - of - death recode. women who died of other causes (n = 2, 982) were censored as were those who were lost to follow up or still alive on december 31, 2009. competing risks for other causes of death were analyzed using 19752009 seer 9 data and kaplan - meier survival curves. for the ocs analysis, women who died from thyroid cancer or other noncancer causes, were lost to follow up, or were still alive at the end of the study period were censored. for the ncs analysis, women who died from cancer, were lost to follow up, or were still alive at the end of the study period were censored. log - rank tests were conducted to evaluate differences in the kaplan - meier survival curves. the proportional hazards assumption was assessed for age group, histology group, race / ethnicity, stage, marital status, metro / non - metro county designations, and surgery and radiation treatment. all analyses were performed using sas statistical software, version 9.2 (released march 2008). characteristics of women diagnosed with ptc and ftc from 1975 to 2009 by age group are presented in table 1. a total of 35,053 women inclusion criteria, of these 41.5% (n = 14, 540) were diagnosed before the age of 40 (median age at diagnoses was 31 years) and 58.5% (n = 20, 513) were diagnosed at the age of 40 or older (median age at diagnosis was 52 years). ptcs represented a large majority of cases (89%), and younger women were more likely to be diagnosed with ptc (90.9%) than older women (87.2%). seventy - five percent of female dtcs occurred among nh whites for both age groups. younger women were less likely to be married at the time of diagnosis (58.3%) as compared to older women (66.2%) and were less likely to be diagnosed with subsequent primary cancers (6.4%) than older women (12.4%). stage, tumor size, and treatment differences between women < 40 and 40 + were also significant. younger women were less likely to be diagnosed with localized disease (60.9%) than older women (66.7%) and were only slightly more likely to have surgery and/or radiation as part of their first course treatment (99.4% surgery, 45.8% radiation) than older women (98.5% surgery, 43.9% radiation). associations by type of surgery received (lobectomy, thyroidectomy, or nonspecific surgery) were assessed for a subsample of women diagnosed from 1992 to 2009. we found that younger women were more likely to have thyroidectomies (78.9% versus 74.4%) whereas older women were more likely to have lobectomies (23.5% versus 20.2%) (results not shown). geographic distributions varied significantly by age group, with san francisco - oakland, iowa, atlanta, and utah registries contributing more younger cases than older cases, while connecticut and detroit contributed more older cases than younger cases. younger women were slightly more likely to reside in metro counties (89.4%) when compared to older women (88.0%). younger women were less likely to live in high - poverty counties (lowest ses tertile) (14.0%) as compared to older women (15.7%). as expected, more deaths were observed for older women (17.6%) than younger women (2.8%). median follow - up time for women < 40 was approximately 11 years and median follow - up time for women 40 + was shorter at just over 7 years. log - rank tests revealed significant survival differences between age groups for both ptc and ftc (p < 0.001) (figure 1). younger women consistently showed better os and dss than older women. to test for proportional hazards, an interaction term between time and key variables was included in the os and dss models, including age group, histologic group, race, stage, marital status, metro / non - metro, surgery, radiation, and subsequent cancers. the interaction between time and age group (< 40 and 40 +) was significant (p < 0.0001), and thus we stratified all multivariate cox ph regression models by age group. no other interactions were significant, indicating that proportional hazards assumptions were met for all other variables in our analysis. we found that age at diagnosis modified many of the hrs for death in the os model as significance and magnitude varied by age group (table 2). unlike older women, differences in os between women who were diagnosed at regional stage and women diagnosed at localized stage were not statistically different in the < 40 age group. the difference between distant stage and localized stage was significant for both age groups, but was substantially lower in women < 40 (hr = 1.96, 95% ci 1.233.07) as compared to women 40 + (hr = 4.96, 95% ci 4.405.59). nh black women had a significantly higher risk of death as compared to nh white women in both age groups. however, in younger women, the racial disparity was larger as nh black women had double the hazard rate of their nh white counterparts (hr = 2.04, 95% ci 1.452.87 ; 40 + hr = 1.21, 95% ci 1.071.37). the risk for unmarried women as compared to married women was also higher in both age groups, although the effect size was smaller for women < 40 (hr = 1.26, 95% 1.031.54) as compared to women 40 + (hr = 2.14, 95% ci 2.002.30). younger women who did not have surgery had 3.65 times the hazard rate of death (95% ci 1.1411.72) as compared to women in the same age group who had surgery, and again, the magnitude of the association for younger women was smaller when compared to older women (hr = 4.97, 95% ci 4.175.93). women < 40 who were diagnosed with subsequent primary cancers had 4.63 times the risk of death (95% ci 3.765.71) than women with only one primary cancer of the thyroid. this is nearly 2.8 times higher than the hr for women who were diagnosed at age 40 and older (hr = 1.67, 95% ci 1.551.80). interestingly, women who did not have radiation as part of their first - course treatment did not have a higher risk of death, and this was consistent for both age groups. there were substantially fewer deaths from thyroid cancer among younger women (n = 63, 0.4%) than older women (n = 962, 4.7%) (table 2). histological subtype, race / ethnicity, marital status, and subsequent primary cancers were not significantly associated with thyroid cancer deaths in women < 40. however, similar to older women, the effect of distant stage at diagnosis is much more pronounced (hr = 7.8, 95% 3.9816.07) for dss than it was for os. radiation treatment remains insignificant for women < 40, but the effect of not having surgery was substantially larger for dss as younger women who did not have surgery were 15 times more likely to die from thyroid cancer than women who had surgery (hr = 15.14, 95% ci 3.3069.49). after controlling for a number of potential risk factors, geographic differences in survival these women had a 20% greater risk of death from any cause as compared to women who lived in metro areas (hr = 1.20, 95% ci 1.091.32) (results not shown). when we evaluated registry - specific differences in os and dss between younger and older women, we found strong, significant differences between age groups for all registries as younger women were consistently and significantly at lower risk of death as compared to older women (results not shown). we found little variation in hrs across registries after controlling for other risk factors (results not shown). high county - level poverty (tertile 1, low ses) was only a significant risk factor for os among older women, conferring a 24% increased risk of death as compared to women living in counties with the lowest poverty levels (tertile 3, high ses) (hr = 1.24, 95% ci 1.111.38) (table 3). area - based poverty was not a significant risk factor for young women diagnosed with thyroid cancer for both os and dss. log - rank tests for, kaplan - meier survival curves for ocs and ncs revealed significant survival differences between age groups (p < 0.001) as the < 40 group experienced significantly better survival outcomes for both ocs and ncs (results not shown). competing risk analysis revealed significantly greater race / ethnic disparities in the ncs model as compared to what we found in the os model (figure 2). disparities were more pronounced for ncs among young nh black women in both age groups (< 40 hr = 3.36, 95% ci 2.175.22 ; 40 + hr = 1.44, 95% ci 1.241.69). we also found elevated risks for ncs among young apis (hr = 1.82, 95% ci 1.142.88) as compared to nh whites, which did not exist in os and dss models. the distribution of causes of death by age group is described in table 4. the largest percentage of deaths were due to non - cancer - related causes, namely, heart disease (< 40 21.6% ; 40 + 37.1%). although younger women in our study were less likely to be diagnosed with subsequent cancers (table 2), they had a larger percentage of deaths due to other types of cancers (n = 113, 27.8%) as compared to older women (n = 648, 18.0%). this finding persisted in our multivariate ocs model as being diagnosed with a subsequent primary cancer was the only factor that was significantly associated with deaths from other cancer causes among young women (results not shown). rising incidence trends in thyroid cancer and disparities with respect to age and gender have gained considerable attention in recent years [35, 41, 42 ], leading to a growing number of investigations into prognostic clinical and epidemiological determinants of survival from the disease [6, 11, 15, 20, 22 ]. this study expands previous research by first describing the long - term survival of women diagnosed with dtc under the age of 40 and exploring potential geographic and socioeconomic risk factors. then we explore long - term survival outcomes in the context of competing risks. the use of 40 as the age cutoff is a recognized departure from previous thyroid studies [6, 11, 14, 29, 31, 43, 44 ], save ronckers. ; however, there is limited consistency in age grouping across studies. furthermore, we found compelling statistical evidence vis - - vis effect modification, which suggests that factors associated with thyroid cancer survival affect women differently when they are diagnosed under the age of 40 as compared to women who are diagnosed at age 40 and older. however, none examined how age at diagnosis might modify stage - specific survival outcomes. in our study, we found that even after controlling for histological subtype and other key demographic and clinical factors, young women with regional stage disease were not significantly different from women with localized disease in terms of thyroid cancer survival. this was not true for women who were diagnosed with regional disease at age 40 and older. we also found that although younger women with distant - stage thyroid cancer were nearly eight times more likely to die from their disease than women of the same age diagnosed with localized disease, the association of late stage diagnosis among older women was much more pronounced, conferring a nearly 20-fold increase in risk for thyroid cancer death. this is consistent with known age disparities in thyroid cancer survival which led the american joint committee on cancer to incorporate age as a determining factor in the staging of thyroid cancer (i.e., women diagnosed under the age of 45 were assumed to have early stage disease). this has been criticized for often leading to systematic bias in stage - related outcomes. however, this potential source of bias was not a problem in our study because we used seer historic stage a to define stage at diagnosis, which does not follow ajcc age - specific staging guidelines. since staging methodologies are not likely to account for this finding, our results suggest that either younger women may be better equipped to fight their cancer biologically through more robust immune systems or financially through better access to healthcare or that older women have more aggressive tumors which are not adequately captured in the seer staging or morphology coding systems. aggressive forms of thyroid cancer are often measured histologically (e.g., anaplastic or medullary thyroid cancers). our study did not include anaplastic or medullary thyroid cancers which are often more fatal and occur in older women. aggressive forms of thyroid cancer may also be measured with other tumor features including size, extension, nodal involvement, or multifocal attributes. we were limited in our ability to account for other tumor features in our study because of the inconsistency in how these features were reported in seer from 1975 to 2009 and tumor size was not recorded for thyroid cancer in seer until 1983. however, we may have indirectly accounted for tumor size by using the seer staging system, which includes tumor size in its algorithm. future research may explore these additional risk factors as well as account for more aggressive forms of papillary thyroid cancers (e.g., diffuse sclerosing and tall cell variants) on the risk of thyroid cancer deaths and use a shorter diagnostic time period when these variables were consistently recorded (e.g., 19882009). our study also identified additional risk factors which were not examined in previous studies, namely, marital status and the diagnosis of subsequent cancers. it is well established that being married confers a survival benefit for many illnesses, including cancer [36, 37, 48 ]. the interesting aspect of marital status in our study, however, is that not being married seems to have a larger, negative association among women who were diagnosed after the age of 40. the reason for this finding is not clear but may point to social factors that likely affect older women in different ways (i.e., the absence of social support in combination with competing demands of career and children). previous studies have found that younger women have an increased risk of subsequent primary cancers when compared to older women [14, 2431 ]. we found that women diagnosed at age 40 or older were more likely to be diagnosed with subsequent cancers than women diagnosed before age 40 in our bivariate analysis. the difference between our study and others is likely due to differences in the age cut points. our multivariate analysis, in fact, showed that the diagnosis of subsequent cancers was significantly associated with poorer overall survival among young women, conferring a nearly 5-fold increase in risk of death from any cause. our study further showed that the diagnosis of subsequent cancers was the only significant risk factor for other (nonthyroid) cancer deaths, ceteris paribus. although it is reasonable to hypothesize that women were in fact dying from their subsequent cancer, our study did not explore this relationship. the lack of other significant associations could also be a result of small numbers as only 0.8% of women who were diagnosed with thyroid cancer before the age of 40 died from another cancer and only 3.2% of women who were diagnosed with thyroid cancer at age 40 or older died from other cancers. however, this may also suggest that there may be other unmeasured and potentially confounding risk factors that were not accounted for in our study (e.g., comorbidities, family history of cancer, insurance status, healthcare access, etc). although we failed to find systematic geographic variations and ses effects on survival in younger women, we know of no other study to date that has evaluated the simultaneous effects of these variables in addition to age and other prognostic factors on thyroid cancer survival. we consider these findings as preliminary because we used seer registry as a proxy for geography and county - level poverty as a proxy for individual - level ses. our data are also limited in terms of geographic / demographic scope (i.e., seer 12) and time (i.e., 19922009), which limits the generalizability of our findings. nevertheless, all registries showed a substantial decrease in risk of death in younger women as compared to older women after controlling for other known risk factors. therefore, we suggest that despite potential regional / geographic differences in healthcare utilization and access, age remains one of the strongest prognostic factors of thyroid cancer survival. future research should attempt to describe geographic and ses effects using more precise measures including more granular geographic levels and ses at the individual level. previous reports considering racial disparities in thyroid cancer survival are mixed [10, 15 ]. although hollenbeak. reported that african americans have significantly poorer survival from thyroid cancer as compared to whites, they attributed the higher risk to higher rates of anaplastic and follicular thyroid cancer types and larger tumors. yu and colleagues found that for papillary thyroid cancers, blacks had slightly lower observed 5-year survival (91.6%) when compared to other race / ethnicity groups which had survival rates between 94% and 95%. however, yu. did not use multivariate methods to control for potential risk factors. in our study, we found that while substantial racial disparities in long - term overall survival exist between non - hispanic white and non - hispanic black women at any age, the racial disparities were much more pronounced among young women and for other (noncancer) causes of death. the racial disparities for noncancer causes of death were also found among young asian / pacific islander women. these findings provide some evidence to suggest that the racial disparities in survival after thyroid cancer are due to competing risks from comorbid conditions (e.g., heart disease), which tend to be higher in minority populations. unfortunately, the low number of deaths from thyroid cancer, particularly for those under the age of 40, leads to poorer precision in the models and wide confidence intervals. our findings may also have limited generalizability to the larger us population as the seer 9 and seer 12 registries represent approximately 10% and 12% [49, 50 ] of the us population, respectively. and although seer registries are geographically diverse, populations within their catchment area may come from more urban areas and have a larger proportion of foreign - born residents. another limitation of our study was our inability to control for insurance status, which has been associated with cancer incidence [9, 16, 17 ], early detection [5255 ], and survival outcomes [5658 ]. a final caveat to our findings is our inability to control for the entire spectrum of treatment protocols (e.g., chemotherapy, hormone treatment, radiation dosing, etc.) as these data were not available in the seer database. future research might include a broader range of treatment modalities and protocols to assess long - term survival outcomes. despite the high overall survival rate for thyroid cancer, there remains significant variability in long - term survival outcomes among young women who are diagnosed with this prevalent disease before the age of 40. this study provides strong evidence that age at diagnosis remains one of the strongest prognostic factors for thyroid cancer survival. based on our analysis of competing risks, we found that women who were diagnosed with thyroid cancer at any age were more likely to die from other causes, including other cancers, suggesting that young, female thyroid cancer survivors may benefit from more directed efforts to ensure that they receive effective care for comorbid conditions including heart disease and multiple primary cancers to reduce mortality from other causes. | background. differentiated thyroid cancers (dtcs) are one of the most common and survivable cancers diagnosed in women. we examine factors associated with long - term survival and competing risks of death in women diagnosed with dtc under the age of 40 (< 40) and aged 40 and older (40 +). methods. seer data was used to identify dtcs diagnosed in women from 1975 to 2009. we examined overall (os), disease - specific (dss), other cancer (ocs), and non - cancer - related (ncs) survival using multivariate cox proportional hazards modeling. results. observed survival was 97.2% for < 40 (n = 14,540) and 82.5% for 40 + (n = 20,513). distant stage (hr = 1.96, 95% ci 1.233.07), non - hispanic black (hr = 2.04, 95% ci 1.452.87), being unmarried (hr = 1.26, 95% 1.031.54), and subsequent primary cancers (hr = 4.63, 95% ci 3.765.71) were significant for os in women < 40. age was an effect modifier for all survival outcomes. racial disparities in ncs were most pronounced for young non - hispanic black women (hr = 3.36, 95% ci 2.175.22). women in both age groups were more likely to die from other causes. conclusions. age at diagnosis remains one of the strongest prognostic factors for thyroid cancer survival. more directed efforts to ensure effective care for comorbid conditions are needed to reduce mortality from other causes. |
recent progress in molecular biology has revealed the molecular basis in the pathogenesis of various diseases. one such therapy is antivascular endothelial growth factor (anti - vegf) therapy, which is now widely used to treat age - related macular degeneration (amd) and cancer. its role in treating amd is to regulate ocular vascular lesions and prevent secondary damage to the neural retinal cells, which are critical for visual function. the first research into vegf was reported in the 1970s, and in 2004 the fda approved the first anti - vegf drug for clinical use in human eyes. basic research on neurotrophic regulation also began in the 1970s, but clinical trials started only recently. molecular - targeting therapies for retinal neuroprotection are on the horizon, and further studies are needed to understand the molecular mechanisms in retinal diseases and to explore new treatment approaches. in the treatment of retinal diseases, developing neuroprotective therapies for neural retinal cells deserves special emphasis ; these cells have a very limited regenerative capacity and are critical to vision. the neural retinal cells derive from the monolayer of the neural tube during embryogenesis and are part of the central nervous system. damage to these cells occurs in common diseases such as chorioretinal inflammation and diabetic retinopathy, as well as in less - common conditions, like retinitis pigmentosa, a hereditary retinal degeneration with mutated genes in the retinal cells. diabetes chronically affects it, even in the absence of obvious microangiopathy [68 ] : patients experience a gradual loss of visual function even when diabetic neovascularization is well regulated by vitreous surgery and/or anti - vegf therapy. in amd, local retinal inflammation is involved in the process of vision loss ; association of inflammatory molecules is reported in both early and late stage amd. however, the investigation of the molecular mechanisms of retinal neuropathogenesis is in its early stages. here, we describe the molecular mechanism of neurodegeneration that we recently reported in animal models of innate chorioretinal inflammation (endotoxin - induced uveitis) and diabetic retinopathy, and compare our findings with studies from other fields to obtain additional clues to the pathogenesis of retinal diseases. clinical reports show that il-6 in the vitreous fluid increases not only in uveitis but also in diabetic retinopathy [11, 12 ], retinal vein occlusion, and retinal detachment. research with experimental animals has shown that diffusible factors, il-6 and other proteins in the il-6 family, such as leukemia inhibitory factor (lif) and ciliary neurotrophic factor (cntf), are expressed in the retina. both il-6 and lif are found in mller glial cells, and cntf is found in the retinal ganglion cells and astrocytes around the vessels. these endogenous il-6 family proteins are upregulated during inflammation and function to promote pathogenesis of the vascular system. il-6 family proteins use cytokine - specific receptors to activate a transmembrane receptor, gp130, which then recruits janus kinase (jak) to activate transcription factor signal transducer and activator of transcription 3 (stat3). stat3 then regulates various molecules at the transcriptional level, including suppressor of cytokine signaling 3 (socs3). socs3 acts as a negative feedback modulator of stat3 by inhibiting jak and subsequent stat3 activation (figure 1). in the retina, socs3 is expressed in the photoreceptor cells, mller glial cells, and retinal ganglion cells, and it inhibits stat3 activation in these cells [21, 22 ]. since stat3 activation induces further stat3-activating factors, such as the il-6 family ligands, the balance between stat3 activation and socs3 level is one of the key determinants of an inflammatory reaction [23, 24 ]. this balance between stat3 and socs3 also plays an important role during the development of the retina ; activated stat3 inhibits the photoreceptor - specific transcription factor crx at the transcriptional level, which in turn inhibits downstream photoreceptor - specific markers such as rhodopsin. retina - specific conditional knockout mice of socs3, -cre socs3 flox / flox mice (socs3cko), induce increase in the endogenous stat3 activation, and show delay in the initiation of rhodopsin expression at the transcriptional level. stat3 is activated in the embryonic retina but is shut down by socs3 which appears in the neonatal retina, thereby allowing rod photoreceptor cell differentiation. therefore, the timing of rod photoreceptor cell differentiation is fine - tuned by the initiation of socs3 expression and downregulation of stat3 activation. although stat3 activation in the socs3cko retina is still upregulated in the adulthood, the rhodopsin level in the socs3cko mice is compensated for by as - yet - unknown mechanisms and matches that of wild - type mice. a murine model of accelerated innate immunity, the endotoxin - induced uveitis model (eiu model), was used to further analyze the role of the gp130-stat3-socs3 loop in adult retinal inflammation. in this model, inflammatory cytokine, il-6 is upregulated, leading to stat3 activation [21, 26, 27 ] in the retina. this induction does not cause retinal cellular apoptosis, but it does reduce visual function [21, 26, 27 ] : rhodopsin protein levels decrease, and the rod photoreceptor outer segments (oss), where rhodopsin is concentrated, are shortened. the scotopic electroretinogram (erg) a - wave amplitude, which represents rod photoreceptor cell function, also decreases. stat3 activation correlates with the rhodopsin reduction in the adult retina, as in the developing retina. surprisingly, though, neither rhodopsin mrna nor its upstream regulator, crx, decreases in the adult retina during inflammation. this suggests that a different mechanism is involved in rhodopsin and crx regulation in the adult retina than in the developing retina [22, 25 ]. the role of activated stat3 in retinal dysfunction during inflammation has been analyzed using socs3cko mice. in these socs3-deficient mice, stat3 activation can increase greatly in the retina (figures 2(a) and 2(c)). thus, we have hypothesized that the mechanism of rhodopsin reduction during inflammation might be enhanced in these cells. as expected, the eiu models generated in the adult socs3cko mice showed a relative depletion of rhodopsin protein (figures 2(b) and 2(d)), followed by os shortening. the subsequent rod photoreceptor cell dysfunction, as measured by scotopic erg, was prolonged. this model also revealed that during inflammation, rhodopsin reduction is not regulated at the transcriptional level, but by a post - transcriptional inhibitory mechanism. the reduction in rhodopsin protein levels is rapid and global, starting only several hours from the onset of inflammation. under stress conditions, massive protein degradation through the ubiquitin - proteasome system (ups) is known to increase. a genetically abnormal rhodopsin protein that causes autosomal dominant retinitis pigmentosa, p23h, interacts with the ups and forms aggresomes aggresomes are inclusion bodies of accumulated waste proteins, formed when cellular degradation machinery is impaired or overwhelmed, and they are a pathologic finding in neurodegenerative diseases. in the case of p23h, the rhodopsin protein folds abnormally and accumulates rather than following the normal process of elimination from the cell. this finding hinted that genetically normal rhodopsin protein might also be degraded extensively following the excessive induction of the ups by inflammation. moreover, ubiquitin is present in the rod os under control conditions, thus it can rapidly degrade rhodopsin as needed. elevated levels of ubiquitin - conjugated rhodopsin are followed by rhodopsin depletion in the socs3cko eiu mouse model. the same process occurs in wild - type mice, but it is more rapid and more severe in the socs3cko mice, in which stat3 activation is increased. therefore, the activated stat3 level correlates with the ubiquitination and degradation of rhodopsin. this has been confirmed in vitro by using jak inhibitor to inhibit il-6-induced stat3 activation. it is illustrated by the preservation of rhodopsin levels under il-6-induced stat3 activation, when a stat3-dependent ubiquitin e3 ligase, ubiquitin - protein ligase e3 component n - recognin 1 (ubr1), is inhibited through the small inhibitory rna (sirna) system. we propose that it contributes to rhodopsin protein degradation during inflammation, especially given that inflammatory cytokines, including il-6, lif, and cntf, induce the ubiquitin - conjugation of rhodopsin protein and ubr1 expression in the rod photoreceptor cells, resulting in excessive rhodopsin degradation and disturbed visual functioning (figure 3). stat3 can be activated not only through the gp130 receptor, but also through an inflammatory diffusible factor, angiotensin ii. an angiotensin ii type 1 receptor blocker (arb) suppresses stat3 activation during inflammation directly, or indirectly, inhibiting il-6 production, thereby preserving rhodopsin levels and visual function. angiotensin ii also induces oxidative stress, which can induce the ubiquitination of specific proteins. the antioxidant lutein, which suppresses oxidative stress and the induction of reactive oxygen species (ros) during inflammation, also reduces stat3 activation, thus preserving the rhodopsin level and visual function during inflammation. therefore, the mechanism of visual dysfunction is, at least in part, explained by the excessive degradation of the essential protein during inflammation. in diabetic retinopathy, the main findings include microangiopathy [33, 34 ] and neurodegeneration [35, 36 ]. visual dysfunction begins before vascular abnormalities become obvious [68 ], and inner retinal dysfunction is reflected in changes in erg oscillatory potentials (ops) (figure 4). however, little is known about the molecular mechanism of diabetes - related neuronal degeneration. our recent analyses using a streptozotocin- (stz-) induced type 1 diabetes model shed light on this critical issue [35, 36 ]. both diabetes and hypertension are involved in metabolic syndrome, in which angiotensin ii signaling plays an important role. arbs have been approved for treatment of not only high blood pressure, but also diabetes - related renal failure. angiotensin ii is converted from angiotensinogen in a stepwise fashion by enzymes, including renin, angiotensin converting enzyme, and others. angiotensin ii can bind to either the angiotensin ii type 1 receptor (at1r) or type 2 receptor (at2r) on the cell surface, which in turn activates several contextually - dependent intracellular signals. these components of the rennin angiotensin system (ras) are all present physiologically in the retina, and are upregulated in pathological conditions, as we have shown in the murine model retina of stz - induced diabetes. at1r is coexpressed with the major synaptic vesicle protein synaptophysin in the inner layers of the retina. this is consistent with several previous reports showing the synaptic expression of at1r in the brain [38, 39 ]. synaptophysin, a synapse marker, is reduced in the postmortem brains of patients who had had neurodegenerative diseases such as parkinson 's disease and alzheimer 's disease. given that the ops in erg originate from inner retinal neurons bearing at1r, these erg changes may represent angiotensin ii - induced synaptophysin dysregulation and the resulting damage to visual function. we have verified this hypothesis by administering arb (either telmisartan or valsartan) to stz - induced diabetic mice. arbs protect the expression of synaptophysin protein and ops in the diabetic retina (figure 4). interestingly, synaptophysin mrna is not reduced in the diabetic retina, indicating that the protein 's reduction is regulated post - transcriptionally. post - transcriptional synaptophysin reduction caused by angiotensin ii exposure was reproduced in a rat neuronal cell line, pc12. in this system, synaptophysin protein degradation is inhibited by the proteasome inhibitor mg132, but not the lysosome inhibitor e64. at1r and its downstream extracellular signal - related protein kinase (erk) activation induce synaptophysin degradation, and at1r increases the ubiquitin - conjugated synaptophysin protein levels. angiotensin ii signaling activates erk in the diabetic retina in vivo, suggesting that the at1r - erk pathway is responsible for diabetes - induced pathogenic protein degradation through the ups. synaptophysin protein may be degraded by the mammalian homolog of drosophila seven in absentia (sina), an e3-ligase selective for synaptophysin named seven in absentia homologue (siah). since drosophila sina is regulated by erk signaling, the siah may also be regulated by erk activation, which is increased in the diabetic retina. erk activation and the resulting reduction of synaptophysin in the diabetic retina is also inhibited by an antioxidant, lutein, which indicates that angiotensin ii signaling and oxidative stress may share a role in the pathogenesis of diabetic retinopathy. not only that the reduction of synaptophysin, a synaptic protein, impairs the transmission of neuronal and visual signals, but impairment of synaptic activity itself inhibits neuronal cell survival [34, 35 ]. synaptic activity, that is, the neuronal electric stimuli, directly increases the levels of intracellular calcium ion in neurons, which promotes cell survival. moreover, brain - derived neurotrophic factor, bdnf, a neuronal survival factor, is regulated by neuronal synaptic activity [37, 38 ]. taken together, these findings suggest that the synaptophysin levels and the related neuronal synaptic activity function together to influence neuronal survival and neuronal network activity. the reduction of synaptophysin levels and neuronal activity, observed 1 month after the onset of diabetic retinopathy, is later followed by the apoptosis of retinal ganglion cells and inner retinal cells. therefore, one part of the neurodegenerative mechanism in the diabetic retina is explained by the excessive degradation of a protein that is essential for visual function (figure 5). the ups is a rapid and effective method of degrading specific proteins, and in many cases a protein is degraded only in response to a particular cellular signal or event. this process involves the coordinated actions of three enzymes a generally distributed e1 ubiquitin - activating enzyme, several more specific e2 ubiquitin - conjugating enzymes, and highly specific e3 ubiquitin ligases for the targeted protein. the ups involvement in pathogenesis has led to interest in targeting proteasomes as a therapeutic approach in several fields. ups is involved in cardiomyocyte cell pathogenesis : oxidized and ubiquitinated proteins are observed in rat hearts after cardiac ischemia / reperfusion injury [43, 44 ]. muscle wasting due to ups activation is also reported in cases of chronic kidney disease, diabetes, high angiotensin ii levels, and sepsis, all of which cause inflammation, inhibit insulin signaling, and promote glucocorticoid expression to induce protein degradation [4547 ]. this pathway can be blocked by overexpressing igf-1, which inhibits atrogin-1, an e3-ligase acting for muscle atrophy, through pi3k / akt [46, 48 ]. in the retina, innate inflammation activates the il-6-stat3 pathway, and diabetes activates the angiotensin ii - erk pathway. both pathways induce the ups, most likely through inducing a specific e3-ligase. moreover, both pathogenic conditions induce oxidative stress [27, 36 ], which oxidizes and unfolds proteins, after which they are easily ubiquitinated and pushed into the ups pathway. in contrast, modification of the 20s proteasome subunits by the lipid peroxidation product 4-hydroxy-2-nonenal (hne), which occurs in cardiac ischemia / reperfusion, results in the selective inactivation of 20s activity. thus, modified and ubiquitinated proteins may accumulate to induce cell death in some pathological conditions. the ups degrades numerous proteins, including apoptotic proteins, and regulates multiple signaling pathways. in human - dilated cardiomyopathy, the increased expression of the proapoptosis regulator p53 has recently been associated with ups dysregulation and accumulation of polyubiquitinated proteins. therefore, the above findings show that the ups, a selective and bulk protein degradation system, may be modified through multiple pathways. this system excessively degrades proteins necessary for tissue - specific function and/or cell survival, causing tissue pathogenesis. however, if this system is overwhelmed and/or dysregulated, modified proteins can accumulate and damage the cells. in inflammation, protein degradation damages tissue function, while it may also protect tissue from pathogenic protein accumulations. since the ups acts on specific proteins, regulating it may improve the prognosis. bortezomib, a dipeptide boronic acid, is the first fda - approved proteasome inhibitor for the treatment of multiple myeloma. bortezomib directly induces cell - cycle arrest and apoptosis, and it targets the tumor microenvironment. combination chemotherapy regimens using bortezomib have been developed that provide high rates of long - lasting remissions. interestingly, in patients treated with bortezomib, proteasome inhibition improves myocardial ischemia / reperfusion injuries, prevents postischemic ventricular tachyarrhythmias, promotes cardiac hypertrophy regression, and reverses diabetes - induced vascular endothelial dysfunction [52, 53 ]. proteasome inhibition can be also applied locally. in a balloon injury model of the rat carotid artery, a locally administered proteasome inhibitor, in addition to bortezomib, another proteasome inhibitor, sorafenib has been also approved by fda for advanced cancer therapy, and another candidate reagent is now under trial. in view of the potential therapeutic benefit of ups regulation inflammatory retinal diseases, including diabetic retinopathy, induce inflammatory cytokines that influence protein metabolism. excessive degradation of tissue function essential proteins is an important factor in retinal neuronal dysfunction. further analyses of the mechanisms that impair visual function may lead us to new therapeutic approaches for retinal neuroprotection. | understanding pathogenesis at the molecular level is the first step toward developing new therapeutic approaches. here, we review the molecular mechanisms of visual dysfunction in two common diseases, innate chorioretinal inflammation and diabetic retinopathy, and the role of the ubiquitin - proteasome system (ups) in both processes. in innate chorioretinal inflammation, interleukin-6 family ligands induce stat3 activation in photoreceptors, which causes ups - mediated excessive degradation of the visual substance, rhodopsin. in diabetic retinopathy, angiotensin ii type 1 receptor (at1r) signaling activates erk in the inner layers of the retina, causing ups - mediated excessive degradation of the synaptic vesicle protein, synaptophysin. this latter effect may decrease synaptic activity, in turn adversely affecting neuronal survival. both mechanisms involve increased ups activity and the subsequent excessive degradation of a protein required for visual function. finally, we review the therapeutic potential of regulating the ups to protect tissue function, citing examples from clinical applications in other medical fields. |
professional musicians have been used over the last 15 years as a model for brain plasticity. why are musicians so interesting for plasticity research ? first of all, they are experts in playing musical instruments. to play the demanding two three - second segments of the 11th variation from the sixth paganini etude by franz liszt, for example, requires the production of 30 notes per second. ericsson and colleagues were among the first to show how much professional musicians do in fact practice. the authors showed that professional pianists and violinists practice for 7,500 hours before reaching the age of 18 years, whereas music teachers can look back on a total practice time of approximately 3,500 hours. this difference was unaffected by the quality of musical education since all musicians in this study had graduated from the prestigious berlin academy of music. thus, the amount of practice is one of the most important factors influencing musical expertise, at least in terms of the skill required to play a musical instrument. if musicians practice that much, it is hypothesised, they should show some kind of neuroanatomical and neurophysiological adaptations. professional, semi - professional, and non - professional musicians have now been studied extensively in terms of the neuroanatomical and neurophysiological underpinnings of their expertise. in principle, three different approaches to studying plastic processes in musicians are possible : (a) the first approach is cross - sectional in nature and mostly employs quasi - experimental designs (post - test - only designs with non - equivalent groups in the terminology of cook and campbell). with this design, musicians and non - musicians are studied at the same point in time in terms of anatomical or functional brain measures. this approach has been widely used because it is relatively easy to collect the data. differences between both groups are attributed mostly to the different learning histories of musicians and non - musicians. however, the interpretation of these data is limited since this approach does not allow the inference of strong causation because it can not be ruled out that selection differences between the two groups or the different treatments (here, music lessons) are responsible for the results. to enhance the interpretability of such designs, several research groups have employed pretest measures related to musical expertise to control for pretest between - group differences this design, which is called the untreated control group design with proxy pretest measures, allows stronger causation about the influence of musical training. (b) the second approach used in this research context consists of short - term longitudinal studies in which subjects have undergone a specific training intervention. these studies are typically designed according to a pre - post design, and the subjects are enrolled in training programs lasting from several hours to several months. (c) finally, long - term longitudinal studies in which subjects have undergone a longer (at least a period of years) training are also used. longitudinal studies are more complicated in terms of organisation of the experiments, they take longer, and they are more expensive. in addition, longitudinal studies are repeated measurements studies, implying some methodological problems (for example, unwanted practice effects). to understand the influence of music practice on brain plasticity a general finding of the studies published thus far is that nearly all of those brain areas involved in the control of musical expertise (motor cortex, auditory cortex, cerebellum, and other areas) show specific anatomical and functional features in professional and semi - professional musicians. in the following, i will review most of the recent papers (after 2002) supporting the idea of brain plasticity driven by musical expertise and musical training. recently, hyde and colleagues published a paper strongly supporting the idea of use - dependent brain plasticity driven by musical training. in summary, this study demonstrates that 6-year - old children receiving instrumental musical training for 15 months (compared with children receiving non - musical training) not only learned to play their musical instrument but also showed changed anatomical features in brain areas known to be involved in the control of playing a musical instrument. most of these brain areas are part of the cortical motor system, but there were also structural changes in the auditory system and in the corpus callosum. this is the first longitudinal study demonstrating brain plasticity in children in the context of learning a musical instrument. although longitudinal studies are the gold standard in plasticity research, several cross - sectional studies demonstrating specific anatomical features in musicians have recently been published. omega sign (indicative of a larger hand motor area) on both hemispheres, whereas violinists showed the omega sign on only the right hemisphere controlling the left hand. this specific anatomical feature is possibly related to the fact that pianists practice a lot with both hands, whereas violinists practice a lot with their left hand (manipulating the strings) and their right arm (manipulating the bow). thus, violinists might drive only the right - sided hand motor area, whereas pianists drive the hand motor areas on both hemispheres. this interesting finding is in strong concordance with older studies reporting specific anatomical features in the hand motor area in pianists and violinists. using a voxel - based morphometry approach, gaser and schlaug identified grey matter volume differences in motor, auditory, and visual - spatial brain regions when comparing professional musicians (keyboard players in this study) with a matched group of amateur musicians and non - musicians. most interestingly, they found a strong association between structural differences (grey matter density), musician status, and practice intensity, supporting the view that practice (in this case, practicing to play a musical instrument) has an impact on brain anatomy. increased grey matter density (and volume) is currently taken as evidence of an increase in capillary density as well as smaller changes in synapse and glial cell density. thus, these changes might reflect neuroanatomical adaptations in order to improve the cognitive and motor functions controlled by these particular brain areas. most recently, a swedish group used diffusion tensor imaging (dti) to measure the integrity of fiber tracts (association fibers and commissures) in eight professional pianists and found a strong positive correlation between the measure of fractional anisotropy (fa) (indicating the integrity of the fiber system) and time spent practicing the piano. thus, the pianists who practiced more often showed higher fa values (indicating a higher integrity of the fiber system). this finding is of outstanding importance because it brings to light morphometric differences even within a highly specialised group of skilled pianists and indicates that these differences are due to practice time (specialisation of the specialised). in 2002, schneider and colleagues, of heidelberg, germany, reported a remarkable anatomical finding in musicians. using magnetoencephalography (meg) and sophisticated anatomical analyses, the authors found neurophysiological and anatomical differences between musicians and non - musicians. first, the neurophysiological activity in the primary auditory cortex 19 - 30 ms after tone onset was more than 100% larger. in addition, the grey matter volume of the anteromedial part of heschl s gyrus (which covers most of the primary auditory cortex) was 130% larger in musicians. this study is one of the first to indicate that both the morphology and neurophysiology of heschl s gyrus have an essential influence on musical aptitude., they found a strong relationship between the strategy used in processing complex tones and anatomical features in the primary auditory cortex. professional musicians who preferentially analyse the fundamental pitch (the fundamental tone, abbreviated f0 or f0, is the lowest frequency in a harmonic series) of complex tones were found to have a leftward asymmetry of grey matter volume in heschl s gyrus, whereas those who prefer to analyse the spectral pitch of complex tones show a rightward asymmetry of grey matter volume of heschl s gyrus. thus, a marked anatomical feature of the auditory system correlated with a particular tone - processing strategy within a group of professional musicians. patricia sluming and colleagues, of liverpool, uk, published a paper in which they reported anatomical differences in broca s area between musicians and non - musicians. in particular, the authors reported increased grey matter in broca s area in the left inferior frontal gyrus in musicians. in addition, they observed significant age - related volume reductions in cerebral hemispheres, dorsolateral prefrontal cortex bilaterally, and grey matter density in the left inferior frontal gyrus in controls but not in musicians ! in other words, musicians showed no or a smaller decrease in grey matter density in the frontal cortex compared with non - musicians with increasing age. (this is very important for aging research since the volume of the frontal cortex has been shown to correlate negatively with age.) this anatomical study suggests that orchestral musical performance might promote use - dependent retention, and possibly expansion, of grey matter within broca s area (a brain area that is responsible for speech production, language processing, and language comprehension as well as controlling facial neurons ; it is named after pierre paul broca, who discovered the area after studying the postmortem brain of a patient with a speech impairment). in addition, this study emphasises the significant point that shared neural networks (within broca s area) are involved in the control of language and music. in a more recent study, the same group showed that broca s area is also involved in the control of mental rotation, but only in musicians. they relate this extraordinary finding to the sight - reading skills of musicians. in sight reading broca s area might be involved in the control of this specific inter - relationship. the most recent study to use dti techniques was published by imfeld.. these authors measured the training effects on fa in the corticospinal tract (cst) of professional musicians and control non - musicians and found significantly lower fa values in both the left and the right cst in the musician group. diffusivity, a parameter indicating the amount of water that diffuses along and across the axon, was negatively correlated with the onset of musical training in childhood in the musician group. a subsequently performed median split into an early- and a late - onset musician group (median of 7 years) revealed increased diffusivity in the cst of the early - onset group as compared with both the late - onset group and the controls. in conclusion, dti was successfully applied in revealing plastic changes in white matter architecture of the cst in professional musicians. the present results challenge the notion that increased myelination induced by sensorimotor practice leads to an increase in fa, as has been suggested previously. instead, training - induced changes in diffusion characteristics of the axonal membrane may lead to increased radial diffusivity reflected in decreased fa values. however, this issue deserves more intensive discussion about the methodological aspects associated with fa and diffusivity measurements. besides the above - mentioned specific anatomical features in musicians recently, lappe. demonstrated particular changes with respect to the neurophysiological responses of the auditory cortex in non - musicians who trained for 2 weeks to play the piano. these authors randomly assigned subjects to one of two groups : one group learned to play a musical sequence on the piano, whereas the control group listened to the music that had been played by the other group. the authors demonstrated training - induced cortical plasticity using the musically elicited mismatch negativity (mmnm) from meg measurements before and after training. the mmnm is a neurophysiological response reflecting the pre - attentive processing of auditory stimuli. the subjects who learned to play piano showed significant enlargement of mmnm after training compared with the group who only listened to the music. thus, practicing to play the piano improves not only hand motor skills but also the auditory representation of the musical tones that are generated by the piano keys. thus, a strong crossmodal link between motor commands and the representation of auditory information is established, causing a stronger representation of musical information in the auditory cortex. several years ago, bangert and altenmller demonstrated a similar finding using electroencephalogaphy (eeg). they identified changed activations in frontal brain regions of subjects who had just 20 minutes of piano training, but only in the learning conditions during which the subjects could easily associate a particular piano key with a note. in situations during which this association was random effects of training have also been shown to be instrument - specific [23 - 25 ], and the eeg responses of children taking music lessons have been shown to change differently over the course of a year compared with those of children not studying music. thus, in summary, musicians or musically experienced subjects respond differently to musical stimuli even if top - down factors like attention are controlled for. there is also ample evidence of change in the auditory system due to musical practice and in the entire sensorimotor system [28 - 32 ]. interestingly, most of the recent findings indicate that even neurophysiological responses at the level of the brainstem are dependent on experience - dependent influences. neural activity generated from the brainstem can be measured using frequency following responses (ffrs). the ffr is an electrophysiological scalp - recorded electrical response that reflects processing stages of auditory information at the level of the brainstem. specifically, wong. first showed music - related plasticity in ffrs elicited by speech. later, musacchia. found that musicians had more robust ffrs to auditory and audiovisual speech and music sounds. the latter study also strengthened the notion that musical experience shapes not only auditory processing but multisensory mechanisms as well. however, both studies indicate that playing music enhances the fidelity of the earliest stage of auditory response, not only to musical stimuli but also to speech and multisensory cues. more recently, krishnan. analysed the ffrs from chinese and english subjects in response to four mandarin tonal contours presented in a non - speech context. the ffr analysis revealed that the chinese group exhibited stronger representation of multiple pitch - relevant harmonics relative to the english group across all four tones. the authors concluded that long - term experience (here, experience with mandarin) enhanced the sensitivity to linguistically relevant variations in pitch. thus, specific language experience changes the ffr in a manner similar to that of music experience. recently, hyde and colleagues published a paper strongly supporting the idea of use - dependent brain plasticity driven by musical training. in summary, this study demonstrates that 6-year - old children receiving instrumental musical training for 15 months (compared with children receiving non - musical training) not only learned to play their musical instrument but also showed changed anatomical features in brain areas known to be involved in the control of playing a musical instrument. most of these brain areas are part of the cortical motor system, but there were also structural changes in the auditory system and in the corpus callosum. this is the first longitudinal study demonstrating brain plasticity in children in the context of learning a musical instrument. although longitudinal studies are the gold standard in plasticity research, several cross - sectional studies demonstrating specific anatomical features in musicians have recently been published. omega sign (indicative of a larger hand motor area) on both hemispheres, whereas violinists showed the omega sign on only the right hemisphere controlling the left hand. this specific anatomical feature is possibly related to the fact that pianists practice a lot with both hands, whereas violinists practice a lot with their left hand (manipulating the strings) and their right arm (manipulating the bow). thus, violinists might drive only the right - sided hand motor area, whereas pianists drive the hand motor areas on both hemispheres. this interesting finding is in strong concordance with older studies reporting specific anatomical features in the hand motor area in pianists and violinists. using a voxel - based morphometry approach, gaser and schlaug identified grey matter volume differences in motor, auditory, and visual - spatial brain regions when comparing professional musicians (keyboard players in this study) with a matched group of amateur musicians and non - musicians. most interestingly, they found a strong association between structural differences (grey matter density), musician status, and practice intensity, supporting the view that practice (in this case, practicing to play a musical instrument) has an impact on brain anatomy. increased grey matter density (and volume) is currently taken as evidence of an increase in capillary density as well as smaller changes in synapse and glial cell density. thus, these changes might reflect neuroanatomical adaptations in order to improve the cognitive and motor functions controlled by these particular brain areas. most recently, a swedish group used diffusion tensor imaging (dti) to measure the integrity of fiber tracts (association fibers and commissures) in eight professional pianists and found a strong positive correlation between the measure of fractional anisotropy (fa) (indicating the integrity of the fiber system) and time spent practicing the piano. thus, the pianists who practiced more often showed higher fa values (indicating a higher integrity of the fiber system). this finding is of outstanding importance because it brings to light morphometric differences even within a highly specialised group of skilled pianists and indicates that these differences are due to practice time (specialisation of the specialised). in 2002, schneider and colleagues, of heidelberg, germany, reported a remarkable anatomical finding in musicians. using magnetoencephalography (meg) and sophisticated anatomical analyses, the authors found neurophysiological and anatomical differences between musicians and non - musicians. first, the neurophysiological activity in the primary auditory cortex 19 - 30 ms after tone onset was more than 100% larger. in addition, the grey matter volume of the anteromedial part of heschl s gyrus (which covers most of the primary auditory cortex) was 130% larger in musicians. this study is one of the first to indicate that both the morphology and neurophysiology of heschl s gyrus have an essential influence on musical aptitude., they found a strong relationship between the strategy used in processing complex tones and anatomical features in the primary auditory cortex. professional musicians who preferentially analyse the fundamental pitch (the fundamental tone, abbreviated f0 or f0, is the lowest frequency in a harmonic series) of complex tones were found to have a leftward asymmetry of grey matter volume in heschl s gyrus, whereas those who prefer to analyse the spectral pitch of complex tones show a rightward asymmetry of grey matter volume of heschl s gyrus. thus, a marked anatomical feature of the auditory system correlated with a particular tone - processing strategy within a group of professional musicians. patricia sluming and colleagues, of liverpool, uk, published a paper in which they reported anatomical differences in broca s area between musicians and non - musicians. in particular, the authors reported increased grey matter in broca s area in the left inferior frontal gyrus in musicians. in addition, they observed significant age - related volume reductions in cerebral hemispheres, dorsolateral prefrontal cortex bilaterally, and grey matter density in the left inferior frontal gyrus in controls but not in musicians ! in other words, musicians showed no or a smaller decrease in grey matter density in the frontal cortex compared with non - musicians with increasing age. (this is very important for aging research since the volume of the frontal cortex has been shown to correlate negatively with age.) this anatomical study suggests that orchestral musical performance might promote use - dependent retention, and possibly expansion, of grey matter within broca s area (a brain area that is responsible for speech production, language processing, and language comprehension as well as controlling facial neurons ; it is named after pierre paul broca, who discovered the area after studying the postmortem brain of a patient with a speech impairment). in addition, this study emphasises the significant point that shared neural networks (within broca s area) are involved in the control of language and music. in a more recent study, the same group showed that broca s area is also involved in the control of mental rotation, but only in musicians. they relate this extraordinary finding to the sight - reading skills of musicians. in sight reading broca s area might be involved in the control of this specific inter - relationship. the most recent study to use dti techniques was published by imfeld.. these authors measured the training effects on fa in the corticospinal tract (cst) of professional musicians and control non - musicians and found significantly lower fa values in both the left and the right cst in the musician group. diffusivity, a parameter indicating the amount of water that diffuses along and across the axon, was negatively correlated with the onset of musical training in childhood in the musician group. a subsequently performed median split into an early- and a late - onset musician group (median of 7 years) revealed increased diffusivity in the cst of the early - onset group as compared with both the late - onset group and the controls. in conclusion, dti was successfully applied in revealing plastic changes in white matter architecture of the cst in professional musicians. the present results challenge the notion that increased myelination induced by sensorimotor practice leads to an increase in fa, as has been suggested previously. instead, training - induced changes in diffusion characteristics of the axonal membrane may lead to increased radial diffusivity reflected in decreased fa values. however, this issue deserves more intensive discussion about the methodological aspects associated with fa and diffusivity measurements. besides the above - mentioned specific anatomical features in musicians, several recent (and older) studies have shown specific neurophysiological adaptations. recently, lappe. demonstrated particular changes with respect to the neurophysiological responses of the auditory cortex in non - musicians who trained for 2 weeks to play the piano. these authors randomly assigned subjects to one of two groups : one group learned to play a musical sequence on the piano, whereas the control group listened to the music that had been played by the other group. the authors demonstrated training - induced cortical plasticity using the musically elicited mismatch negativity (mmnm) from meg measurements before and after training. the mmnm is a neurophysiological response reflecting the pre - attentive processing of auditory stimuli. the subjects who learned to play piano showed significant enlargement of mmnm after training compared with the group who only listened to the music. thus, practicing to play the piano improves not only hand motor skills but also the auditory representation of the musical tones that are generated by the piano keys. thus, a strong crossmodal link between motor commands and the representation of auditory information is established, causing a stronger representation of musical information in the auditory cortex. several years ago, bangert and altenmller demonstrated a similar finding using electroencephalogaphy (eeg). they identified changed activations in frontal brain regions of subjects who had just 20 minutes of piano training, but only in the learning conditions during which the subjects could easily associate a particular piano key with a note. in situations during which this association was random effects of training have also been shown to be instrument - specific [23 - 25 ], and the eeg responses of children taking music lessons have been shown to change differently over the course of a year compared with those of children not studying music. thus, in summary, musicians or musically experienced subjects respond differently to musical stimuli even if top - down factors like attention are controlled for. there is also ample evidence of change in the auditory system due to musical practice and in the entire sensorimotor system [28 - 32 ]. interestingly, most of the recent findings indicate that even neurophysiological responses at the level of the brainstem are dependent on experience - dependent influences. neural activity generated from the brainstem can be measured using frequency following responses (ffrs). the ffr is an electrophysiological scalp - recorded electrical response that reflects processing stages of auditory information at the level of the brainstem. specifically, wong. first showed music - related plasticity in ffrs elicited by speech. later, musacchia. found that musicians had more robust ffrs to auditory and audiovisual speech and music sounds. the latter study also strengthened the notion that musical experience shapes not only auditory processing but multisensory mechanisms as well. however, both studies indicate that playing music enhances the fidelity of the earliest stage of auditory response, not only to musical stimuli but also to speech and multisensory cues analysed the ffrs from chinese and english subjects in response to four mandarin tonal contours presented in a non - speech context. the ffr analysis revealed that the chinese group exhibited stronger representation of multiple pitch - relevant harmonics relative to the english group across all four tones. the authors concluded that long - term experience (here, experience with mandarin) enhanced the sensitivity to linguistically relevant variations in pitch. thus, specific language experience changes the ffr in a manner similar to that of music experience. the preceding findings give rise to the question of whether there is transfer from musical to non - musical skills. a well - trained auditory system might support the perception of auditory speech information and thus auditory speech information might be processed more efficiently. in addition, when learning to play a musical instrument, the trainee also practices attention, planning functions, memory, and self - discipline. it is thus hypothesised that musical experience would positively influence executive functions, language functions, or even intelligence in general., one paper demonstrates that extended musical experience enhances executive control on a non - verbal spatial task and auditory tasks. glenn schellenberg uncovered a greater iq increase in children enrolled in music classes compared with well - matched children who received no musical lessons, and ho. uncovered an enhancement of verbal memory skills (but not visual memory skills) in children enrolled in musical lessons. there is therefore mounting evidence on the behavioural level of positive transfer from musical expertise to non - musical domains. recently, moreno. established that musical training (not longer than 6 months) improves non - musical functions such as reading and linguistic perception. this study is one of the very few longitudinal studies to have been conducted in the context of musical training. if music has such a strong influence on brain plasticity, this raises the question of whether this effect can be used to enhance brain plasticity and cognitive performance in general and clinical settings. in a recent single - blind randomised examined whether daily music listening enhances the recovery of cognitive functions and mood after stroke. this study demonstrates that recovery of verbal memory and focused attention improved significantly and substantially in the group of patients who listened to their favourite music on a daily basis compared with patients who listened to audio books or received no listening material. besides the cognitive improvement in the context of listening to music, there was a substantial mood improvement in the patients who listened to music. thus, music could be used as a non - invasive tool for neuropsychological and neurological therapies. in addition, musical elements could be used to improve specific cognitive functions for which positive transfer effects have been demonstrated. for example, reading and writing skills as well as memory functions are possible candidates for functions that might benefit from musical training elements. evidence shows that writing and reading can be improved when dyslexic children learn to associate graphemes and phonemes with musical notes and that many memory elements are linked to music. hopefully, the current trend in the use of musicians as a model for brain plasticity will continue in future experiments and extend to the field of neuropsychological rehabilitation. | music is becoming more and more of an issue in the cognitive neurosciences. a major finding in this research area is that musical practice is associated with structural and functional plasticity of the brain. in this brief review, i will give an overview of the most recent findings of this research area. |
fifteen years earlier, she had undergone left modified radical mastectomy for infiltrating ductal carcinoma of the breast (pathologic stating of t1n0m0, according to the american joint committee on cancer [ajcc ] tumor - node - metastasis [tnm ] classification for breast cancer). thereafter, she received chemotherapy twice and underwent radiation therapy (rt, 55 gy) for recurrence at the cervical, axillary, and internal mammary lymph nodes. a thorough work - up for dysphagia revealed squamous cell carcinoma at the mid - thoracic portion of the esophagus 26 to 30 cm from the upper incisor. transthoracic esophagectomy with mediastinal lymphadenectomy and reconstruction using a gastric tube according to the ivor lewis procedure was performed in the usual manner with hand - sewn anastomosis. because the apical portion of the right upper lobe was fibrotic and tightly attached to the parietal pleura, it was injured during dissection and wedge resection was done. the final pathologic report cited moderately differentiated squamous cell carcinoma that had invaded to the adventitia without metastasis to mediastinal (0/12) or perigastric lymph nodes (0/30) ; pathologic staging of t3n0m0 (stage iib) according to the 7th edition of the ajcc tnm classification for esophageal cancer. in the wedge - resected portion of the lung, atrophy and diffuse sclerosis was noted. serous fluid was drained through a chest tube at the rate of approximately 300 to 400 ml / day. on postoperative day 3, the patient complained of a sudden onset of dyspnea and continuous cough with a marked inspiratory and expiratory wheezing sound. on the chest x - ray, upper mediastinal widening was detected, but it was first thought to be related to the gastric conduit and was not different from the chest x - ray checked before. because respiratory distress was aggravated remarkably, endotracheal intubation followed by mechanical ventilation was applied, but her respiratory symptoms did not improve much in spite of the fact that the endotracheal tube was placed appropriately (fig. 2) with intravenous contrast demonstrated a large mediastinal fluid collection from the thoracic inlet to the inferior pulmonary vein level that caused marked luminal compression on the trachea and the gastric conduit. mediastinitis with abscess formation due to leakage at the anastomosis site was first considered, but leakage was not detected through a chest tube by using the methylene blue dye swallowing test. on explorative thoracotomy, inflammatory signs were not prominent in the pleural cavity. while mobilizing the gastric conduit, the clear serous fluid spilled out from the space between the trachea and the gastric conduit. even after draining the fluid completely, the space was not obliterated, which was assumed to be caused by rt - induced thickened and fibrotic mediastinal pleura. the esophagogastric anastomosis portion and the gastric conduit were preserved well without any infection or dehiscence. no leakage from the mediastinum was found after instillation of 100 ml of milk through a levin tube. upon placing the chest tube into this space, we considered the operation completed. the patient s respiratory - related symptoms all improved, and she was ready to be transferred to the general ward the next day. eight days after esophagectomy, a gastrografin (berlimed sa, madrid, spain) swallow study revealed that minor leakage was suspected at the esophagogastric anastomosis site. enteral feeding through a jejunostomy tube was started, and the next day, about 1,000 ml of a turbid fluid was drained, which was confirmed as chyle by a biochemical analysis of triglyceride contents. total parenteral nutrition was started again, and ten days later, drainage of chyle was no longer observed., pyeongtaek, korea) was delivered for aggravated focal dehiscence at the anastomosis site on the follow - up ct scan. the patient was discharged 2 months later ; then, she was eating a regular diet and had a mini pigtail drainage catheter (10fr ; uresil, skokie, il, usa) for empyema in the apex. although airway obstruction is a well - recognized complication of the anterior mediastinal mass in infants and children whose airways are compressible, it is quite rare in the adult population after esophageal surgery, and few such cases have been reported. three cases of tracheal compression have developed after transhiatal esophagectomy (the) with a gastric pull - up procedure [35 ]. the distended transposed stomach compressed the trachea in a 58-year - old male patient of achalasia, and another case of tracheal compression developed in a 61-year - old male patient with esophageal cancer who had undergone neo - adjuvant chemoradiotherapy. ikeda. reported a case of tracheal compression after the in a 66-year - old female patient with ankylosing spondylitis, implying that the possible causes were the small distance between the sternum and the vertebrae, and the loss of flexibility of the thorax and the mediastinum. because the is composed of blunt dissection and unintentional repositioning of the stomach within a limited space, the membranous portion of the trachea can be compressed by a transposed stomach, particularly when the space provides little capacity for them. it is more likely to occur when the mediastinal pleura around the esophagus is thickened or fibrotic. in contrast, during the ivor lewis operation, because the mediastinal pleura over the esophagus is dissected and the transposed gastric tube is rearranged in the space of the resected esophagus and the right pleural cavity, the trachea will not usually be compressed by the stomach, except upon the sudden onset of distension by bleeding. one case of tracheal compression after the ivor lewis operation was reported, which occurred due to intramural hematoma of the gastric tube in a patient of anticoagulation therapy for atrial fibrillation. in addition, an airway can be compressed by mediastinitis induced by intramural perforation and a peritracheal abscess, and mediastinal chyloma after a successful conservative treatment of chylothorax. in these cases, regretfully, the author did not perform a biochemical analysis during re - operation to confirm the diagnosis, but the possibility of chyloma was very high because chylothorax developed eventually, although the volume of drainage was limited and the nature had been clear until jejunostomy feeding despite instillation of milk during the re - operation. the chest ct scan was the most valuable test for the exact diagnosis, because the serial chest x - ray did not show tracheal obstruction even when respiratory distress developed. however, above all, the possibility of airway obstruction should always be considered when a patient with mediastinal pathology complains of the sudden onset of respiratory deterioration. the decision on how to manage the obstruction should be made as soon as possible, because complete obstruction can occur suddenly despite an appropriately positioned tracheal tube and mechanical ventilation when the endotracheal tube does not pass through the narrowed portion. in the case reported here, exploration was decided upon be cause a long segment of the trachea was compressed and mediastinal abscess with leakage was suspected. this case illustrates that the possibility of tracheal compression be considered in a patient who complains of respiratory deterioration after esophagectomy, even without abnormal findings on the serial chest x - ray. the author recently experienced a case of tracheal compression by loculated effusion after the ivor lewis operation and reports on this case along with a review of the related literature. | airway obstruction after esophageal surgery is quite rare, and few such cases have been reported. a 57-year - old woman who underwent the ivor lewis procedure for esophageal carcinoma complained of a sudden onset of severe dyspnea on postoperative day 3. chest computed tomography scan revealed that the collection of a large volume of mediastinal fluid caused marked luminal compression on the trachea and the gastric conduit. explorative thoracotomy revealed a clear serous fluid in the space between the trachea and the gastric conduit, and all respiratory symptoms were relieved after the fluid was drained. the possibility of tracheal compression by loculated effusion, such as chyloma, should be considered in a patient who complains of respiratory deterioration after esophageal surgery. |
s. le g. and b.k.f. are supported by the intramural research program of the national cancer institute, national institutes of health. k.j.p. was supported by the homing plus/2012 - 6/12 grant of the foundation for polish science and the national science center, poland (grant 2011/01/d / nz1/03478). | replication of retroviruses and transposition of endogenous retroelements exploits a unique mechanism of post - transcriptional regulation as a means of exporting their incompletely - spliced mrnas (which serve as both the genomic rna and the template for protein synthesis). following discovery of the rev response element (rre) that mediates nucleocytoplasmic export of the full - length and singly - spliced human immunodeficiency virus type 1 (hiv-1) genome, equivalent cis - acting regulatory elements have been characterized for both complex and simple retroviruses and retroelements, together with the obligate viral and host proteins with which they interact. the exception to this is the gammaretrovirus family of simple retroviruses, exemplified by reticuloendotheliosis virus (rev), murine leukemia virus (mlv) and xenotropic mlv - related retrovirus (xmrv). in this commentary, we discuss our recent data that reported structural and functional data on the mlv / xmrv post - transcriptional regulatory element (designated the pte). the pte was characterized by a highly - structured region of multiple stem - loops (sl1 sl7) overlapping the pro and 5 portion of the pol open reading frames, comprising a bipartite export signal whose structures are separated by 1400 nt. in addition, structural probing suggested that sl3 nucleotides were involved in pseudoknot formation. these data, when compared with rna transport elements of complex retroviruses (hiv) and simple murine retrotransposons (musd), collectively present an emerging picture that long - range tertiary interactions are critical mediators of their biological function. |
testicular tumors are rare and account for 1 - 1.5% of all the tumors affecting men but are the commonest tumors in 15 - 35 years of age. ninety to ninety five percent of testicular tumors are germ cell tumors (gcts), 52 - 56% of which are seminomas. with the current multimodality approach, gcts are considered as one of the most curable solid neoplasms with > 90% survival rates. chemotherapy is the usual first - line treatment for stage iia - iib gcts with bulky disease or stage iic - iii disease after high inguinal orchidectomy. postchemotherapy, 60 - 80% of seminomatous and 30% of nonseminomatous gcts (nsgcts) have residual masses, most commonly in the retroperitoneum. postchemotherapy retroperitoneal lymph node dissection (pc - rplnd) is indicated in patients with normal serum markers with radiologically identifiable disease in the retroperitoneum [especially in nsgct > 1 cm and seminoma > 3 cm with fluorodeoxyglucose (fdg) uptake ]. various studies show that postchemotherapy residual masses comprise necrosis in 50%, viable tumor in 15%, and teratoma in 35% of cases with nsgct and viable tumor in 10% and necrosis in 90% of patients with seminoma. thus, there is a significant rate of nontherapeutic exploration. in the absence of established imaging or nomogram parameters that can accurately predict the histology of residual masses thus, pc - rplnd is both diagnostic by providing definite histology and therapeutic by achieving local control. this is especially true for nsgct because a teratoma may be present in the retroperitoneal mass that is chemoresistant and has potential for local growth, invasion, and transformation. further, postchemotherapy residual masses can be considered chemotherapy resistant and they are better served by surgery. with growing experience of such surgical procedures refinement in surgical technique and better understanding of the anatomy, the morbidity rates are decreasing, further supporting its widespread role and incorporation into a therapeutic protocol. we present a tertiary care center experience of the role and postoperative outcomes of rplnd in patients with retroperitoneal residual masses after primary or salvage chemotherapy. from june 2003 to july 2012, a total of 70 patients underwent rplnd for gct, of whom 45 had received chemotherapy. patients who had residual, resectable retroperitoneal masses after chemotherapy, normal postchemotherapy tumor markers [alpha - fetoprotein and beta - human chorionic gonadotropin (beta - hcg) ] and no previous rplnd were included in this study. patients who underwent primary rplnd, had inadequate follow - up or incomplete data were excluded. all patients underwent detailed physical examination, contrast - enhanced computed tomography (cect) of the chest and abdomen, and tumor marker assessment preoperatively and clinical stage was assigned according to the american joint committee on cancer (ajcc) staging system and the international germ cell cancer collaborative group (igcccg) tumor risk categorization. the mean age of our patient group was 26.8 years (16 - 50 years) with primary testicular tumor on the right side in 18 (51.4%) patients and bilateral tumor in three (8.5%) patients. mixed gct was the most prevalent histology of primary tumor present in 19 (54.5%) patients. thirteen (37.1%) patients had teratoma in the primary tumor, either alone (in 6 patients - 17.1%) or as a component of mixed gct (in 7 patients - 20%). seminoma was present in four (11.4%) patients, all of whom had undescended testes. characteristics of primary testicular tumor the most common site of retroperitoneal mass was the paraaortic region (14 patients - 40%) and the mean size of retroperitoneal mass was 8.8 cm (range 2.2 cm-20.4 cm) before chemotherapy and 5.4 cm (range 1.2 cm-14.6 cm) postchemotherapy. characteristics of retroperitoneal mass all patients received cisplatin - based chemotherapy either as induction (29 patients) or salvage therapy (six patients). twenty - two (62.8%) patients received four cycles of bep (bleomycin, etopside, cisplatin), five (14.2%) patients received three cycles of bep, and two (5.7%) patients received four cycles of ep (etopside and cisplatin) as induction therapy. salvage chemotherapy was given to six patients, five (14.2%) received four cycles of vip (vinblastin, ifosfamide, cisplatin), and one (2.8%) received four cycles of tip (paclitaxel, ifosfamide, cisplatin). two patients were given radiotherapy in addition to four cycles of bep as induction therapy. rplnd was performed by abdominal or thoracoabdominal approach in standard bilateral infrahilar template and nerve sparing was done whenever possible. standard template extended from renal vessels superiorly to bifurcation of common iliac vessels inferiorly and up to ureters laterally and anterior spinal ligament and psoas muscle fascia posteriorly. split and roll technique was used to dissect the lymph node packet. in patients with small volume residual disease and minimal desmoplastic reaction, nerve - sparing technique with preservation of sympathetic chains, postganglionic fibers, and hypogastric postoperatively, all patients were scheduled for regular follow - up every 3 months with physical examination, serum tumor markers, chest x - ray, and cect of the abdomen and pelvis for the first 2 years and then every 6 months for the next 2 years and then annually. the median follow - up was of 33 months (range 9 months-60 months) and data were censored on january 1, 2014. all patients received cisplatin - based chemotherapy either as induction (29 patients) or salvage therapy (six patients). twenty - two (62.8%) patients received four cycles of bep (bleomycin, etopside, cisplatin), five (14.2%) patients received three cycles of bep, and two (5.7%) patients received four cycles of ep (etopside and cisplatin) as induction therapy. salvage chemotherapy was given to six patients, five (14.2%) received four cycles of vip (vinblastin, ifosfamide, cisplatin), and one (2.8%) received four cycles of tip (paclitaxel, ifosfamide, cisplatin). two patients were given radiotherapy in addition to four cycles of bep as induction therapy. rplnd was performed by abdominal or thoracoabdominal approach in standard bilateral infrahilar template and nerve sparing was done whenever possible. standard template extended from renal vessels superiorly to bifurcation of common iliac vessels inferiorly and up to ureters laterally and anterior spinal ligament and psoas muscle fascia posteriorly. split and roll technique was used to dissect the lymph node packet. in patients with small volume residual disease and minimal desmoplastic reaction, nerve - sparing technique with preservation of sympathetic chains, postganglionic fibers, and hypogastric postoperatively, all patients were scheduled for regular follow - up every 3 months with physical examination, serum tumor markers, chest x - ray, and cect of the abdomen and pelvis for the first 2 years and then every 6 months for the next 2 years and then annually. the median follow - up was of 33 months (range 9 months-60 months) and data were censored on january 1, 2014. all patients received cisplatin - based chemotherapy either as induction (29 patients) or salvage therapy (six patients). twenty - two (62.8%) patients received four cycles of bep (bleomycin, etopside, cisplatin), five (14.2%) patients received three cycles of bep, and two (5.7%) patients received four cycles of ep (etopside and cisplatin) as induction therapy. salvage chemotherapy was given to six patients, five (14.2%) received four cycles of vip (vinblastin, ifosfamide, cisplatin), and one (2.8%) received four cycles of tip (paclitaxel, ifosfamide, cisplatin). two patients were given radiotherapy in addition to four cycles of bep as induction therapy. rplnd was performed by abdominal or thoracoabdominal approach in standard bilateral infrahilar template and nerve sparing was done whenever possible. standard template extended from renal vessels superiorly to bifurcation of common iliac vessels inferiorly and up to ureters laterally and anterior spinal ligament and psoas muscle fascia posteriorly. split and roll technique was used to dissect the lymph node packet. in patients with small volume residual disease and minimal desmoplastic reaction, nerve - sparing technique with preservation of sympathetic chains, postganglionic fibers, and hypogastric postoperatively, all patients were scheduled for regular follow - up every 3 months with physical examination, serum tumor markers, chest x - ray, and cect of the abdomen and pelvis for the first 2 years and then every 6 months for the next 2 years and then annually. the median follow - up was of 33 months (range 9 months-60 months) and data were censored on january 1, 2014. all our patients underwent pc - rplnd by an open approach ; an abdominal incision was used in 32 (91.4%) and thoracoabdominal incision in three (8.5%) patients. standard template rplnd (as described) was performed in all the patients with three patients requiring extension of template in suprahilar area for residual masses. nerve sparing was possible in seven patients (20%) (unilateral sparing in five and bilateral sparing in two) with low volume stage iia / b disease. fourteen patients (40%) required 15 adjunctive procedures, the most common being nephrectomy in nine (25.7%) patients, which was more common on the left side (six patients). adjunctive procedures were more common in patients with larger retroperitoneal masses (> 5 cm) (9 out of 12 patients with > 5 cm residual mass), patients who presented with stage iii disease (six out of 10 patients) or have received salvage chemotherapy (four out of six patients) or had fibrosis as the final histology (six out of six patients). most of the surgical complications were of clavien grade i or ii (the most common being blood transfusion in 28.5% patients) and required conservative management. only two (5.7%) patients required surgical intervention for clavien grade iii complications. one patient who developed adhesive intestinal obstruction in the immediate postoperative period was re - explored on postoperative day 12 after a failed trial of conservative management. intraoperatively, there was a band at terminal ileum with twisting of the small bowel requiring adhesiolysis and detorsion. another patient developed paralytic ileus after drain removal on postoperative day 5 and ultrasonography of the patient revealed a 19 cm 10 cm localized collection in the right paracolic gutter extending into the pelvis that turned out to be of serous nature on diagnostic tapping. ultrasound - guided percutaneous drain was placed under local anesthesia and the ileus resolved after 2 days. the drain was removed after 5 days when serial abdominal ultrasounds documented no residual collection. complications were more common in patients with larger postchemotherapy masses (> 5 cm) and fibrosis as the final histology. characteristics of surgical procedures surgical complications the histopathology of rplnd specimen was fibrosis or necrosis in 17 (48.5%) patients, teratoma in 12 (34.2%) patients, and viable tumor in six (17.1%) patients. on retrospect, among these six cases with viable tumor, two had lung metastasis and three were at high risk according to the igcccg risk classification, and the clinical stage was of either stage iic (three cases) or stage iii (three cases). four of these patients had received salvage chemotherapy and the retroperitoneal mass size was > 5 cm in three patients. the median follow - up was of 33 months (ranging 9 - 60 months). antegrade ejaculation was preserved in five out of 28 (17.8%) patients with standard rplnd and in five out of seven (71.4%) patients with nerve - sparing procedure. seven (20%) patients had recurrence over a median follow - up of 33 months. three patients had recurrence in the retroperitoneum ; bilateral complete rplnd was performed in two patients and extended rplnd in one. three patients had distant recurrence, one had bony recurrence, and the other two had pulmonary recurrence. out of these four patients with retroperitoneal recurrence, the histopathology of the resected residual mass was viable tumor in two, teratoma in one, and necrosis in one patient. five of these seven patients with recurrences succumbed to their disease and two were alive with stable disease at the last follow - up. surgical resection of residual retroperitoneal lymph node mass represents an integral part of the multimodality treatment for patients with advanced testicular cancer undergoing systemic chemotherapy. the objective is to remove persistent retroperitoneal lymph nodes that may have mature teratoma in about 40 - 50% or viable tumor in 10 - 20% of cases. in our series, out of 35 patients, 26 presented with advanced disease (stage iic in 16 and stage iii in 10) and 11 patients were in the poor risk category according to the igccc tumor risk categorization. the postchemotherapy mean retroperitoneal tumor size was 5.4 cm (range 1.2 cm-14.6 cm). bulky retroperitoneal mass necessitated bilateral classical rplnd in 32 patients with extended dissection in three cases. fourteen patients required adjunctive procedures and the complication rate was 37.1%. at a median follow - up of 33 months nerve sparing may be used in pc - rplnd although the rates of antegrade ejaculation are lower than that for primary rplnd as complete resection of the often large, adherent mass supersedes ejaculatory concerns. in our series, nerve - sparing procedure was possible in seven patients with low volume iia / iib disease with preservation of antegrade ejaculation in five (71.4%) patients without any disease recurrence with a median follow - up of 42 months. in 28 patients who had undergone bilateral standard template rplnd, this compares favorably with 79 - 89% antegrade ejaculation rates and 98% 5-year relapse free survival rates reported in the literature. the small number of nerve - sparing procedures in our series was attributed to the advanced stage of presentation and bulky retroperitoneal disease. in pc - rplnd, 20 - 25% of the patients require adjunctive procedures for completeness of the resection, nephrectomy being the most common amongst them. in our series, 14 (40%) patients underwent adjunctive procedure and among them, nine (25.7%) underwent nephrectomy. four patients required resection of adjacent structures and two required separation of the ureter from the desmoplastic mass (ureterolysis). of those who underwent adjunctive nephrectomy, four patients received salvage chemotherapy, three had advanced stage (iii) disease, and two had bulky retroperitoneal disease (retroperitoneal tumor size > 10 cm). the higher proportion of patients with adverse features in our pc - rplnd series may explain the higher rate of adjunctive nephrectomy unlike the usual 25% reported in the literature. in our series, histopathological analysis of the nephrectomy specimen revealed residual disease in six (66.6%) patients, justifying the need for adjunctive nephrectomy in these patients. the complication rates for postchemotherapy surgery are higher than that for primary rplnd. in our series, the complication rate was 37.1% that compares well with the reported rates of 20 - 35% in various series. most of the complications were of clavien grade i and ii, which included blood transfusion, postoperative fever, superficial wound infection, and paralytic ileus. the higher complication rate in our series is attributed to the advanced stage of presentation, bulky retroperitoneal disease, and high number of patients with salvage chemotherapy with severe desmoplastic reaction, all of which are known risk factors. luz. observed that patients with fibrosis in the residual mass had more complications and ended up undergoing nonnerve - sparing procedures. these findings pose a dilemma when offering pc - rplnd in residual masses as almost half of the patients will have necrosis and nontherapeutic resections with higher complications. we observed seven recurrences including three retroperitoneal, three distant, and one combined. among these 7 patients with recurrence all belonged to the igccc high risk category and 4 patients had a viable tumor in residual mass and received postoperative chemotherapy. fox. noted that if viable gct was present in the retroperitoneal mass and is entirely resected, two additional cycles of adjuvant chemotherapy confer a disease - free survival of 70%. in our series among the seven patients with recurrences (local or distant), five died due to disseminated neoplasia and the estimated 5-year overall survival of pc - rplnd after induction and salvage chemotherapy has been reported as 85 - 95% and 44 - 61%, respectively. in our series, the estimated 5-year overall survival rate after pc - rplnd in our series was 83.75% and approaches the results of previously reported series. pc - rplnd is a relatively safe procedure in experienced hands with an acceptable morbidity rate. half of the residual masses have teratoma or viable tumor, thereby, justifying surgical resection. complete removal of post - chemotherapy residual mass remains the standard of care and allows improved oncologic and functional outcomes. | introduction : we aimed to study the outcomes of retroperitoneal lymph node dissection (rplnd) in postchemotherapy residual masses in advanced testicular germ cell tumor (gct) in the indian population.patients and methods : we retrospectively analyzed 35 patients who underwent postchemotherapy rplnd at our institute after primary (29 patients) or salvage (6 patients) chemotherapy over a period of 9 years (june 2003 to july 2012).results : the mean age of our patients was 26.8 years. 18 (51.42%) presented with primary tumor in the right testis and 3 (8.51%) had bilateral tumors. mixed gct was the most common histology among 19 (54.3%) patients. 14 (40%) patients had the residual mass in para - aortic location, which was the most common site. 14 (40%) patients required an adjunctive procedure, most commonly nephrectomy which was required in 9 out of 14 (25.7%). we recorded 25 complications, mostly clavien - dindo grade ii. histopathology of residual mass was necrosis in 17 (48.57%), teratoma in 12 (34.28%), and viable tumor in 6 (17.14%) patients.conclusion:nearly half of the patients had either teratoma or viable tumor, thus justifying the surgical resection of postchemotherapy residual mass. although nearly half of the patients had complications, they were adequately managed and there was no mortality. thus, postchemotherapy rplnd can be a useful procedure in multimodality approach to gct in carefully selected patients. |
we report the discovery of hd5-cd, an unprecedented c2-symmetric -barrel - like covalent dimer of the cysteine - rich host - defense peptide human defensin 5 (hd5). dimerization results from intermonomer disulfide exchange between the canonical -defensin cysii cysiv (cys5cys20) bonds located at the hydrophobic interface. this disulfide - locked dimeric assembly provides a new element of structural diversity for cysteine - rich peptides as well as increased protease resistance, broad - spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen acinetobacter baumannii. |
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this study was conducted under institutional review board approval from wake forest university school of medicine. identification, clinical characteristics, and recruitment of african american patients and controls have been previously described in detail (22). briefly, 1,033 unrelated african american patients with type 2 diabetes were recruited from dialysis facilities. type 2 diabetes was diagnosed in african americans who reported developing type 2 diabetes after the age of 25 years and who did not receive only insulin therapy since diagnosis. in addition, cases had to have at least one of the following three criteria for inclusion : 1) type 2 diabetes diagnosed at least 5 years before initiating renal replacement therapy, 2) background or greater diabetic retinopathy, and/or 3) 100 mg / dl proteinuria on urinalysis in the absence of other causes of nephropathy. an additional 1,106 unrelated african americans without a current diagnosis of diabetes or renal disease were recruited from the community and internal medicine clinics as controls. all type 2 diabetic cases and nondiabetic controls were born in north carolina, south carolina, georgia, tennessee, or virginia. dna extraction was performed using the puregene system (gentra systems, minneapolis, mn). the dna screening panel was composed of 96 african american subjects : 48 type 2 diabetic cases and 48 controls. dna sequencing reactions were performed using bigdye terminator v.1.1 cycle sequencing kits and analyzed on the applied biosystems 3730xl dna analyzer (applied biosystems, foster city, ca). a search of the region sequenced was performed at dbsnp (www.ncbi.nlm.nih.gov) to record all previously identified polymorphisms reported in the region. genotyping of dg10s478 was performed by fragment length analysis on an abi prism dna analyzer 3700 with previously published primers (21) in a manner similar to that previously described (23). snp genotyping was performed on the iplex massarray genotyping platform (sequenom, san diego, ca). for all snps, the genotyping success rate was greater than 95%. in preliminary analyses, snps in the tcf7l2 gene region 10 kb (c10:114689999114926060) were imputed using data from hapmap phase ii hybrid panel (1:1, yri : ceu ; 108 snps) and the 1000 genomes yri pilot 1 dataset (497 snps) to circumvent limited coverage of the genetic diversity in this specific region by the affymetrix 6.0 array. snps were imputed in 965 type 2 diabetic end - stage renal disease (esrd) cases and 1,029 controls with high - quality score (rsq - hat 0.3) using the software mach (www.sph.umich.edu/csg/abecasis) (24). four common snps identified from direct sequence analysis had minor allele frequencies (maf) 0.05 and failed assay design on the sequenom platform. with the use of the resequencing genotype data obtained from the 96 african american samples as reference, these snps were imputed in the remaining 2,043 samples with high - quality score (rsq - hat 0.5) using the software mach (www.sph.umich.edu/csg/abecasis) (24). snps were tested for departure from hardy - weinberg equilibrium (hwe) using an exact test of hwe proportions for the combined group of cases and controls and then for cases only and controls only (25). haplotype block structure was established using haploview 4.1 (26), defining blocks using the method from gabriel. unadjusted measures of ld and association were assessed using the software snpgwa (http://www.phs.wfubmc.edu) (28). snpgwa computes ld statistics, d and r, for each pair of tandem snps. snpgwa also performs multiple tests of association including the overall two - degree of freedom test (genotype), dominant model, recessive model, additive model (cochran - armitage trend test), and the corresponding lack of fit to the additive model. odds ratios, 95% confidence intervals, and p values were computed for each model of association. assuming a log additive model, x = (1 f) + 2f(1 f) + f where is the estimated odds ratio (or) and f is the risk allele frequency. ancestry estimates were determined from 70 biallelic admixture informative markers (aims) as previously described (16,29). briefly, aims were selected to maximize european and african allele frequency differences and sample all non - acrocentric arms of the autosomal genome. reference population allele frequencies were derived by genotyping 44 african (yoruba from ibadan, nigeria) and 39 european americans. individual ancestral proportions were generated for each subject using frappe (30), an expectation - maximization algorithm, under a two - population model. the individual ancestral estimates were used as covariates in the association analyses. to test whether snps were independently associated with type 2 diabetes, we performed an omnibus test for the haplotype association using plink (31). we further adjusted the omnibus test by controlling for one of the snps at a time. an insignificant conditional test suggests that the conditioned snp is sufficient to explain the haplotype association and there is a single, rather than multiple, association signals at the haplotype. this study was conducted under institutional review board approval from wake forest university school of medicine. identification, clinical characteristics, and recruitment of african american patients and controls have been previously described in detail (22). briefly, 1,033 unrelated african american patients with type 2 diabetes were recruited from dialysis facilities. type 2 diabetes was diagnosed in african americans who reported developing type 2 diabetes after the age of 25 years and who did not receive only insulin therapy since diagnosis. in addition, cases had to have at least one of the following three criteria for inclusion : 1) type 2 diabetes diagnosed at least 5 years before initiating renal replacement therapy, 2) background or greater diabetic retinopathy, and/or 3) 100 mg / dl proteinuria on urinalysis in the absence of other causes of nephropathy. an additional 1,106 unrelated african americans without a current diagnosis of diabetes or renal disease were recruited from the community and internal medicine clinics as controls. all type 2 diabetic cases and nondiabetic controls were born in north carolina, south carolina, georgia, tennessee, or virginia. dna extraction was performed using the puregene system (gentra systems, minneapolis, mn). the dna screening panel was composed of 96 african american subjects : 48 type 2 diabetic cases and 48 controls. dna sequencing reactions were performed using bigdye terminator v.1.1 cycle sequencing kits and analyzed on the applied biosystems 3730xl dna analyzer (applied biosystems, foster city, ca). a search of the region sequenced was performed at dbsnp (www.ncbi.nlm.nih.gov) to record all previously identified polymorphisms reported in the region. genotyping of dg10s478 was performed by fragment length analysis on an abi prism dna analyzer 3700 with previously published primers (21) in a manner similar to that previously described (23). snp genotyping was performed on the iplex massarray genotyping platform (sequenom, san diego, ca). in preliminary analyses, snps in the tcf7l2 gene region 10 kb (c10:114689999114926060) were imputed using data from hapmap phase ii hybrid panel (1:1, yri : ceu ; 108 snps) and the 1000 genomes yri pilot 1 dataset (497 snps) to circumvent limited coverage of the genetic diversity in this specific region by the affymetrix 6.0 array. snps were imputed in 965 type 2 diabetic end - stage renal disease (esrd) cases and 1,029 controls with high - quality score (rsq - hat 0.3) using the software mach (www.sph.umich.edu/csg/abecasis) (24). four common snps identified from direct sequence analysis had minor allele frequencies (maf) 0.05 and failed assay design on the sequenom platform. with the use of the resequencing genotype data obtained from the 96 african american samples as reference, these snps were imputed in the remaining 2,043 samples with high - quality score (rsq - hat 0.5) using the software mach (www.sph.umich.edu/csg/abecasis) (24). snps were tested for departure from hardy - weinberg equilibrium (hwe) using an exact test of hwe proportions for the combined group of cases and controls and then for cases only and controls only (25). haplotype block structure was established using haploview 4.1 (26), defining blocks using the method from gabriel. unadjusted measures of ld and association were assessed using the software snpgwa (http://www.phs.wfubmc.edu) (28). snpgwa computes ld statistics, d and r, for each pair of tandem snps. snpgwa also performs multiple tests of association including the overall two - degree of freedom test (genotype), dominant model, recessive model, additive model (cochran - armitage trend test), and the corresponding lack of fit to the additive model. odds ratios, 95% confidence intervals, and p values were computed for each model of association. assuming a log additive model, x = (1 f) + 2f(1 f) + f where is the estimated odds ratio (or) and f is the risk allele frequency. ancestry estimates were determined from 70 biallelic admixture informative markers (aims) as previously described (16,29). briefly, aims were selected to maximize european and african allele frequency differences and sample all non - acrocentric arms of the autosomal genome. reference population allele frequencies were derived by genotyping 44 african (yoruba from ibadan, nigeria) and 39 european americans. individual ancestral proportions were generated for each subject using frappe (30), an expectation - maximization algorithm, under a two - population model. to test whether snps were independently associated with type 2 diabetes, we performed an omnibus test for the haplotype association using plink (31). we further adjusted the omnibus test by controlling for one of the snps at a time. an insignificant conditional test suggests that the conditioned snp is sufficient to explain the haplotype association and there is a single, rather than multiple, association signals at the haplotype. controls were significantly younger than type 2 diabetic cases (p 0.05 ; aggressive tagging algorithm) with a mean r = 0.73. notably, the snp of greatest interest, rs7903146, is not typed on the affymetrix 6.0 array. this snp is located in a genomic interval that is not tagged well (max r = 0.45), resulting in only nominal evidence of association in the affymetrix 6.0 analysis. to circumvent limited coverage of the genetic diversity in this specific region, imputation was used. using data from hapmap phase ii hybrid panel (1:1, yri : ceu), no variants were identified with the same magnitude of significance as rs7903146. in a separate analysis, 497 snps were imputed from the 1000 genomes yri pilot 1 dataset (supplementary fig. two variants were identified (rs33998771, chr10:114740378) proximal to our region with significant p values (3.58 10 and 3.29 10, respectively) similar to that of rs7903146. to test whether these snps (rs33998771, chr10:114740378, rs7903146) were independently associated with type 2 diabetes, we performed an omnibus test for the haplotype association controlling for one snp at a time. four common haplotypes (act, att, ttt, and ttc) accounted for 99.9% of all haplotypes. these haplotype frequencies were significantly different between type 2 diabetic cases and controls (0.042, 0.019, 0.268, and 0.672, respectively for cases ; 0.020, 0.016, 0.229, and 0.735, respectively for controls, omnibus test p = 5.4 10), with the haplotypes ttc and act strongly associated with protection and risk for type 2 diabetes, respectively (p 0.80). snp rs7903146 and microsatellite dg10s478, depicted as gray circles, were typed independently in the same set of samples. estimated recombination rates from hapmap are plotted in the background to depict the ld structure in the region. (a high - quality color representation of this figure is available in the online issue.) fourteen additional snps were genotyped in an expanded set of type 2 diabetic cases and controls within this interval and analyzed for association as summarized in table 2. the core region of association was between snps rs4132115 and rs7903146 (admixture - adjusted additive p values ranging from 0.012 to 2.38 10). this region encompasses a 4.3-kb ld block in the yri population (hapmap phase ii yri data), which is bounded by the two snps rs7901695 and the previously associated rs7903146 (16). single snp genotypic association results for snps in the tcf7l2 gene showing association with type 2 diabetes esrd inconsistent with hwe in cases. in addition, the previously associated (21) microsatellite marker dg10s478, which lies 38 kb distal to and outside of this ld block, was typed, and the results of the analysis are presented in table 3. allele sizes and frequencies were consistent with prior genotyping in samples from african populations (15). evidence of association was observed with the protective alleles 4 and 16 (p = 0.043 and 5.02 10, respectively) and the risk allele 8 (p = 0.022). dg10s478 allelic association with type 2 diabetes esrd in african americans the core region of association (c10:114744078114748339) 2 kb was analyzed by direct sequence analysis in 96 samples (48 type 2 diabetic cases and 48 controls). a total of 46 snps were identified, of which 72% (33/46) were novel and 17% (8/46) had a maf > 5%. when genotyped in the expanded cohort, five of the novel snps were found to be monomorphic and 10 could not be typed on the sequenom platform because of the repetitive nature of the region. these 10 were genotyped via direct sequence analysis on a subset of 96 type 2 diabetic cases and 96 controls. four snps were common, and these sequencing data were paired with existing data to be used as the known set of haplotypes for imputation in the remaining cohort. in addition, rs61875120 was common but yielded poor quality imputation (rsq = 0.31) and was therefore genotyped by direct sequence analysis. the remaining five snps had low maf (maf 0.74 ; fig. 2) and associated with disease susceptibility (or = 1.301.37) under an additive model. three common haplotypes (cct, ccc, and ttc) accounted for 99.6% of all haplotypes. these haplotype frequencies were significantly different between type 2 diabetic cases and controls (0.342, 0.056, and 0.602, respectively for cases ; 0.278, 0.058, and 0.664, respectively for controls, omnibus test p = 3.7 10). omnibus analysis revealed that the haplotype association was lost after adjusting for rs7903146 (p = 0.85), whereas modest association remained by conditioning rs34872471 or rs35198068 (p = 0.05), suggesting that rs7903146 alone is sufficient to explain the overall haplotype association. single snp genotypic association results for snps identified by direct sequence analysis in the tcf7l2 gene showing association with type 2 diabetes esrd low maf in 96 samples resulting in poor imputation.. a : haploview - generated ld map of the 40 snps identified by direct sequence analysis (c10:114742846114750274) in african american controls (n = 1,106). regions of high ld (d = 1 and logarithm of the odds [lod ] > 2) are shown in dark red. markers with lower ld (0.45 2) are shown in dark through light red, with the color intensity decreasing with decreasing d value. regions of low ld and low lod scores (lod 0.05) identified by direct sequence analysis (c10:114742846114750274) in african american controls (n = 1,106). regions of high ld (d = 1 and lod > 2) are shown in dark red. markers with lower ld (0.45 2) are shown in light red, with the color intensity decreasing with decreasing d value. regions of low ld and low lod scores (lod 0.05 ; aggressive tagging algorithm) with a mean r = 0.73. notably, the snp of greatest interest, rs7903146, is not typed on the affymetrix 6.0 array. this snp is located in a genomic interval that is not tagged well (max r = 0.45), resulting in only nominal evidence of association in the affymetrix 6.0 analysis. to circumvent limited coverage of the genetic diversity in this specific region, imputation was used. using data from hapmap phase ii hybrid panel (1:1, yri : ceu), 108 no variants were identified with the same magnitude of significance as rs7903146. in a separate analysis, 497 snps were imputed from the 1000 genomes yri pilot 1 dataset (supplementary fig. two variants were identified (rs33998771, chr10:114740378) proximal to our region with significant p values (3.58 10 and 3.29 10, respectively) similar to that of rs7903146. to test whether these snps (rs33998771, chr10:114740378, rs7903146) were independently associated with type 2 diabetes, we performed an omnibus test for the haplotype association controlling for one snp at a time. four common haplotypes (act, att, ttt, and ttc) accounted for 99.9% of all haplotypes. these haplotype frequencies were significantly different between type 2 diabetic cases and controls (0.042, 0.019, 0.268, and 0.672, respectively for cases ; 0.020, 0.016, 0.229, and 0.735, respectively for controls, omnibus test p = 5.4 10), with the haplotypes ttc and act strongly associated with protection and risk for type 2 diabetes, respectively (p 0.80). snp rs7903146 and microsatellite dg10s478, depicted as gray circles, were typed independently in the same set of samples. estimated recombination rates from hapmap are plotted in the background to depict the ld structure in the region. (a high - quality color representation of this figure is available in the online issue.) fourteen additional snps were genotyped in an expanded set of type 2 diabetic cases and controls within this interval and analyzed for association as summarized in table 2. the core region of association was between snps rs4132115 and rs7903146 (admixture - adjusted additive p values ranging from 0.012 to 2.38 10). this region encompasses a 4.3-kb ld block in the yri population (hapmap phase ii yri data), which is bounded by the two snps rs7901695 and the previously associated rs7903146 (16). single snp genotypic association results for snps in the tcf7l2 gene showing association with type 2 diabetes esrd inconsistent with hwe in cases. in addition, the previously associated (21) microsatellite marker dg10s478, which lies 38 kb distal to and outside of this ld block, was typed, and the results of the analysis are presented in table 3. allele sizes and frequencies were consistent with prior genotyping in samples from african populations (15). evidence of association was observed with the protective alleles 4 and 16 (p = 0.043 and 5.02 10, respectively) and the risk allele 8 (p = 0.022). the core region of association (c10:114744078114748339) 2 kb was analyzed by direct sequence analysis in 96 samples (48 type 2 diabetic cases and 48 controls). a total of 46 snps were identified, of which 72% (33/46) were novel and 17% (8/46) had a maf > 5%. when genotyped in the expanded cohort, five of the novel snps were found to be monomorphic and 10 could not be typed on the sequenom platform because of the repetitive nature of the region. these 10 were genotyped via direct sequence analysis on a subset of 96 type 2 diabetic cases and 96 controls. four snps were common, and these sequencing data were paired with existing data to be used as the known set of haplotypes for imputation in the remaining cohort. in addition, rs61875120 was common but yielded poor quality imputation (rsq = 0.31) and was therefore genotyped by direct sequence analysis. the remaining five snps had low maf (maf 0.74 ; fig. 2) and associated with disease susceptibility (or = 1.301.37) under an additive model. three common haplotypes (cct, ccc, and ttc) accounted for 99.6% of all haplotypes. these haplotype frequencies were significantly different between type 2 diabetic cases and controls (0.342, 0.056, and 0.602, respectively for cases ; 0.278, 0.058, and 0.664, respectively for controls, omnibus test p = 3.7 10). omnibus analysis revealed that the haplotype association was lost after adjusting for rs7903146 (p = 0.85), whereas modest association remained by conditioning rs34872471 or rs35198068 (p = 0.05), suggesting that rs7903146 alone is sufficient to explain the overall haplotype association. single snp genotypic association results for snps identified by direct sequence analysis in the tcf7l2 gene showing association with type 2 diabetes esrd low maf in 96 samples resulting in poor imputation.. a : haploview - generated ld map of the 40 snps identified by direct sequence analysis (c10:114742846114750274) in african american controls (n = 1,106). regions of high ld (d = 1 and logarithm of the odds [lod ] > markers with lower ld (0.45 2) are shown in dark through light red, with the color intensity decreasing with decreasing d value. regions of low ld and low lod scores (lod 0.05) identified by direct sequence analysis (c10:114742846114750274) in african american controls (n = 1,106). regions of high ld (d = 1 and lod > 2) are shown in dark red. markers with lower ld (0.45 2) are shown in light red, with the color intensity decreasing with decreasing d value. regions of low ld and low lod scores (lod 0.74 ; fig. 1) and associated with disease susceptibility (or = 1.301.37). this analysis suggests that association at rs34872471 and rs35198068 was the result of correlation with the true signal from rs7903146. although this study has eliminated the possibility of additional common variants (maf > 0.01) contributing to type 2 diabetes susceptibility within the fine - mapped interval (c10:114744078114748339 2 kb) of the tcf7l2 locus, four variants (ivs3 + 42245, ivs3 + 42428, ivs3 + 43487, and ivs4 43007) were found to have maf < 0.01. to date these variants have not been identified by other ongoing studies, i.e., the 1000 genomes project, suggesting they are private mutations. additionally, given the low maf, these variants are not likely to explain the association observed at the tcf7l2 locus, but we can not rule out the possibility that these and other unidentified rare variants contribute to disease susceptibility. if this were so, effect sizes of such rare variants would have to be in a range unprecedented for noncoding variants. as a result of fine - mapping the tcf7l2 locus to determine the region most likely to harbor susceptibility variants, dg10s478 is located 41 kb proximal to the critical interval defined in the african american population and is in weak ld with rs7903146 (d = 0.35, r = 0.07). only a single common allele of dg10s478 is nominally associated with type 2 diabetes, with the strongest association, which is protective, seen with low maf variants. this study represents the first comprehensive evaluation of variation within the tcf7l2 gene in a large african american population. taking advantage of the ld structure in our african - derived sample of african americans, we were able to reduce the genomic interval of association to 4.3 kb and exclude the possible contribution of the previously identified microsatellite marker to type 2 diabetes susceptibility. our analysis identified three snps, rs34872471, rs35198068 (imputed), and rs7903146, which were highly associated with type 2 diabetes ; all had p values that were two orders of magnitude stronger than other snps. snp rs7903146 remains the most significantly associated variant within the tcf7l2 gene with a calculated par of 17.4%. we can not exclude this possibility with total confidence, but the assessment of markers in tcf7l2 by direct genotyping, imputation, and then through the use of the 1000 genomes data using conditional analysis suggests the likelihood that such a common variant exists is low. evaluation of long range ld on chromosome 10 shows little evidence for a remote variant (data not shown). an alternative is the possibility that a rare variant of large effect in ld with rs7903146 is the actual functional variant. although theoretically possible (33), we have recently shown empirically that it is easy to differentiate between a rare functional variant with large effect and a common variant in ld (34). thus, fine - mapping at the tcf7l2 locus using an african ancestry population has statistically implicated rs7903146 as the causal variant. (20) have implicated rs7903146 as a functional variant by mapping sequence variants to open chromatin sites. they found that rs7903146 is located in islet - selective open chromatin, and human islet samples heterozygous for rs7903146 showed allelic imbalance in islet enhancer activity. thus genetic and functional studies make a consistent case for a functional role for rs7903146. | objectivevariation in the transcription factor 7-like 2 (tcf7l2) locus is associated with type 2 diabetes across multiple ethnicities. the aim of this study was to elucidate which variant in tcf7l2 confers diabetes susceptibility in african americans.research design and methodsthrough the evaluation of tagging single nucleotide polymorphisms (snps), type 2 diabetes susceptibility was limited to a 4.3-kb interval, which contains the yri (african) linkage disequilibrium (ld) block containing rs7903146. to better define the relationship between type 2 diabetes risk and genetic variation we resequenced this 4.3-kb region in 96 african american dnas. thirty - three novel and 13 known snps were identified : 20 with minor allele frequencies (maf) > 0.05 and 12 with maf > 0.10. these polymorphisms and the previously identified dg10s478 microsatellite were evaluated in african american type 2 diabetic cases (n = 1,033) and controls (n = 1,106).resultsvariants identified from direct sequencing and databases were genotyped or imputed. fifteen snps showed association with type 2 diabetes (p < 0.05) with rs7903146 being the most significant (p = 6.32 106). results of imputation, haplotype, and conditional analysis of snps were consistent with rs7903146 being the trait - defining snp. analysis of the dg10s478 microsatellite, which is outside the 4.3-kb ld block, revealed consistent association of risk allele 8 with type 2 diabetes (odds ratio [or ] = 1.33 ; p = 0.022) as reported in european populations ; however, allele 16 (maf = 0.016 cases and 0.032 controls) was strongly associated with reduced risk (or = 0.39 ; p = 5.02 105) in contrast with previous studies.conclusionsin african americans, these observations suggest that rs7903146 is the trait - defining polymorphism associated with type 2 diabetes risk. collectively, these results support ethnic differences in type 2 diabetes associations. |
diabetes mellitus (dm) is a chronic metabolic disease which is characterized by hyperglycemia, absolute or relative deficiencies in insulin secretion, and/or insulin action. the new classification proposed by the american diabetes association in 1997 was based on the pathogenesis of the disease and comprises four categories : type 1 dm (dm1), type 2 dm (dm2), other types, and gestational diabetes. dm1 is mostly due to genetic disorders and an autoimmune disease resulting in the selective destruction of -cells in the pancreas that leads to insulin loss. dm2, also called noninsulin - dependent diabetes mellitus (niddm), is characterized by insulin resistance with significant metabolic dysfunction including obesity, impaired insulin function and secretion, and increased endogenous glucose output. although the two types of diabetes have distinct etiologies, they lead to similar diabetic complications, and both of them are related to oxidative stress status. it is demonstrated that activation of oxidative stress pathways plays a key role in the development of not only the late complications (such as cardiovascular disease, nephropathy, retinopathy, and amputations) in dm1 and dm2, but also in the early stage such as insulin resistance. there are many sources of reactive oxygen species (ros) production in diabetes including mitochondrial and nonmitochondrial origins : nadph oxidase, xanthine oxidase, uncoupled enos, lipoxygenase, cyclooxygenase, cytochrome p450 enzymes, and other hemoproteins, and mitochondrion is thought to be the main source of ros generation site in dm. the mechanisms about ros and dm development were shown in figure 1. the uncoupling proteins (ucps) are a family of mitochondrial transport proteins located in the inner mitochondrial membrane. these anion - carrier proteins transport protons (h) to the mitochondrial matrix and in turn dissipate the proton motive force as heat and uncouple the substrate oxidation from the production of atp. ucp1 is mainly expressed in brown adipose tissue (bat), which is responsible for thermogenesis in newborns. ucp2 is widely distributed in several tissues including the spleen, kidney, immune system, pancreas, and central nervous system, whereas ucp3 is mainly restricted to the skeletal muscle, and ucp4 and ucp5/bmcp1 are mainly expressed in the brain. besides the nonshivering thermogenesis function of ucp1, functions of the other ucps are still unclear. ucp2 is reported to be involved in glucose and lipid metabolism to control immune cell activation by modulating mapk pathways and the production of mitochondrial ros, and a neuroprotective role is also suggested based on the regulation of mitochondria membrane potential, production of ros, preservation of calcium homeostasis, modulation of neuronal activity, and eventually inhibition of cellular damage. ucp3 is suggested to be involved in mediating energy expenditure via uncoupling, especially in fatty acid metabolism, and it seems to protect mitochondria against lipid - induced oxidative stress, which makes this protein a potential player in the development of dm2. fewer studies focused on the physiologic roles of ucp4 and ucp5, and the protection against oxidative stress and mitochondrial dysfunction are also reported. although the physiological functions of ucps are still not been completely elucidated, their abilities of reducing mitochondrial ros formation are widely accepted. that is, high membrane potential of mitochondria will induce ros production and thus oxidative damage ; these ros may activate ucps and therefore cause a mild uncoupling and (as a negative feedback) will prevent further superoxide production and decrease oxidative damage (figure 2). this antioxidative activity of ucps makes it logical to search any benefit on dm through counteracting the oxidative stress appeared in dm and the complications. on the other hand, it is also found that ucp2 is a negative regulator of insulin secretion ; the superoxide - mediated activation of ucp2 causes pancreatic -cell dysfunction. the vast majority of diabetes are type 1 or type 2 (dm2 represents at least 80 percent and dm1 accounts for about 510 percent), and increasing numbers of studies focused on the roles of ucps on dm or the complications. we will review the progress in the relations between ucps (ucp1, ucp2, and ucp3) and dm1 and/or dm2 in the following. the relationship between ucp1 and dm has already been reported long before the other ucps were discovered in 1997. studies revealed that ucp1 mrna and protein concentrations in bat were regulated by insulin [12, 13 ]. as ucp1 has been proved to decrease membrane potential, downregulate ros generation, and increase energy expenditure, so ucp1 gene is regarded as a candidate gene for obesity, dm2, or related traits. the role of ucp1 in the development of obesity and dm2 has been reviewed in 2010 and 2012 [14, 15 ] ; the authors focused on the polymorphisms 3826a / g, 1766a / g, and 112a / c in the promoter region, ala64thr in exon 2, and met299leu in exon 5 of ucp1 gene and pointed out that they are possibly associated with obesity, lipid / lipoprotein - related disease, and/or dm2. the 3826a / g polymorphism of ucp1 was further reported to be associated with diabetic retinopathy (dr) in dm1 group and the ucp1 gene expression was increased in human retina ; while the same polymorphism of ucp1 (3826a / g) was not found the association with dm2 with european ancestry in another study, although a significant association between the ucp2 ala55val and ucp3 55c / t polymorphisms and increased susceptibility for dm2 were detected in asians in the same study. the 3826a / g polymorphism influenced ucp1 gene expression : g allele carriers had higher ucp1 cdna and protein concentrations than a / a carriers. and more interestingly, g allele carriers exhibited increased mnsod2 expression, which suggested that this allele could be a marker of oxidative stress. as oxidative stress is related to dr, so this deleterious polymorphism in ucp1 gene is suggested to be a risk factor for dr (multivariate analysis confirmed that the g / g genotype was an independent risk factor for dr). ucp1 had been thought to be expressed only in rodents and human infants for a long time ; however, ucp1 protein and/or its mrna expression were detected in human white adipose tissue, skeletal muscle, longitudinal smooth muscle layers, retinal cells, and islet cells recently [18, 19 ], although the physiological functions of ucp1 in these tissues and organs are not established as well as in bat. in 2013, the adult human neck brown fat is further reported with the anatomical localization, gene expression profiling, and functional characterization. the imbalance between energy intake and expenditure is the underlying cause of obesity and dm. bat consumes fuel for thermogenesis through tissue - specific ucp1 ; it was once thought that bat had a functional role in rodents and human infants only, but it has been recently shown that in response to mild cold exposure adult human bat consumes more glucose per gram than any other tissues. in addition to this nonshivering thermogenesis, human bat may also combat weight gain by becoming more active in the setting of increased whole - body energy intake. this suggests that activation of human bat could be used as a safe treatment for obesity and metabolic dysregulation and further help to cure dm. in view of the ucp1 gene expressed in the other tissues, more attention may be paid to the role of ucp1 in muscle tissue, islet cells, and thymus function in the future. ucp2 is the most widely distributed ucp and highly expressed in pancreatic -cells in dm, so it is the most frequently studied one concerning its role in dm. ross activate ucp2, which results in proton leak across the mitochondrial inner membrane, and this leads to reduced -cell atp synthesis and content, which is a critical parameter in regulating glucose - stimulated insulin secretion (gsis). the recent reviews about ucp2 and dm were published in 2009 and 2011 [2325 ]. early in 2001, just 4 years after the discovery of ucp2, it was reported that ucp2 negatively regulated insulin secretion and was a major link between obesity, -cell dysfunction, and dm2. in a model of obesity - induced diabetes mice (ob / ob mice) the ucp2-deficient mice had higher islet atp levels and increased gsis ; this indicated that ucp2 negatively regulates insulin secretion. the ob / ob mice lacking ucp2 had restored first - phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia. in recent years, the mitochondrial ros generation or oxidative stress in -cell was found to be a prominent role in the effect of ucp2 on insulin secretion, although the consequences were not always consistent. in a study published in 2009, all of the three highly congenic strain backgrounds (c57bl/6j, a / j, 129/svimj) ucp2/ mice exhibited increased oxidative stress, and the gsis in ucp2/ islets of each congenic strain was significantly decreased. ucp2 knockdown in ins-1e insulinoma cells improved the gsis, and this can be annulled completely by the cell - permeative antioxidant mntmpyp. in the islets of -cell - specific ucp2 knockout mice, all of these three studies described a ros - related pathway about the role of ucp2 and gsis. that is to say, the ucp2 knockdown will cause elevation of ros and/or oxidative stress and then the enhancement of gsis. a study about the associations between polymorphisms in ucp2 and ucp3 with dm2 was carried out in korea and found that the ucp2 5331g > a and ucp3 2078c > t polymorphisms are susceptibility markers for dm2 among koreans. among the other three studies, no significant association of the ucp2 866g / a polymorphism with dm2 risk the study about asian indians indicated that ala55val polymorphism at ucp2 and 55c / t polymorphism at ucp3 are associated with a significantly reduced risk of developing dm2. while these correlations are different between europeans and asian descent : neither the ucp2 ala55val nor the ucp3 55c / t polymorphism showed any significant association with dm2 risk in europeans (or 1.04, 95% ci 0.98, 1.09 for ala55val ; or 1.04, 95% ci 1.00, 1.09 for 55c / t) ; and in contrast, a statistically significant association was observed for both polymorphisms in participants of asian descent (or 1.23, 95% ci 1.12, 1.36 for ala55val ; or 1.15, 95% ci 1.03, 1.28 for 55c / t). in a study of the relationship between ucp2 polymorphisms and proliferative diabetic retinopathy (pdr), three ucp2 polymorphisms were selected (866g / a (rs659366), ala55val (rs660339), and 45 bp insertion / deletion (ins / del)), and the haplotype [a val ins ] was an independent risk factor for pdr in both types 1 and 2 diabetic groups. three studies focused on the relationship between 866g / a polymorphism and obesity in a balinese population, ischemic stroke in chinese dm2 patients, and obesity in danes and showed a significant association between them [3537 ]. american ginseng stimulates insulin production and prevents apoptosis through downregulation of ucp2 in cultured -cells. inhibited ucp2 expression by an antisense oligonucleotide can reverse diet - induced dm mice models by the effects on both insulin secretion and action. microrna-15a positively regulates insulin synthesis by directly targeting and inhibiting ucp2 gene expression. a chinese medicine, kaiyuqingre formula, improves insulin secretion via decreasing the overexpression of ucp2 in cultured ins-1 cells. korean red ginseng promoted the expression of insulin and downregulated the expression of ucp2 in the spontaneously diabetic goto - kakizaki rats. most of the studies about the role of ucp2 in dm focused on the ucp2 functions in -cells, and the results indicated a deleterious effect of ucp2 on dm. as ucp2 is widely expressed in many tissues, the antioxidative activities (downregulating mitochondrial ros generation) of this protein should be evaluated in the future. much fewer studies focused on the role of ucp3 in dm, for it was thought to be expressed restrictedly in skeletal muscle for a long time. ucp3 mrna and protein levels are decreased in skeletal muscle of patients with dm2 compared to healthy control subjects. ucp3 protein content is reduced in prediabetic subjects (i.e., impaired glucose tolerance) and dm2, and eight weeks of rosiglitazone treatment significantly increases insulin sensitivity and restores skeletal muscle ucp3 protein in diabetic patients. similar to ucp2, ucp3 was found to be expressed in pancreatic -cells in 2008, where it also influenced insulin secretion, although the physiological function of ucp3 in -cells is still not known. ucp3 mrna is expressed in human islets ; the relative abundance of ucp2 mrna was 8.1-fold higher (p a and ucp3 55c > t variants on prospective risk of dm2 and found that these variations in the ucp2-ucp3 gene cluster are associated with an increased risk of dm2. in 2008, a report focused on 14 tag single nucleotide polymorphisms (tsnp) (rs637028, rs653263, rs622064, rs2306820, rs673494, rs655717, rs643064, rs660339, rs659366, rs591758, rs668514, rs647126, rs1800006, and rs1800849), for each of the 14 tsnps across the genomic region of the ucp2-ucp3 gene cluster ; they did not observe significant effects on dm2. however, haplotype - based analyses suggest that a haplotype set defined by rs591758, rs668514, rs647126, and rs1800006 was significantly associated with dm2 risk in caucasian women only, especially among those who were overweight. this may be emphasizing that the dm2 development is driven by a multifactor model more than a single - factor one. in a finnish diabetes prevention study, three variants in the ucp2 gene, one variant in the ucp2-ucp3 intergenic region, and five variants in the ucp3 gene were explored. the authors concluded that the genetic variations in the ucp2-ucp3 gene cluster may act as a modifier increasing serum lipid levels and indices of abdominal obesity and may thereby also contribute to the metabolic aberrations observed in obesity and dm2. another study analyzed other 14 tsnps (rs622064, rs2306820, rs655717, rs660339, rs17132534, rs659366, rs668514, rs3741135, rs1626521, rs2734827, rs3781907, rs1800849, rs1685333, and rs826071) in ucp2-ucp3 gene cluster and could not find an association of any of the 14 tsnps tested with dm2 risk. this result is similar to the other 14 tsnps study mentioned previously, and they may both imply the same fact that the development of dm2 is a multifactor style ; any one of the 14 tsnps is not enough to affect the dm2. it is important to illustrate how the polymorphisms of ucp genes are involved in the development of dm, and this may help to develop new strategies for dm prevention and/or treatment. the present data indicated that all of the three ucps (ucp1, ucp2, and ucp3) have some kind of relationships with the development of dm (figure 3). as dm is a multifactorial disease, the ethnic differences, gender, genomic factors, age, nutritional characteristics, lifestyle, and even environmental factors are all related to the outcomes. the single ucp or single ucp gene variation may not be enough to affect the results. more importantly, the physiological function of ucps, at least ucp2 and ucp3, is still unclear, which may limit this kind of study very much. so, it is better to keep on exploring the physiological function of ucps in the future, and based on this, the synergetic studies on the multisite of ucps variations may help to elucidate the relationship between ucps and dm. | uncoupling proteins (ucps) are anion carriers expressed in the mitochondrial inner membrane that uncouple oxygen consumption by the respiratory chain from atp synthesis. the physiological functions of ucps have long been debated since the new ucps (ucp2 to 5) were discovered, and the role of ucps in the pathogeneses of diabetes mellitus is one of the hottest topics. ucps are thought to be activated by superoxide and then decrease mitochondrial free radicals generation ; this may provide a protective effect on diabetes mellitus that is under the oxidative stress conditions. ucp1 is considered to be a candidate gene for diabetes because of its role in thermogenesis and energy expenditure. ucp2 is expressed in several tissues and acts in the negative regulation of insulin secretion by -cells and in fatty acid metabolism. ucp3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. several gene polymorphisms of ucp1, ucp2, and ucp3 were reported to be associated with diabetes. the progress in the role of ucp1, ucp2, and ucp3 on diabetes mellitus is summarized in this review. |
antiphospholipid antibodies are circulating immunoglobulins that are associated with both venous and arterial thrombotic events. the antiphospholipid antibody syndrome is defined by 2 major criteria : the occurrence of at least 1 clinical thrombotic event or pregnancy morbidity (either 1 otherwise unexplained fetal death after 10 weeks gestation, 1 premature birth before 34 weeks gestation because of preeclampsia, eclampsia or placental insufficiency, or 3 unexplained pregnancy losses before 10 weeks gestation), and the presence of antiphospholipid antibodies measured on 2 or more occasions at least 12 weeks apart. venous thrombosis is the most common systemic manifestation of the antiphospholipid antibody syndrome, but within the central nervous system, arterial thrombosis is more common than venous thrombosis. arterial stroke is the presenting symptom in 13% of patients with the antiphospholipid antibody syndrome [2, 3 ]. imaging studies of patients with the antiphospholipid antibody syndrome have emphasized the parenchymal changes noted on ct and mri. these studies have shown that subcortical infarcts and hyperintense white matter foci are common in patients presenting both with and without clinical strokes. fewer studies have reported on the angiographic findings in stroke patients with the antiphospholipid antibody syndrome. these studies have shown predominantly intracranial stem or branch occlusions and irregularities of the cerebral vessel walls consistent with vasculopathy [4, 5 ]. these angiographic findings suggest a link between the antiphospholipid antibody syndrome and cerebral arteriopathies and raise the possibility that arteriopathies and a hypercoagulable state are both implicated in the increased risk of arterial stroke. whereas most arteriopathies in stroke patients with the antiphospholipid antibody syndrome involve the intracranial circulation, occasionally the extracranial internal carotid artery is affected. little is known about this manifestation and about the natural progression of stenosis of the extracranial internal carotid artery in patients with the antiphospholipid antibody syndrome. we describe a young patient who experienced progressive steno - occlusive disease of her bilateral extracranial internal carotid arteries in the setting of antiphospholipid antibodies. a 39-year - old woman was transferred to our hospital after being found unconscious at home. she was confused, but was able to give a history of left - sided weakness with decreased use of her left hand and difficulty bearing weight on her left leg that had begun abruptly 3 days prior to presentation. she had had one prior outside hospital admission 1 year earlier for a left middle cerebral artery stroke that presented with expressive aphasia and right arm and leg weakness. limited mri at that time revealed 2 areas of restricted diffusion in the left parietal lobe with increased flair signal and associated cortical / subcortical enhancement. axial t1-weighted images showed the absence of a flow void in the left internal carotid artery, while the right internal carotid artery appeared patent based on the presence of a normal flow void. the patient underwent stereotactic biopsy of the left parietal lesion to rule out a neoplasm. she was discharged to inpatient rehabilitation, and over the subsequent months her speech and motor function completely recovered. three years prior to her current presentation, the patient had had a miscarriage at 12 weeks gestation. her history was also significant for intravenous methamphetamine use, ending 13 years prior to presentation, and an 18 pack - year smoking history. she did not use birth control pills and did not have a history of deep venous thrombosis or pulmonary embolism. her family history was remarkable for systemic lupus erythematosus in her father and a brother who was diagnosed with thrombotic thrombocytopenia purpura. on examination, her blood pressure was 126/80 mm hg, and her heart rate was 91 beats / min and regular. she was alert and oriented to person, but not place, time, or situation. her speech was fluent, with intact repetition and naming and without aphasia or dysarthria. cranial nerve examination was significant for a left homonymous hemianopsia and left lower facial weakness. her motor exam revealed left hemiparesis, left hyperreflexia, and a left babinski sign. she had intact sensation of light touch bilaterally, but extinction on the left with double simultaneous stimuli. abnormal coagulation studies included a prolonged activated partial thromboplastin time at 42.6 s (normal range 25.137.6 s) which did not correct with a 1:1 mix with normal plasma, a thromboplastin inhibition test that was strongly positive for lupus anticoagulant, and a dilute russell 's viper venom test that was prolonged at 64.3 s (normal 2946 s) which corrected with the addition of phospholipids. other coagulation studies, which included anticardiolipin and anti-2 glycoprotein antibodies, were within normal limits. erythrocyte sedimentation rate (55 mm / h) and c - reactive protein (5.7 mg / l) were moderately elevated, and the antinuclear antibody assay was elevated at 1:160. other studies were normal or negative, including complete blood count, platelet count, renal, liver, and thyroid functions, hemoglobin a1c, and ldl cholesterol. a ct scan showed areas of evolving infarction in the right anterior and middle cerebral artery distributions. ct angiography showed bilateral internal carotid artery occlusions extending from the level of the bifurcation to the supraclinoid segments. mri showed areas of acute infarction within the right middle cerebral artery - anterior cerebral artery watershed territory and chronic infarcts in the left middle cerebral artery - anterior cerebral artery watershed territory (fig. 1). conventional angiography confirmed bilateral internal carotid artery occlusions in the proximal cervical segments, with reconstitution of the carotid arteries at the supraclinoid segments via the ethmoidal branches of the distal internal maxillary arteries and the ophthalmic arteries (fig. the posterior circulation was normal, with extensive collaterals to the anterior circulation via the posterior communicating arteries. no abnormalities concerning for vasculopathy or dissection were noted in the aortic arch or in the carotid, vertebral, subclavian, renal, or femoral arteries. our patient met clinical and laboratory criteria for a diagnosis of the antiphospholipid antibody syndrome. she was young, had a characteristic history of prior spontaneous abortion at more than 10 weeks gestation, and had a strongly positive thromboplastin inhibitor test consistent with lupus anticoagulant antibodies. her presentation with strokes secondary to bilateral carotid artery occlusions suggests an association between the antiphospholipid antibody syndrome and a vasculopathy involving the extracranial segments of the internal carotid arteries. the causal relationship between the antiphospholipid antibody syndrome and bilateral carotid occlusions is further supported by the absence of reasonable alternative causes of carotid occlusions in this young patient. specifically, there was no history of head or neck trauma and no evidence of dissection or atherosclerosis on imaging. she was a smoker, but she had no history of hypertension, hyperlipidemia, or other vascular risk factors. her medical history indicates that vasculopathy of the internal carotid arteries in the setting of the antiphospholipid antibody syndrome can be rapidly progressive. mri at that time was notable for an absence of a flow void in the left internal carotid artery, consistent with a left carotid occlusion, and the presence of a flow void in the right internal carotid artery, indicating patency of that vessel. within 1 year, however, she presented with a right middle cerebral artery stroke, and workup revealed progression to bilateral carotid artery occlusions. in summary, the findings suggest that the antiphospholipid antibody syndrome may be implicated in pathological changes that can result in occlusions of the proximal segments of the internal carotid arteries. consequently, young stroke patients who present with unexplained internal carotid artery occlusions may benefit from testing for the presence of antiphospholipid antibodies. | a 39-year - old woman presented with a right - hemispheric stroke 1 year after she had suffered a left - hemispheric stroke. her diagnostic workup was notable for bilateral occlusions of the internal carotid arteries at their origins and a positive lupus anticoagulant antibody test. there was no evidence of carotid dissection or another identifiable cause for her carotid occlusions. these findings suggest that the antiphospholipid antibody syndrome may be implicated in the pathological changes that resulted in occlusions of the extracranial internal carotid arteries. young stroke patients who present with unexplained internal carotid artery occlusions may benefit from testing for the presence of antiphospholipid antibodies. |
we report a rare case with simultaneous onset of amn and central retinal vein occlusion (crvo), which is a retinal circulation disorder a 44-year - old woman complained of central visual loss of the left eye for the previous 2 weeks. fundoscopic examination revealed a wedge - shaped, dark reddish - brown lesion at the macula, and crvo - like retinal hemorrhages os. the patient s scanning laser ophthalmoscopy infrared imaging result led to a diagnosis of amn. two weeks after corticosteroid pulse therapy, her visual acuity improved to 1.2 os, with improvement of macular findings and humphrey perimetry. when the dose of oral corticosteroid was decreased, the amn lesion worsened, with recurrence of retinal hemorrhages. these results suggest that circulatory disorders almost simultaneously occurred in choroidal and retinal vessels, resulting in the onset of both amn and crvo. acute macular neuroretinopathy (amn) is a rare disease characterized by a wedge - shaped, dark reddish - brown lesion at the macula.1 scanning laser ophthalmoscopy (slo) infrared imaging clearly reveals these lesions as hyporeflectivity, which is a characteristic finding in amn, and this abnormal finding is present even though amn lesions are not biomicroscopically evident.2 the development of spectral - domain optical coherence tomography (oct) demonstrated that amn lesions mainly affect the outer retina and not the inner retina.2 recently, it was reported that hyper - reflective lesions at the level of the outer plexiform and outer nuclear layers on oct temporally appeared despite an intact photoreceptor inner / outer segment junction (is / os) in patients with early - stage amn ; thereafter, the is / os was affected at the same sites.3 ischemia in the deep inner retinal capillary vessels was speculated as the cause of these newly discovered early oct lesions,3 because retinal circulation provides photoreceptors with 10% of their oxygen supply. approximately 10% of amn patients appear to be complicated by intraretinal hemorrhages, which are inner retinal disorders.4,5 however, the relationship between these conditions is unknown. herein, we describe the case of a patient who developed amn and central retinal vein occlusion (crvo). a 44-year - old woman noticed central visual loss with photopsia in her left eye for the previous 2 weeks. the patient s medical and family histories were unremarkable ; however, she took oral contraceptives for menstrual irregularities. the patient s best - corrected visual acuity (bcva) was 1.2 in the right eye (od) and 0.5 in the left eye (os). fundoscopic examination revealed a wedge - shaped, dark reddish - brown lesion at the macula, brush- or blot - like intraretinal hemorrhages that spread in a pandirectional manner from the optic disc, a mildly swollen optic disc, and dilatation and tortuosity of the retinal veins (figure 1a). slo infrared imaging revealed that the dark area corresponded to the dark reddish lesion (figure 1b). fluorescein angiography (fa) revealed hypofluorescence corresponding to the macular lesion and retinal hemorrhages in the initial phase (figure 1c), and retinal phlebitis and the leakages from the optic disc in the late phase (figure 1d). indocyanine green angiography (icga) revealed a normal appearance during the initial phase, and spotted hypofluorescence at the macular area during the late phase (figure 1e). humphrey threshold 102 perimetry showed a central scotoma (mean deviation [md ] value : 9.40db) corresponding to the lesion area (figure 1f). oct showed loss of the photoreceptor is / os corresponding to the dark reddish lesion (figure 1 g). no macular edema was evident, possibly because the relatively mild occlusion of retinal veins appeared to be restricted mainly to the superotemporal branch and two nasal branches. systemic screening comprising serological analysis or antibody titres for infectious diseases, including syphilis ; quantiferon ; serum autoantibodies, including antiphospholipid antibody ; full blood counts ; erythrocyte sedimentation rate ; serum lipids ; serum and urine glucose ; hemoglobin a1c ; and endogenous coagulation function revealed no abnormalities in the patient. the patient was diagnosed with amn and crvo os and was followed up with withdrawal of oral contraceptives and no medication. two months after the first visit, the area of the macular lesion and bcva remained almost unchanged, although most of the retinal hemorrhages spontaneously disappeared (figure 2a), together with the resolution of retinal phlebitis and the leakages from the optic disc on late - phase fa (figure 2b). after informed consent was obtained, corticosteroids were initially administered as per the following schedule : intravenous methyl - prednisolone (mpsl) at 1,000 mg / day for 3 days, oral psl at 30 mg / day for 7 days, and intravenous mpsl at 1,000 mg / day for 3 days. oral psl (30 mg / day) was then administrated and tapered by 10 mg / day every 1 month. the dark reddish - brown lesion had shrunk (figure 2c), and the slo dark area was reduced (figure 2d). humphrey perimetry showed that the central scotoma had shrunk, with marked improvement of md value (3.58db) (figure 2e), and that the discontinuity of the is / os at the macula had improved (figure 2f). three months after treatment (during oral psl administration at 15 mg / day), her bcva was reduced to 0.6 os, the macular lesion and md value (7.42db) worsened, and the intraretinal hemorrhages recurred. the patient received an increased oral psl dose (30 mg / day) and a posterior subtenon injection of triamcinolone acetonide (40 mg) os. the amn lesion again improved, with the disappearance of the retinal hemorrhages, and bcva and md values increased to 0.9 and 6.08db, respectively, 6 months after the initial treatment. thereafter, psl was tapered by 5 mg / day every 23 months and was continued for 18 months. sixty months after the first visit, the patient s bcva and md values were 0.8 and 3.88db, respectively. the dark reddish lesion was faintly observed without a dark area on slo infrared imaging (figure 3a and b). late - phase fa and icga showed resolution of the hypofluorescent lesions at the macula (figure 3c and d). oct revealed complete recovery of the is / os, with partial discontinuity of the cone outer segment tip line (figure 3e, arrows). the lesions and visual functions improved at an early stage after systemic corticosteroid therapy. to our knowledge, this is the first reported amn case with crvo. amn with intraretinal hemorrhages was previously reported in six patients;4,5 however, the extent of retinal hemorrhage was mild in those cases. retinal capillary disorders following the incidental complication of systemic hypertension was inferred as the cause of intraretinal hemorrhages associated with amn.4,5 however, our patient had no history of arteriosclerotic diseases, including hypertension. amn lesions can show hypofluorescence on fa and icga,6 as observed in the present case. we recently used laser speckle flowgraphy to demonstrate reduced choroidal blood flow velocity at the lesion site during the acute phase of amn.6 in the present case, we could not evaluate choroidal circulation impairment using this technique, because of the patient s poor fixation. nevertheless, the hypofluorescent areas observed on fa and icga at the lesion site suggest choroidal hypoperfusion in this case, because there were no obvious findings on oct responsible for blocked fluorescence within the amn lesion. these observations suggest that choroidal circulation impairment is related to the pathogenesis of amn. on the other hand, crvo is caused by circulation disturbances in retinal veins following the stenosis or occlusion of the central retinal vein near the lamina cribrosa from unknown causes. crvo is associated with diseases causing arteriosclerosis in the elderly, whereas it is thought to be associated with vasculitis in young adults.7,8 when an amn lesion recurred, retinal hemorrhages also appeared again. therefore, we infer that the onset of both amn and crvo in the present case was due to circulatory disturbances that occurred almost simultaneously in both choroidal and retinal vessels. inflammatory mechanisms are speculated because the patient had crvo with retinal vasculitis and because the functional and morphological abnormalities improved from the early stage after systemic corticosteroid administration, similar to findings in our recently reported case.6 however, thrombosis following oral contraceptive use9,10 or other causes can not be excluded, because in a large case series of 588 patients with retinal vein occlusion, six of nine patients aged younger than 35 years were taking oral contraceptive pills.10 further studies regarding retinal and choroidal blood flow are needed in amn patients. in the literature review, amn lesions showed no spontaneous improvement in 65% of patients.11 at present, therapeutic strategies for amn remain to be established. in this case, the is / os improved from the early stage after systemic corticosteroid therapy, and finally completely recovered, as in our recently reported case.6 thus, these results suggest the potential efficacy of systemic corticosteroid therapy for some amn patients, and warrant future prospective studies to examine this intervention. the results suggest that circulatory disorders in the retina and/or choroid relate to the etiology of amn. | purposethe precise mechanism causing acute macular neuroretinopathy (amn) is still unknown. a recent report suggested that choroidal circulation impairment correlates with its pathogenesis. we report a rare case with simultaneous onset of amn and central retinal vein occlusion (crvo), which is a retinal circulation disordermethodscase report.resultsa 44-year - old woman complained of central visual loss of the left eye for the previous 2 weeks. the patient s visual acuity was 0.5 in the left eye (os). fundoscopic examination revealed a wedge - shaped, dark reddish - brown lesion at the macula, and crvo - like retinal hemorrhages os. fluorescein angiography revealed retinal vasculitis and hypofluorescence corresponding to the macular lesion. the patient s scanning laser ophthalmoscopy infrared imaging result led to a diagnosis of amn. two weeks after corticosteroid pulse therapy, her visual acuity improved to 1.2 os, with improvement of macular findings and humphrey perimetry. when the dose of oral corticosteroid was decreased, the amn lesion worsened, with recurrence of retinal hemorrhages. visual functions improved again after an increased dose of corticosteroid.conclusionthese results suggest that circulatory disorders almost simultaneously occurred in choroidal and retinal vessels, resulting in the onset of both amn and crvo. |
it is known that swimming is a non - weight - bearing activity that can increase aerobic capacity and lean body mass, however, it has no positive effects on bone mineral density (bmd) [1 - 5 ]. the loss of bone volume and quality due to a lack of mechanical stimuli can be explained by wolff s law. bone loss may induce osteoporosis later in life, a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to increased susceptibility to fractures. osteoporosis has become a poignant health problem, particularly for women, and it has long been known that female athletes, especially swimmers, have menstrual irregularity [9 - 11 ]. females are more vulnerable to bone loss as a result of having smaller skeletons compared with males, and the estrogen patterns that accompany menarche and menopause. hence, low bmd associated with menstrual irregularity in female athletes may cause severe health problems later in life. previous cross - sectional studies have compared the bmd of competitive swimmers to other competitive athletes, showing that the bmd of collegiate swimmers is significantly lower than that of their counterparts who participate in impact exercise and equal to or lower than that of non - athletic controls. the results of these studies indicate that swimming is not beneficial for the promotion, maintenance, or increase of bmd. conversely, several studies have addressed that there is no significant difference between swimmers and controls with respect to bmd [14 - 16 ]. as shown in a previous study, bone - exercise - nutrition interaction exists, and therefore, studies that evaluate the effect of dietary supplementation in swimmers are needed in order to find out the possible interaction between training and bone health. nutritional aspects such as calcium, magnesium, and vitamin d may also affect bone health in swimmers. to date, swim training still seems to have conflicting effects on bone health maintenance in female athletes. therefore, the present review focuses on swim training, dietary supplementation, and bmd in athletes from prior representative research from the 1990s to date, with a view to elucidating the effect of swim training on bmd in the athletic population. the international society of clinical densitometry (iscd) recommends the use of z - scores rather than t - scores, because they compare bmd with age- and sex - matched controls. iscd defines a z - score of -2 or below as low bmd under the expected age, and recommends that a diagnosis of osteoporosis be made if additional risk factors for poor bone health are identified. low bmd as a history of nutritional deficiencies, hypoestrogenism, stress fractures, and/or other secondary clinical risk factors for fracture, together with a z - score between -1.0 and -2.0 due to the fact that athletes tend to have 10 - 15% higher bmd than the non - athletic population. although low bmd has been defined as a z - score of -2 or below, there is no report of the characteristics of normal distribution, and therefore, the exact range of low bmd can not be obtained. moreover, a z - score between -1.0 and -2.0 can be applied to members of the entire population including well - trained athletes and untrained individuals. hence, bmd measurement in the athletic population needs to consider characteristics of the sport type and bone - loaded regions. according to wolff s law, in the healthy population bone mass can be increased by physical activities requiring high force and/or generating high impact on bone. in previous studies, several exercise training programs have been used to improve bmd [8, 20 ]. in general, swimming is considered to be an unloaded exercise that has no impact on bmd, whereas exercises that include walking, jumping, running, and stopping such as basketball, running, and gymnastics are considered to be high impact sports that have a positive effect on bmd. however, bmd can be also be directly affected by weight bearing and indirectly affected by repeated muscle contraction. therefore, recent studies have focused on the relationship between bmd and the characteristics of muscle fibers based on genetic factors in animals and humans. although swim training may not actually cause positive phenotypic effects on bmd, regular and long - term exercise is repeatedly performed and thus may appear to exert positive changes in bmd. to support this suggestion, the joint effect of phenotypic activation, considering age and physiological characteristics and major and minor trace minerals affecting bone metabolism, should be conducted in order to elucidate an exact mechanism for bmd change. in the 1990 s, studies examined and compared bmd in swimmers and athletes who participated in impact sports, concluding that swimming does not promote high bmd, based on their attempts to mechanistically explain their results. prior cross - sectional studies have demonstrated higher bmd among athletes who engage in weight - bearing and impact sports when compared with their non - athlete counterparts [23 - 26 ], as well as with their non - weight - bearing athlete counterparts. the goal of the study was to clarify the relationship between athletic training and bone density in young eumenorrheic athletes. swimmers had a significantly lower mean density in the lumbar spine compared with all other groups. the major conclusion of the paper is that the data support the concept that athletes in sports involving impact have a higher bmd than non - athletes and swimmers. taaffe., examined the role of skeletal loading patterns on bmd in eumenorrheic athletes who chronically trained using various forms of skeletal loading ; the intensive impact of gymnastics, the non - weight - bearing of swimming, and the usual activity among controls. they examined 39 athletes who competed in their respective sports at the ncaa division 1 level and 19 non - athletes who exercised three hours or less a week. the study found no difference in bmd among the groups in either the lumbar spine or the whole body. the gymnasts and controls had greater bmd at the greater trochanter and femoral neck than the swimmers. the study concluded that high - impact weight - bearing activity is beneficial for bone accumulation, and the authors proposed that high ground reaction forces and extreme muscle contraction on bone may contribute to the higher bmd in gymnasts., compared the bmd of collegiate female athletes who competed in impact - loading sports (volleyball and gymnastics) and swimming. impact - loading players had greater bmd than swimmers and controls, however, there was no significant difference in bmd between swimmers and controls, at any site. this study also concluded that the prevalence of menstrual dysfunction in some participants does not appear to negatively influence bmd. in the 2000 s, creighton., examined bmd and markers of bone turnover in female athletes with different levels of impact in their sports. they determined the effect of regular training in three different types of competitive sport on bmd, bone formation, and bone resorption in young women. 50 women participated in the study and were separated into three groups based on the degree of impact, which was determined by ground reaction forces associated with their sport. the high impact group was found to have significantly higher bmd at the femoral neck, ward 's triangle, trochanter, and a higher total bmd than the medium impact, non - impact (swimmers), and control groups. markers of bone formation were significantly lower in the swimming group compared with the high and medium impact groups. swimmers had high bone resorption and low bone formation markers compared with the high, medium, and control groups. the study concluded that female athletes involved in high impact sports have the greatest bmd at weight - bearing sites as well as the highest markers for bone formation. duncan and colleagues investigated the influence of different exercise types on bmd in elite female athletes including cyclists, runners, swimmers, triathletes, and controls (15 per group). total and areal bmd using dxa scans were conducted and it was found that running, a weight - bearing exercise, was associated with higher bmd than swimming or cycling, which is in accordance with previous studies. in 2007, mudd and colleagues found that bone mass and sport type were important determinants of bone health in female athletes when comparing impact sports and swimming. this cross - sectional study recruited 99 collegiate female athletes in gymnastics, softball, cross country, field hockey, soccer, crew, and swimming / diving. runners had higher overall bmd values than other sports athletes, and swimmers and divers had a significantly lower leg bmd (1.1170.086 g / cm) than every other sports athlete. this study concluded that greater differences among sports were seen when comparing lumbar, pelvis, and leg bmds, and recommended that a longitudinal study would need to be conducted in order to investigate bone changes in response to training over a longer period of time in female athletes. maimoun and colleagues investigated the bmd of gymnasts (impact activity), swimmers (non - impact activity), and controls using dxa for total and regional measurement. at baseline and the 12-month follow - up, gymnasts showed a significantly higher bmd than swimmers and controls. moreover, the total bmd of swimmers (0.9600.013 g / cm) was lower than that of the controls (0.9950.013 g / cm) at baseline and the 12-month follow - up (0.9910.014 g / cm and 1.0090.013 g / cm, respectively). in accordance with previous findings, this study demonstrated that the osteogenic effect of impact activity was greater than non - impact activity. summarizing previous studies, it has been shown that the loading - induced bmd of impact sports athletes seems to be higher compared with swimmers and/or the non - athletic population. thus, it appears that different bone - loading history and type may highly affect skeletal adaptation in athletes. future studies should consider this point in order to elucidate a better and clearer mechanism of the effect of swim training on bmd. several previous studies on bmd and other markers of bone strength have shown an advantage for swimmers compared with inactive controls and/or other sports athletes. some researchers insist that swim training increases muscle contraction and strain on the skeleton, leading to increased mechanical loading, which would have a positive effect on bmd despite the fact that swimming generally represents a non - weight - bearing sport. matsumoto., examined biomarkers of bone metabolism and bmd in 103 male and female athletes. the subjects were japanese collegiate athletes who specialized in long distance running, judo, and swimming at the national level, however, no controls were used in the study. whole body bmd was measured using dxa, and urine samples were collected for pyriodinoline (pyr) and deoxypyridinoline (dpd, bone resorption markers) using high pressure liquid chromatography (hplc). blood was collected for bone alkaline phosphatase (balp) and carboxyterminal propeptide of type one collagen (picp, bone formation markers) measurement. researchers found that the total body bmd was significantly higher in judo athletes compared with runners and swimmers. male runners had a significantly lower balp level than male judoists, and found in balp among the female athletes. the dpd level of male and female runners and swimmers was significantly lower than that of male and female judoists. however, the pyd level of female swimmers was not significantly different from that of runners or judoists. the conclusion by the researchers in this study was that differences in total body bmd are in part due to the demands of the specific sport, and that they are reflected in the levels of bone metabolic markers. this was the first study of its kind to examine biomarkers of bone metabolism among various types of athletes, although its results are limited and there were no control subjects. biomarkers are best if assessed over time to determine change in bone metabolism with a given stimulus, and bmd is best when examining clinically relevant sites such as the hip and spine. similar to this study, summarizing previous studies shows that swimmers seem to have higher levels of bone turnover biomarkers [33, 34 ], although they do not have a higher bmd compared with other sports athletes and/or controls. greenway., examined the negative effect of long - term swim training participation in 43 swimmers compared with 44 controls. swimmers had a swimming career of over 5 years, and dxa was used to determine the total and regional bmd. there were no differences in bone mass or bmd between the two groups, at any site. the total body z - score of swimmers was 1.24 and of the controls was 1.18. this study concluded that long - term swim training participation did not compromise regional bmd, and swim training coupled with weight - bearing activities may induce positive effects on bmd. it appears that longitudinal swim study investigating other physical activity history is needed to verify the exact effect of swim training on bmd. in 2014, stanforth and colleagues compared the bmd in female athletes of various sports (basketball, soccer, swimming, volleyball) aged 18 - 23. they reported that female swimmers in this study had an increased bmd from baseline to year 3 post - season, although the increased amount was still smaller than that of impact sports athletes. they concluded that differences in bmd between impact and non - impact sports are large, however, this study provides evidence that swim training may increase bmd in female swimmers. akgl and colleagues examined the effect of swim training on bmd in 79 swimmers. all swimmers had engaged in at least 2 years swim training, and dxa was used to measure the total and regional bmd. the bmd of 68 swimmers (86%) was normal and a total of 9 swimmers (11.4%) had low bmd. this study implies that swim training may positively affect bmd, although it did not induce increased bmd in female swimmers. summarizing previous studies, it has been shown that swimming may be preferable for maintaining bone health, although swimming has not been shown to result in an improvement in bmd like impact sports. therefore, swim training combined with other types of training would be beneficial on the bone health of swimmers, in order to ensure a better health condition. it has been stressed that reliable reports concerning the dietary habits of athletes, especially elite swimmers, are needed. several studies have shown significantly higher calcium intake in swimmers than in controls [36 - 38 ]. other nutritional aspects such as magnesium, iron, and vitamin d may also affect the maintenance of bone health in swimmers. a healthy and well - balanced diet, including the proper amount of calcium, vitamin d, magnesium, and iron, may provide sufficient nutrients. in 2009, hoogenboom., determined the nutritional knowledge and eating behaviors of female collegiate swimmers. they proposed that swimming, due to the importance of a lean body weight, is associated with nutritional deficiency, and that this would lead to the development of the female athletic triad ; osteoporosis, menstrual dysfunction, and eating disorders. to determine nutritional intake, they used a 24-hour recall food survey, with 85 collegiate female swimmers participating in the study. they reported a mean daily calcium intake of 1578.88 mg in all participants, which was a higher level compared with the recommended dietary allowance (rda) of calcium (1200 mg / day). dugocka and colleagues investigated bmd using dxa in young female swimmers (n=41, aged 11 - 14 years), considering nutritional aspects using a 3-day food intake recall survey. they reported that swimmers had a higher intake of calcium (602229 mg vs. 454236 mg) and phosphorus (1390420 mg vs. 1027292 mg) compared with non - athletic controls, although both groups had a deficiency in average calcium intake. however, they found that the mean value of bmd in both groups did not differ, indicating that in this study, a higher intake of calcium and phosphorus in swimmers may positively affect bone health when compared to controls who were not active in sports. czeczuk., investigated the dietary habits (calcium intake) of 18 former swimmers and 18 current swimmers, reporting that calcium intake in both groups was sufficient and did not exceed 3/4 of the daily normal. these previous studies reported that calcium consumption in swimmers was higher than the rda and/or controls. in contrast, several previous studies have shown that female swimmers have calcium intakes below the rda and/or other sports athletes and controls. berning., investigated the dietary food records of adolescent male and female swimmers, and reported that over 50% of elite adolescent female swimmers consumed lower calcium and iron intake compared with the rda and other sports athletes. similar to this study, hawley and williams investigated 20 elite swimmers (11 females, 9 males) using a 4-day food intake survey, and found that 55% of swimmers consumed an intake of calcium below the rda and 65% of swimmers had a lower intake of iron. moreover, the mean iron intake of female swimmers was significantly lower than the rda, and 82% of female swimmers consumed a low iron intake in this study. based on previous studies [42 - 44 ], a low iron intake is common in female athletes. greenway., examined the calcium intake for the previous 12 months through a questionnaire, and they also found that calcium intake was low in swimmers and controls. swimmers achieved 83% of the recommended daily intake (rdi), however, when compared with the controls (achieved 66% of the rdi), swimmers consumed a higher amount of calcium. czeczelewski and colleagues conducted a 3-year monitoring of bmd and nutrient intakes in adolescent female swimmers (n=20) and non - athletic controls (n=20). the calcium intake of swimmers was below the recommended values, and throughout the 3 years, the bmd of swimmers decreased, suggesting that this could have been due to insufficient calcium intake. akgl and colleagues examined the dietary information of 79 swimmers using a 3-day food diary, and also reported low calcium intake in the swimmers. moreover, they found that swimmers with a normal bmd consumed a higher amount of calcium (median 700 mg) than the swimmers with a low bmd (median 600 mg). furthermore, 31 (39.2%) swimmers in this study had a vitamin d deficiency. vitamin d supplementation is also becoming more popular, especially in the athletic population, because it may reduce the incidence of stress fractures when combined with calcium, and may also have an ergogenic factor for athletes. summarizing previous studies, many authors have reported common dietary irregularities concerning female adolescent and adult swimmers in relation to swim training. therefore, to elucidate the exact mechanism and association between dietary supplementation and swim training, the international society of clinical densitometry (iscd) recommends the use of z - scores rather than t - scores, because they compare bmd with age- and sex - matched controls. iscd defines a z - score of -2 or below as low bmd under the expected age, and recommends that a diagnosis of osteoporosis be made if additional risk factors for poor bone health are identified. the american college of sports medicine defines the term low bmd as a history of nutritional deficiencies, hypoestrogenism, stress fractures, and/or other secondary clinical risk factors for fracture, together with a z - score between -1.0 and -2.0 due to the fact that athletes tend to have 10 - 15% higher bmd than the non - athletic population. although low bmd has been defined as a z - score of -2 or below, there is no report of the characteristics of normal distribution, and therefore, the exact range of low bmd can not be obtained. moreover, a z - score between -1.0 and -2.0 can be applied to members of the entire population including well - trained athletes and untrained individuals. hence, bmd measurement in the athletic population needs to consider characteristics of the sport type and bone - loaded regions. according to wolff s law, in the healthy population, bone usually responds to stress by increasing its mass and bmd. bone mass can be increased by physical activities requiring high force and/or generating high impact on bone. in previous studies, several exercise training programs have been used to improve bmd [8, 20 ]. in general, swimming is considered to be an unloaded exercise that has no impact on bmd, whereas exercises that include walking, jumping, running, and stopping such as basketball, running, and gymnastics are considered to be high impact sports that have a positive effect on bmd. however, bmd can be also be directly affected by weight bearing and indirectly affected by repeated muscle contraction. therefore, recent studies have focused on the relationship between bmd and the characteristics of muscle fibers based on genetic factors in animals and humans. although swim training may not actually cause positive phenotypic effects on bmd, regular and long - term exercise is repeatedly performed and thus may appear to exert positive changes in bmd. to support this suggestion, the joint effect of phenotypic activation, considering age and physiological characteristics and major and minor trace minerals affecting bone metabolism, should be conducted in order to elucidate an exact mechanism for bmd change. in the 1990 s, studies examined and compared bmd in swimmers and athletes who participated in impact sports, concluding that swimming does not promote high bmd, based on their attempts to mechanistically explain their results. prior cross - sectional studies have demonstrated higher bmd among athletes who engage in weight - bearing and impact sports when compared with their non - athlete counterparts [23 - 26 ], as well as with their non - weight - bearing athlete counterparts. the goal of the study was to clarify the relationship between athletic training and bone density in young eumenorrheic athletes. swimmers had a significantly lower mean density in the lumbar spine compared with all other groups. the major conclusion of the paper is that the data support the concept that athletes in sports involving impact have a higher bmd than non - athletes and swimmers. taaffe., examined the role of skeletal loading patterns on bmd in eumenorrheic athletes who chronically trained using various forms of skeletal loading ; the intensive impact of gymnastics, the non - weight - bearing of swimming, and the usual activity among controls. they examined 39 athletes who competed in their respective sports at the ncaa division 1 level and 19 non - athletes who exercised three hours or less a week. the study found no difference in bmd among the groups in either the lumbar spine or the whole body. the gymnasts and controls had greater bmd at the greater trochanter and femoral neck than the swimmers. the study concluded that high - impact weight - bearing activity is beneficial for bone accumulation, and the authors proposed that high ground reaction forces and extreme muscle contraction on bone may contribute to the higher bmd in gymnasts., compared the bmd of collegiate female athletes who competed in impact - loading sports (volleyball and gymnastics) and swimming. impact - loading players had greater bmd than swimmers and controls, however, there was no significant difference in bmd between swimmers and controls, at any site. this study also concluded that the prevalence of menstrual dysfunction in some participants does not appear to negatively influence bmd. in the 2000 s, creighton., examined bmd and markers of bone turnover in female athletes with different levels of impact in their sports. they determined the effect of regular training in three different types of competitive sport on bmd, bone formation, and bone resorption in young women. 50 women participated in the study and were separated into three groups based on the degree of impact, which was determined by ground reaction forces associated with their sport. the high impact group was found to have significantly higher bmd at the femoral neck, ward 's triangle, trochanter, and a higher total bmd than the medium impact, non - impact (swimmers), and control groups. markers of bone formation were significantly lower in the swimming group compared with the high and medium impact groups. swimmers had high bone resorption and low bone formation markers compared with the high, medium, and control groups. the study concluded that female athletes involved in high impact sports have the greatest bmd at weight - bearing sites as well as the highest markers for bone formation. duncan and colleagues investigated the influence of different exercise types on bmd in elite female athletes including cyclists, runners, swimmers, triathletes, and controls (15 per group). total and areal bmd using dxa scans were conducted and it was found that running, a weight - bearing exercise, was associated with higher bmd than swimming or cycling, which is in accordance with previous studies. in 2007, mudd and colleagues found that bone mass and sport type were important determinants of bone health in female athletes when comparing impact sports and swimming. this cross - sectional study recruited 99 collegiate female athletes in gymnastics, softball, cross country, field hockey, soccer, crew, and swimming / diving. runners had higher overall bmd values than other sports athletes, and swimmers and divers had a significantly lower leg bmd (1.1170.086 g / cm) than every other sports athlete. this study concluded that greater differences among sports were seen when comparing lumbar, pelvis, and leg bmds, and recommended that a longitudinal study would need to be conducted in order to investigate bone changes in response to training over a longer period of time in female athletes. maimoun and colleagues investigated the bmd of gymnasts (impact activity), swimmers (non - impact activity), and controls using dxa for total and regional measurement. at baseline and the 12-month follow - up, gymnasts showed a significantly higher bmd than swimmers and controls. moreover, the total bmd of swimmers (0.9600.013 g / cm) was lower than that of the controls (0.9950.013 g / cm) at baseline and the 12-month follow - up (0.9910.014 g / cm and 1.0090.013 g / cm, respectively). in accordance with previous findings, this study demonstrated that the osteogenic effect of impact activity was greater than non - impact activity. summarizing previous studies, it has been shown that the loading - induced bmd of impact sports athletes seems to be higher compared with swimmers and/or the non - athletic population. thus, it appears that different bone - loading history and type may highly affect skeletal adaptation in athletes. future studies should consider this point in order to elucidate a better and clearer mechanism of the effect of swim training on bmd. several previous studies on bmd and other markers of bone strength have shown an advantage for swimmers compared with inactive controls and/or other sports athletes. some researchers insist that swim training increases muscle contraction and strain on the skeleton, leading to increased mechanical loading, which would have a positive effect on bmd despite the fact that swimming generally represents a non - weight - bearing sport. matsumoto., examined biomarkers of bone metabolism and bmd in 103 male and female athletes. the subjects were japanese collegiate athletes who specialized in long distance running, judo, and swimming at the national level, however, no controls were used in the study. whole body bmd was measured using dxa, and urine samples were collected for pyriodinoline (pyr) and deoxypyridinoline (dpd, bone resorption markers) using high pressure liquid chromatography (hplc). blood was collected for bone alkaline phosphatase (balp) and carboxyterminal propeptide of type one collagen (picp, bone formation markers) measurement. researchers found that the total body bmd was significantly higher in judo athletes compared with runners and swimmers. male runners had a significantly lower balp level than male judoists, and found in balp among the female athletes. the dpd level of male and female runners and swimmers was significantly lower than that of male and female judoists. however, the pyd level of female swimmers was not significantly different from that of runners or judoists. the conclusion by the researchers in this study was that differences in total body bmd are in part due to the demands of the specific sport, and that they are reflected in the levels of bone metabolic markers. this was the first study of its kind to examine biomarkers of bone metabolism among various types of athletes, although its results are limited and there were no control subjects. biomarkers are best if assessed over time to determine change in bone metabolism with a given stimulus, and bmd is best when examining clinically relevant sites such as the hip and spine. similar to this study, summarizing previous studies shows that swimmers seem to have higher levels of bone turnover biomarkers [33, 34 ], although they do not have a higher bmd compared with other sports athletes and/or controls. greenway., examined the negative effect of long - term swim training participation in 43 swimmers compared with 44 controls. swimmers had a swimming career of over 5 years, and dxa was used to determine the total and regional bmd. there were no differences in bone mass or bmd between the two groups, at any site. the total body z - score of swimmers was 1.24 and of the controls was 1.18. this study concluded that long - term swim training participation did not compromise regional bmd, and swim training coupled with weight - bearing activities may induce positive effects on bmd. it appears that longitudinal swim study investigating other physical activity history is needed to verify the exact effect of swim training on bmd. in 2014, stanforth and colleagues compared the bmd in female athletes of various sports (basketball, soccer, swimming, volleyball) aged 18 - 23. they reported that female swimmers in this study had an increased bmd from baseline to year 3 post - season, although the increased amount was still smaller than that of impact sports athletes. they concluded that differences in bmd between impact and non - impact sports are large, however, this study provides evidence that swim training may increase bmd in female swimmers. akgl and colleagues examined the effect of swim training on bmd in 79 swimmers. all swimmers had engaged in at least 2 years swim training, and dxa was used to measure the total and regional bmd. the bmd of 68 swimmers (86%) was normal and a total of 9 swimmers (11.4%) had low bmd. this study implies that swim training may positively affect bmd, although it did not induce increased bmd in female swimmers. summarizing previous studies, it has been shown that swimming may be preferable for maintaining bone health, although swimming has not been shown to result in an improvement in bmd like impact sports. in addition to swim training, extra strength training may also positively affect bmd. therefore, swim training combined with other types of training would be beneficial on the bone health of swimmers, in order to ensure a better health condition. it has been stressed that reliable reports concerning the dietary habits of athletes, especially elite swimmers, are needed. several studies have shown significantly higher calcium intake in swimmers than in controls [36 - 38 ]. other nutritional aspects such as magnesium, iron, and vitamin d may also affect the maintenance of bone health in swimmers. a healthy and well - balanced diet, including the proper amount of calcium, vitamin d, magnesium, and iron, may provide sufficient nutrients. in 2009, hoogenboom., determined the nutritional knowledge and eating behaviors of female collegiate swimmers. they proposed that swimming, due to the importance of a lean body weight, is associated with nutritional deficiency, and that this would lead to the development of the female athletic triad ; osteoporosis, menstrual dysfunction, and eating disorders. to determine nutritional intake, they used a 24-hour recall food survey, with 85 collegiate female swimmers participating in the study. they reported a mean daily calcium intake of 1578.88 mg in all participants, which was a higher level compared with the recommended dietary allowance (rda) of calcium (1200 mg / day). dugocka and colleagues investigated bmd using dxa in young female swimmers (n=41, aged 11 - 14 years), considering nutritional aspects using a 3-day food intake recall survey. they reported that swimmers had a higher intake of calcium (602229 mg vs. 454236 mg) and phosphorus (1390420 mg vs. 1027292 mg) compared with non - athletic controls, although both groups had a deficiency in average calcium intake. however, they found that the mean value of bmd in both groups did not differ, indicating that in this study, a higher intake of calcium and phosphorus in swimmers may positively affect bone health when compared to controls who were not active in sports. czeczuk., investigated the dietary habits (calcium intake) of 18 former swimmers and 18 current swimmers, reporting that calcium intake in both groups was sufficient and did not exceed 3/4 of the daily normal. these previous studies reported that calcium consumption in swimmers was higher than the rda and/or controls. in contrast, several previous studies have shown that female swimmers have calcium intakes below the rda and/or other sports athletes and controls. berning., investigated the dietary food records of adolescent male and female swimmers, and reported that over 50% of elite adolescent female swimmers consumed lower calcium and iron intake compared with the rda and other sports athletes. similar to this study, hawley and williams investigated 20 elite swimmers (11 females, 9 males) using a 4-day food intake survey, and found that 55% of swimmers consumed an intake of calcium below the rda and 65% of swimmers had a lower intake of iron. moreover, the mean iron intake of female swimmers was significantly lower than the rda, and 82% of female swimmers consumed a low iron intake in this study. based on previous studies [42 - 44 ], a low iron intake is common in female athletes., examined the calcium intake for the previous 12 months through a questionnaire, and they also found that calcium intake was low in swimmers and controls. swimmers achieved 83% of the recommended daily intake (rdi), however, when compared with the controls (achieved 66% of the rdi), swimmers consumed a higher amount of calcium. czeczelewski and colleagues conducted a 3-year monitoring of bmd and nutrient intakes in adolescent female swimmers (n=20) and non - athletic controls (n=20). the calcium intake of swimmers was below the recommended values, and throughout the 3 years, the bmd of swimmers decreased, suggesting that this could have been due to insufficient calcium intake. akgl and colleagues examined the dietary information of 79 swimmers using a 3-day food diary, and also reported low calcium intake in the swimmers. moreover, they found that swimmers with a normal bmd consumed a higher amount of calcium (median 700 mg) than the swimmers with a low bmd (median 600 mg). furthermore, 31 (39.2%) swimmers in this study had a vitamin d deficiency. vitamin d supplementation is also becoming more popular, especially in the athletic population, because it may reduce the incidence of stress fractures when combined with calcium, and may also have an ergogenic factor for athletes. summarizing previous studies, many authors have reported common dietary irregularities concerning female adolescent and adult swimmers in relation to swim training. therefore, to elucidate the exact mechanism and association between dietary supplementation and swim training, the close and precise monitoring of nutritional habits of swimmers should be conducted. upon the review of previous studies, it is obvious that the majority of studies did not collect physical activity data on swimmers activities outside of their swimming activities. these extra activities may have some influence on the bmd of swimmers, and therefore, future studies need to examine additional physical activity history data in addition to swim training. this additional information may help explain why swimmers ' bmd tends to be lower than the bmd of the controls in many studies. if a swimmer participated at a young age in impact - based sports, such as running or gymnastics, this may be reflected in their current bmd. a thorough knowledge of swimmers ' past physical activity can help to better understand the results seen with dxa and biomarker analysis. since bone adaptation to exercise is limited to loaded regions, exercise types should be carefully chosen. nutritional intake of calcium, magnesium, and vitamin d for swimmers also needs to be considered when conducting training and bmd interaction studies. the compilation of results in this review suggests that further exercise intervention studies are needed in the attempt to introduce various exercise programs to female swimmers, in order to determine the optimal exercise prescription for bone health. moreover, longitudinal studies and randomized control trials are necessary to better understand the association of swim training with bmd in female athletes. | [purpose]the present paper reviews the physiological adaptation to swim training and dietary supplementation relating to bone mineral density (bmd) in female swimmers. swim training still seems to have conflicting effects on bone health maintenance in athletes.[methods]this review article focuses on swim training combined with dietary supplementation with respect to bmd in female athletes.[results]upon review of previous studies, it became obvious that the majority of studies did not collect physical activity data on the swimmers outside of their swimming activities. these activities may have some influence on the bmd of swimmers and therefore, future studies need to examine additional physical activity history data as well as swim training. this additional information may help to explain why swimmers ' bmd tends to be lower than the bmd of control individuals in many studies. moreover, dietary supplementation such as calcium, magnesium, and vitamin d also affect bone health in swimmers, and it is extremely important to evaluate bmd in the context of dietary supplementation.[conclusion]a review of the literature suggests that exercise intervention studies, including longitudinal and randomized control trials, need to attempt to introduce various exercise programs to female swimmers in order to determine the optimal exercise prescription for bone health. |
micrornas (mirnas), a family of small noncoding rnas of 22 nt in length, constitute an important class of regulators that are involved in diverse cellular processes such as developmental control, apoptosis, cell differentiation and proliferation (1). they are also implicated in various disease processes thus emerging as potential targets of therapeutic intervention (2,3). significant efforts have been made to identify mirnas and their target mrnas during last several years. sanger institute 's mirbase serves as the central depository of mirnas that are experimentally validated (4). the current release, version 10.0, contains over 5000 mirnas from various organisms including 533 human and 442 mouse mirnas. however, the function of each mirna is mostly unknown except a few mirnas so far, and diverse experimental and computational approaches are being applied to elucidate their functional significance (5,6). mirnas are involved in the regulation of protein expression primarily by binding to one or more target sites on an mrna transcript and inhibiting translation. thus, identification of target mrnas is of utmost importance aspect in understanding mirna function. computational prediction of target genes in animal has proven challenging mainly due to imperfect base pairing and the limited length of binding sites (7). pictar (8) and targetscans (9) are two prominent programs that utilize cross - species conservation and the near - perfect complementarity between the 5 ' seed region of mirna and the binding sites of target mrna for the prediction of target mrnas. their genome - wide analysis results are available in the ucsc genome browser database (10). also of utility is tarbase which is a manually curated collection of experimentally tested mirna targets in eight organisms (11). recent databases on mirnas tend to combine the compilation of mirna with target prediction modules. mirbase has added the target prediction feature as well using the miranda algorithm (12). argonaute provides compiled information on mirnas of human, mouse and rat (13). argonaute and mirnamap provide the expression profiles of known mirnas although the coverage to date is rather limited. mirgen is a new addition to this list with many convenient features (15). it supports more organisms, diverse types of gene i d, and most of the leading target prediction programs including miranda, pictar, targetscans, diana - microt (16). a unique interface allows users to find the clusters of mirnas at any interval of chromosomes. still, the current state is that most databases available in public so far are simple collection of mirna - related information such as mirna itself and target binding. even though several databases include expression information of mirnas, the coverage is quite limited, failing to integrate most of the high - throughput experimental results (11,13). here, we introduce an integrated database and web interface for functional annotation of mirnas that encompasses expression, function, pathway, disease terms as well as mirna targeting. three prediction programs (miranda, pictar and targetscans) are used for target prediction and their result may be combined in a boolean logic. mechanistic understanding of mirna functions has relied on the properties of a single key target gene in a regulatory or signaling pathway so far. however, since an animal mirna is expected to target several hundred genes on average (9), it is possible that a single mirna targets several genes of related functions. even though it remains to be determined whether or not simultaneous targeting of related genes is the norm and provides a coordinated control mechanism as often seen in the transcriptional regulation, functional relationship between target genes is certainly valuable information in exploring functional significance of each mirna. in this context, mirgator provides a utility for statistical enrichment test of mirna targets in a number of annotation categories such as the gene ontology (go) function (17), genmapp and kegg pathways (18,19), and various diseases. expression profile of mirnas is an important part of functional annotation and we imported the mirna - related expression data from the gene expression omnibus (geo) database (20). several reports have described correlated expression of mirnas and their target genes (21,22). expression pattern of each mirna can be readily compared with that of target mrnas and proteins. importantly, expression correlation between two types of data is also calculated as an estimate for the effect of mirna binding. reciprocal expression pattern between mirna and mrna / protein can be an indirect evidence of mirna targeting. web implementation supports diverse workflows that include search by mirna(s) or target gene(s), search by functional categories and expression profiling of mirnas in geo. figure 1 shows the overview of database integration, analysis flow and web implementation of mirgator. functional analysis begins with target prediction. we used the miranda, pictar and targetscans programs whose genome - wide predictions can be downloaded from the mirbase website and the ucsc genome browser tracks. the lists of target mrnas are tested for statistical over - representation in any functional nodes using hypergeometric distribution of fisher 's exact test. implemented functional categories include the go, kegg / genmapp / biocarta pathways (18,19) and disease ontology of ingenuity pathway analysis. figure 1.schematic overview of mirgator system. schematic overview of mirgator system. since gene expression is an important part of functional annotation, we integrated various mirna - related expression data from the geo database and built a compendium of mirna expression data in a similar fashion to the oncomine cancer profiling database (23). simple interface was developed to visualize the expression profile of mirnas and to address the issue of differential regulation in various situations. prediction of target transcripts often yields false targets even with the state - of - the - art algorithms due to imperfect base pairing and the short length of binding sites. examining expression correlation of mirna and the predicted target mrnas / proteins may provide clues of genuine targeting or indirect regulation. we collected genome - wide expression data of mirna, mrna and protein for matching tissues / samples and evaluated the expression correlation coefficients for all mirna target pairs. microrna binding to target mrna, a bipartite relationship in graph theory, is represented as a advantages of table representation become obvious when multiple mirnas and target genes are simultaneously examined. for example, co - regulation by multiple mirnas can be easily explored by examining common targets. candidate regulatory mirnas can be obtained by providing list of genes from pathway databases or from microarray clustering results. the concepts of mirna targeting, functional enrichment analyses and expression correlation are closely related subjects. we built an integrated module with diverse biological questions into consideration as described in later section. all results are pre - calculated and stored in the database to speed - up the response. many databases of diverse characteristics are closely integrated in mirgator as listed in figure 1. the ucsc genome maps of the ncbi build 35 (hg17) and the ncbi build 35 (mm7) were used for the human and mouse genomes, respectively. genome - wide prediction results from the pictar and targetscans programs were obtained from the ucsc genome browser database. target genes from miranda 4.0 were obtained from the mirbase website where the most up - to - date information were available. downloaded targets on the current genomes (hg18 and mm8) were lifted back to the previous genomes (hg17 and mm7), which substantially increases the mirna coverage. table 1.statistics for various target prediction methodshuman (hg17)mouse (mm7)mirandapictar-4waypictar-5waytargetscansmirandapictar - dogpictar - chickennumber of mirnas470179131139375269249number of target genes15 27491523455770914 76865501492number of binding sites284 714154 89428 87022 837241 791106 0228354average number of target genes per mirna32.551.126.455.539.424.36.0average number of binding sites per mirna60686522016464539434average number of binding sites per gene18.616.98.33.016.416.25.7note : cross - species conservation for each prediction method : miranda (version 4.0) : conserved in at least two species pictar-4way : conserved in 4 species (human, mouse, rat, dog) pictar-5way : conserved in 5 species (human, mouse, rat, dog, chicken) targetscans : conserved in 5 species (human, mouse, rat, dog, chicken) pictar - dog : conserved in 7 species (mouse, rat, rabbit, human, chimp, macaque, dog) pictar - chicken : conserved in 13 species (7 species + cow, armadillo, elephant, tenrec, opossum, chicken). statistics for various target prediction methods note : cross - species conservation for each prediction method : miranda (version 4.0) : conserved in at least two species pictar-4way : conserved in 4 species (human, mouse, rat, dog) pictar-5way : conserved in 5 species (human, mouse, rat, dog, chicken) targetscans : conserved in 5 species (human, mouse, rat, dog, chicken) pictar - dog : conserved in 7 species (mouse, rat, rabbit, human, chimp, macaque, dog) pictar - chicken : conserved in 13 species (7 species + cow, armadillo, elephant, tenrec, opossum, chicken). genome annotation data, mapping genes to nodes of functional classification system, were collected from various resources. gene - to - go mapping was achieved by combining the ucsc kgxref table (known gene to uniprot i d) and goa association table (uniprot i d to go nodes) from the go web site (10,24). genes in the kegg / genmapp / biocarta pathways were obtained from arrayxpath database (25). ipa 's gene to disease mapping from ingenuity systems was used to test disease enrichment of mirna targets. ipa 's disease classification system consists of more than 7000 terms organized in three hierarchical levels of depth. hughes and coworkers (26) generated a series of genome - wide expression data for mouse genome using homogeneous samples. their mrna expression data cover 55 tissues (27) and the proteomic data include 4768 proteins in six organs (28). as for the human genome, golub and coworkers published expression profile of mrna and 217 mirnas in 334 samples (3,29). no global proteomic data in multiple tissues are available for human to the best of our knowledge, and we simply compared the expression profiles of mirna and mrna. thus, the expression correlation analysis for mouse covers mirna, mrna, and proteins, whereas only the expression correlation between mirna and mrna is available for human. twelve mirna - related datasets (566 samples) were downloaded from the geo database. proper normalization process would ideally take the unique features / characteristics of dataset into consideration. analyzing compendium datasets, we used the quantile normalization that performed best in the affymetrix arrays (30) since most mirna microarrays are single channeled (3). each dataset was manually examined to set up 106 two - class comparison studies to find differentially expressed mirnas in various situations. seven studies compared cancer and normal tissues for bladder, breast, colon, eye, kidney, lung and uterus. most other studies were designed to find the tissue - specific or cell - type - specific mirnas. figure 1 shows the overview of database integration, analysis flow and web implementation of mirgator. functional analysis begins with target prediction. we used the miranda, pictar and targetscans programs whose genome - wide predictions can be downloaded from the mirbase website and the ucsc genome browser tracks. the lists of target mrnas are tested for statistical over - representation in any functional nodes using hypergeometric distribution of fisher 's exact test. implemented functional categories include the go, kegg / genmapp / biocarta pathways (18,19) and disease ontology of ingenuity pathway analysis. figure 1.schematic overview of mirgator system. schematic overview of mirgator system. since gene expression is an important part of functional annotation, we integrated various mirna - related expression data from the geo database and built a compendium of mirna expression data in a similar fashion to the oncomine cancer profiling database (23). simple interface was developed to visualize the expression profile of mirnas and to address the issue of differential regulation in various situations. prediction of target transcripts often yields false targets even with the state - of - the - art algorithms due to imperfect base pairing and the short length of binding sites. examining expression correlation of mirna and the predicted target mrnas / proteins may provide clues of genuine targeting or indirect regulation. we collected genome - wide expression data of mirna, mrna and protein for matching tissues / samples and evaluated the expression correlation coefficients for all mirna target pairs. microrna binding to target mrna, a bipartite relationship in graph theory, is represented as a advantages of table representation become obvious when multiple mirnas and target genes are simultaneously examined. for example, co - regulation by multiple mirnas can be easily explored by examining common targets. candidate regulatory mirnas can be obtained by providing list of genes from pathway databases or from microarray clustering results. the concepts of mirna targeting, functional enrichment analyses and expression correlation are closely related subjects. we built an integrated module with diverse biological questions into consideration as described in later section. all results are pre - calculated and stored in the database to speed - up the response. many databases of diverse characteristics are closely integrated in mirgator as listed in figure 1. the ucsc genome maps of the ncbi build 35 (hg17) and the ncbi build 35 (mm7) were used for the human and mouse genomes, respectively. genome - wide prediction results from the pictar and targetscans programs were obtained from the ucsc genome browser database. target genes from miranda 4.0 were obtained from the mirbase website where the most up - to - date information were available. downloaded targets on the current genomes (hg18 and mm8) were lifted back to the previous genomes (hg17 and mm7), which substantially increases the mirna coverage. table 1.statistics for various target prediction methodshuman (hg17)mouse (mm7)mirandapictar-4waypictar-5waytargetscansmirandapictar - dogpictar - chickennumber of mirnas470179131139375269249number of target genes15 27491523455770914 76865501492number of binding sites284 714154 89428 87022 837241 791106 0228354average number of target genes per mirna32.551.126.455.539.424.36.0average number of binding sites per mirna60686522016464539434average number of binding sites per gene18.616.98.33.016.416.25.7note : cross - species conservation for each prediction method : miranda (version 4.0) : conserved in at least two species pictar-4way : conserved in 4 species (human, mouse, rat, dog) pictar-5way : conserved in 5 species (human, mouse, rat, dog, chicken) targetscans : conserved in 5 species (human, mouse, rat, dog, chicken) pictar - dog : conserved in 7 species (mouse, rat, rabbit, human, chimp, macaque, dog) pictar - chicken : conserved in 13 species (7 species + cow, armadillo, elephant, tenrec, opossum, chicken). statistics for various target prediction methods note : cross - species conservation for each prediction method : miranda (version 4.0) : conserved in at least two species pictar-4way : conserved in 4 species (human, mouse, rat, dog) pictar-5way : conserved in 5 species (human, mouse, rat, dog, chicken) targetscans : conserved in 5 species (human, mouse, rat, dog, chicken) pictar - dog : conserved in 7 species (mouse, rat, rabbit, human, chimp, macaque, dog) pictar - chicken : conserved in 13 species (7 species + cow, armadillo, elephant, tenrec, opossum, chicken). genome annotation data, mapping genes to nodes of functional classification system, were collected from various resources. gene - to - go mapping was achieved by combining the ucsc kgxref table (known gene to uniprot i d) and goa association table (uniprot i d to go nodes) from the go web site (10,24). genes in the kegg / genmapp / biocarta pathways were obtained from arrayxpath database (25). ipa 's gene to disease mapping from ingenuity systems was used to test disease enrichment of mirna targets. ipa 's disease classification system consists of more than 7000 terms organized in three hierarchical levels of depth. hughes and coworkers (26) generated a series of genome - wide expression data for mouse genome using homogeneous samples. their mrna expression data cover 55 tissues (27) and the proteomic data include 4768 proteins in six organs (28). as for the human genome, golub and coworkers published expression profile of mrna and 217 mirnas in 334 samples (3,29). no global proteomic data in multiple tissues are available for human to the best of our knowledge, and we simply compared the expression profiles of mirna and mrna. thus, the expression correlation analysis for mouse covers mirna, mrna, and proteins, whereas only the expression correlation between mirna and mrna is available for human. twelve mirna - related datasets (566 samples) were downloaded from the geo database. proper normalization process would ideally take the unique features / characteristics of dataset into consideration. analyzing compendium datasets we used the quantile normalization that performed best in the affymetrix arrays (30) since most mirna microarrays are single channeled (3). each dataset was manually examined to set up 106 two - class comparison studies to find differentially expressed mirnas in various situations. seven studies compared cancer and normal tissues for bladder, breast, colon, eye, kidney, lung and uterus. most other studies were designed to find the tissue - specific or cell - type - specific mirnas. this is the main interface of mirgator for examining target genes, inferred functions and the correlated expression through target prediction. default choice is miranda 4.0 from the mirbase since it covers the most recent compendium of mirnas. other methods are rather outdated with lower coverage (genome - wide calculation performed almost 2 years ago) but their target sites are conserved in more species, which may be of help in filtering out false positives. average number of target genes and binding sites in table 1 would be helpful in estimating reliability of prediction methods. each method produces a different list of target genes and it is often desirable to compare the contents. since the mirna coverage and the extent of conservation are different among prediction methods, we constructed a target summary table that showed the number of target genes for all mirnas according to the prediction methods. this table can be used to pre - examine the number of target genes before actual query. clicking on each number in the table opens up the list of target genes for the prediction method of choice. the main search can be initiated either with mirna(s) or with target gene(s). figure 2 is the collection of screenshots from the target function expression module. it consists of three major parts of mirna target mrna table, functional enrichment analysis of target genes, and expression correlation analysis of target genes. clicking on each number in the table leads to the detailed information on target binding and the expression correlation pattern for corresponding mirna we support sorting target mrnas according to the number of binding sites, which would allow users to concentrate on mrnas with multiple binding sites preferentially. another advantage is that common target genes of multiple mirnas can be easily recognized in this mirna input of multiple genes can be used to find the regulatory mirnas for the given set of genes. if genes belonging to a specific biological pathway are provided as an input, mirnas with multiple target genes within the pathway of interest may be identified. similar approach can be applied to find the regulatory mirnas for gene clusters obtained from mrna expression profiling. functional enrichment analysis of target genes can be performed in three categories go, pathway and disease terms. simple hypergeometric test of over - representation in each term was carried out for all terms in go, pathway and disease classification systems. the output page summarizes the significant nodes for a given p - value, which can be sorted according to various criteria. disease classification of the ingenuity systems inc. was used to test disease implications. since all calculations are pre - computed and stored in the database, the search for mirnas whose target genes are statistically enriched in specific terms a separate module of mirna with inferred function is provided to look for the mirnas with inferred functions in all three functional categories. our web implementation supports any node i d in the go classification and all pathways. as for the disease search expression correlation analysis of target genes gives a table of correlation coefficients between mirna and target mrna as well as mirna and target protein if the data are available. reciprocal expression pattern is expected for genuine targets and the pairs of high correlation between mirna and apparent non - targets may indicate indirect targeting. target genes can be sorted according to the correlation coefficients in descending or ascending order. link to detailed information on target binding and correlated expression pattern is also provided for each mirna mrna pair. the purpose of mirna expression profiling module is to visualize mirna expression and to obtain information on differential regulation in various situations.. simple query of mirna, tissue / organ name, or disease yields a list of relevant comparison studies. once mirna and comparison study are specified, expression pattern of mirna across the samples in the study can be displayed as a bar or box plot. searches other than mirna allow the user to access the list of differentially regulated mirnas in various situations. current implementation supports searches for differentially regulated mirnas in 24 tissues / organs and 28 cell types. comparing expression conditions consists of four types of cancer versus normal, cancer versus cancer, chemical treatment and others. for example, current dataset of comparing cancer versus normal tissues supports seven tissues including bladder, breast, colon, eye, kidney, lung and uterus. this is the main interface of mirgator for examining target genes, inferred functions and the correlated expression through target prediction. default choice is miranda 4.0 from the mirbase since it covers the most recent compendium of mirnas. other methods are rather outdated with lower coverage (genome - wide calculation performed almost 2 years ago) but their target sites are conserved in more species, which may be of help in filtering out false positives. average number of target genes and binding sites in table 1 would be helpful in estimating reliability of prediction methods. each method produces a different list of target genes and it is often desirable to compare the contents. since the mirna coverage and the extent of conservation are different among prediction methods, we constructed a target summary table that showed the number of target genes for all mirnas according to the prediction methods. this table can be used to pre - examine the number of target genes before actual query. clicking on each number in the table opens up the list of target genes for the prediction method of choice. the main search can be initiated either with mirna(s) or with target gene(s). figure 2 is the collection of screenshots from the target function expression module. it consists of three major parts of mirna target mrna table, functional enrichment analysis of target genes, and expression correlation analysis of target genes. clicking on each number in the table leads to the detailed information on target binding and the expression correlation pattern for corresponding mirna we support sorting target mrnas according to the number of binding sites, which would allow users to concentrate on mrnas with multiple binding sites preferentially. another advantage is that common target genes of multiple mirnas can be easily recognized in this mirna input of multiple genes can be used to find the regulatory mirnas for the given set of genes. if genes belonging to a specific biological pathway are provided as an input, mirnas with multiple target genes within the pathway of interest may be identified. similar approach can be applied to find the regulatory mirnas for gene clusters obtained from mrna expression profiling. functional enrichment analysis of target genes can be performed in three categories go, pathway and disease terms. simple hypergeometric test of over - representation in each term was carried out for all terms in go, pathway and disease classification systems. the output page summarizes the significant nodes for a given p - value, which can be sorted according to various criteria. disease classification of the ingenuity systems inc. was used to test disease implications. since all calculations are pre - computed and stored in the database, the search for mirnas whose target genes are statistically enriched in specific terms a separate module of mirna with inferred function is provided to look for the mirnas with inferred functions in all three functional categories. our web implementation supports any node i d in the go classification and all pathways. as for the disease search expression correlation analysis of target genes gives a table of correlation coefficients between mirna and target mrna as well as mirna and target protein if the data are available. reciprocal expression pattern is expected for genuine targets and the pairs of high correlation between mirna and apparent non - targets may indicate indirect targeting. target genes can be sorted according to the correlation coefficients in descending or ascending order. link to detailed information on target binding and correlated expression pattern is also provided for each mirna mrna pair. the purpose of mirna expression profiling module is to visualize mirna expression and to obtain information on differential regulation in various situations. simple query of mirna, tissue / organ name, or disease yields a list of relevant comparison studies. once mirna and comparison study are specified, expression pattern of mirna across the samples in the study can be displayed as a bar or box plot. searches other than mirna allow the user to access the list of differentially regulated mirnas in various situations. current implementation supports searches for differentially regulated mirnas in 24 tissues / organs and 28 cell types. comparing expression conditions consists of four types of cancer versus normal, cancer versus cancer, chemical treatment and others. for example, current dataset of comparing cancer versus normal tissues supports seven tissues including bladder, breast, colon, eye, kidney, lung and uterus. | micrornas (mirnas) constitute an important class of regulators that are involved in various cellular and disease processes. however, the functional significance of each mirna is mostly unknown due to the difficulty in identifying target genes and the lack of genome - wide expression data combining mirnas, mrnas and proteins. we introduce a novel database, mirgator, that integrates the target prediction, functional analysis, gene expression data and genome annotation. mirna function is inferred from the list of target genes predicted by miranda, pictar and targetscans programs. statistical enrichment test of target genes in each term is performed for gene ontology, pathway and disease annotations. associated terms may provide valuable insights for the function of each mirna. for the expression analysis, mirgator integrates public expression data of mirna with those of mrna and protein. expression correlation between mirna and target mrna / proteins is evaluated and their expression patterns can be readily compared. our web implementation supports diverse query types including mirna name, gene symbol, gene ontology, pathway and disease terms. interfaces for exploring common targets or regulatory mirnas and for profiling compendium expression data have been developed as well. currently, mirgator, available at : http://genome.ewha.ac.kr/mirgator/, supports the human and mouse genomes. |
this is an unusual case of a 42-year - old gentleman, admitted to the intensive treatment unit (itu) following an attempted suicide. he was observed to develop significant polyuria following admission to itu and this polyuria resolved when propofol was stopped with no further treatment. this is a rare occurrence of this side effect with this drug, considering how frequently it is used. it may be the case that it occurs more frequently than is known about at present since it may be under - reported, which is why we feel this case report and the discussion it may stimulate are important. a 42-year - old man with a known psychiatric history was admitted in the morning to itu for respiratory and circulatory support following a mixed overdose. he had taken 50 mg of ramipril, 150 mg of olanzapine, and 200 mg of citalopram. he had taken no alcohol, paracetamol, aspirin, or recreational drugs and had not suffered any head injury. on itu he was sedated with 120 mg / h infusion of propofol 1%. in the afternoon, he was noted to be polyuric with a urine output greater than 500 ml / h and his urine was noted to be progressively more dilute [figure 1 ]. catheter bag showing progressively more dilute urine since admission urine and serum osmolarity were measured. the following morning, sedation was stopped and he was extubated successfully. shortly thereafter, urine concentration returned to normal. medline was used to perform searches with combinations of the term diabetes insipidus and the drugs taken in overdose. there were no results found for a medline search of ramipril and diabetes insipidus or citalopram and diabetes insipidus. the search diabetes insipidus and olanzapine gave three results : one case report detailing the case of a 17-year - old who had taken an overdose of olanzapine and developed cranial diabetes insipidus whilst in itu ; a case report of a patient treated for diabetes insipidus secondary to lithium in a patient also taking olanzapine ; and a pilot study of the use of olanzapine in a palliative care setting in which one of the 24 patients developed diabetes insipidus. olanzapine is well absorbed via the oral route and its half - life is approximately 33 h, making it highly unlikely to be the culprit in our case where the polyuria resolved completely less than 36 h following ingestion of the overdose. on reviewing the notes, it was found that the resolution of the polyuria seemed to occur shortly after sedation with propofol was stopped. a further search of diabetes insipidus and propofol was performed. this found one case report linking the use of propofol as an anesthetic with diabetes insipidus. the mechanism of action of propofol (2, 6-diisopropylphenol) as an anesthetic is not fully understood, but it enhances gaba (gamma - aminobutyric acid)-mediated inhibition of adh (anti - diuretic hormone / arginine vasopressin) release in rats, which is a potential mechanism for transient neurogenic diabetes insipidus in humans. the onset of the action of propofol is 30 s and its action is short - lived. the case report linking olanzapine and diabetes insipidus does not state what sedation was used whilst the patient was on itu. this case report shows that it is possible for propofol to induce polyuria in humans as it does in animal studies. why this does not occur more frequently, given how commonly propofol is used in the itu setting, is unclear and should be the subject of further investigation. it is possible that the use of other drugs in conjunction with propofol may have led to the under - recognition of the association with propofol. propofol is often used for the induction of anesthesia rather than maintenance, so polyuria may be less pronounced with smaller doses, attributed to other drugs or to the administration of iv fluids intra - operatively. | case report detailing the occurrence of diabetes insipidus in a 42-year - old man admitted to the intensive treatment unit (itu) following an overdose. whilst on itu, he was sedated with propofol. cessation of treatment with propofol coincided with resolution of the polyuria. animal studies suggest a theoretical mechanism for propofol as the causative agent, but this phenomenon is not commonly seen in humans. |
the rpe is a monolayer of cells between the choroidal circulation and the neural retina. in addition to serving as the outer blood retina barrier, the rpe plays many specialized essential roles in the retina, including phagocytosis of photoreceptor outer segment discs, recycling of retinoids, and participating phototransduction (for review, see [1 - 4 ]). loss of structural and functional integrity in the rpe is associated with many retinal and choroidal diseases, including the dry and wet forms of age - related macular degeneration and diabetic retinopathy [5 - 8 ], leading causes of blindness. to dissect gene functions and to establish a genetic tool for conditional gene activation and inactivation in the rpe, we generated a temporal and spatial gene activation and inactivation system in the rpe using cre / lox and tetracycline - inducible gene expression technologies [9 - 11 ]. in this system, an rpe - specific promoter, the promoter for human vitelliform macular dystrophy-2 (vmd2) gene omim 607854, was used to drive the expression of reverse tetracycline - dependent transactivator (rtta) in the rpe. in the presence of a tetracycline derivative, doxycycline (dox), rtta was capable of binding the tetracycline - responsive element, which in turn, activated the expression of cre recombinase in the rpe. this system was useful for some cre - mediated conditional gene targeting studies that have been conducted independently by several laboratories. however, its potential was not fully exploited, presumably due to a lack of knowledge and procedure for inducing cre expression appropriately in this mouse line. since intravitreal dox delivery is likely to provide a higher level of inducer to the eye, we therefore reexamined the conditions for stringent induction of cre - mediated recombination in inducible rpe - specific cre mice. this report summarizes our recent effort to induce productive cre - mediated recombination in this mouse line with intravitreal dox injection, which will be beneficial to investigators in the field. this study strictly followed the guidelines for the use of animals in ophthalmic and vision research established by the association for research in vision and ophthalmology. all procedures were approved by the institutional animal care and use committees at the university of the oklahoma health sciences center, oklahoma medical research foundation, and dean a. mcgee eye institute. double transgenic mice derived from inducible rpe - specific cre and cre - activatable rosa26 lacz reporter mice were used in analyzing cre expression and function. genotyping for rtta, cre transgene, and the cre - activatable lacz reporter gene was performed according to procedures described previously. louis, mo) was delivered by either feeding (daily gavage feeding at a dose of 0.4 mg / g bodyweight for 2 days) or a single intravitreal injection. intravitreal dox (4 g in 1 l of 1x pbs formulation : 155 mm nacl, 3 mm na2po4, 1.06 mm kh2po4, ph 7.4) delivery was administered with a 33 gauge needle hamilton syringe in post - weanling aged mice, after the mice were subjected to whole body and regional anaesthetization with intraperitoneally injected ketamine / xylazine (10 mg / kg-75 mg / kg body weight) and 0.5% alcaine eye drops, respectively. to prevent the induction of cataract and the disruption of the ocular structure, such as muscle and blood vessels, the needle puncture point was approximately 1 mm from the limbus, at a 45 degree angle. to avoid backflow of material, antibiotic ointment was administered to the eyes to prevent drying and infection while the animals were recovering from the anesthesia. briefly, pupils were dilated with 0.5% tropicamide before the animals were kept in dark overnight. the dark - adopted mice were anesthetized with xylazine / ketamine (10 mg / kg-75 mg / kg body weight, intraperitoneal injection) and were placed on a heating pad to maintain body temperature. the corneal surface was anesthetized with proparacine hydrochloride 1%. a colordome espion erg recording system (diagnosys, lowell, ma) reverse transcription polymerase chain reaction (rt pcr) analysis was performed according to our previous method. briefly, eyeballs were placed in liquid nitrogen immediately after dissection and were stored at 80 c. before the rna samples were prepared, rna samples were prepared with trizol reagent (life technologies, grand island, ny). the first strand cdna was made by random hexamer priming with the superscript first strand synthesis system for rt pcr (life technologies) after brief treatment of rna samples with amplification grade dnase i (life technologies), according to the manufacturer 's instruction. the 411-bp cre mrna was detected with primers (5-agg tgt aga gaa ggc act tag c-3) and (5-cta atc gcc atc ttc cag cag g-3), and the 450 bp -actin mrna was detected with a primer pair (5-gac gag gcg cag agc aag aga gg-3 and 5-ctc ttt gat gtc acg cac gat ttc-3). the rt pcr product was fractionated on 1.2% agarose gel, which was imaged with uvp bioimaging system (upland, ca). immunoblotting for cre with dissected eye cups that contained rpe was performed with an antibody against cre (sigma), according to a previous procedure. -galactosidase (-gal) activity assay, which localized productive cre - mediated recombination in retinal sections, was performed according to our previous procedure. immunostaining for cre - activated--gal on the dissected eye cup was performed with an anti--gal antibody (5 prime-3 prime, boulder, co), according to our previous method. the efficiency of cre - mediated recombination in a particular mouse was calculated by averaging the ratio of cre - positive cells versus total rpe cells from three randomly selected areas (approximately 90 to 120 apart) with approximately 3050 cells in each window. the defined windows in the middle between the optic nerve and one third distance to the edge of the eye cups were deemed the center areas. likewise, those in the middle between the edge of the flatmounts and two thirds distance to optic nerves were defined as peripheral areas. all results were plotted as meanstandard deviation (sd) or standard error (sem). statistical comparison between two samples was performed with the student t test. statistical analysis in a group with more than two samples this study strictly followed the guidelines for the use of animals in ophthalmic and vision research established by the association for research in vision and ophthalmology. all procedures were approved by the institutional animal care and use committees at the university of the oklahoma health sciences center, oklahoma medical research foundation, and dean a. mcgee eye institute. double transgenic mice derived from inducible rpe - specific cre and cre - activatable rosa26 lacz reporter mice were used in analyzing cre expression and function. genotyping for rtta, cre transgene, and the cre - activatable lacz reporter gene was performed according to procedures described previously. louis, mo) was delivered by either feeding (daily gavage feeding at a dose of 0.4 mg / g bodyweight for 2 days) or a single intravitreal injection. intravitreal dox (4 g in 1 l of 1x pbs formulation : 155 mm nacl, 3 mm na2po4, 1.06 mm kh2po4, ph 7.4) delivery was administered with a 33 gauge needle hamilton syringe in post - weanling aged mice, after the mice were subjected to whole body and regional anaesthetization with intraperitoneally injected ketamine / xylazine (10 mg / kg-75 mg / kg body weight) and 0.5% alcaine eye drops, respectively. to prevent the induction of cataract and the disruption of the ocular structure, such as muscle and blood vessels, the needle puncture point was approximately 1 mm from the limbus, at a 45 degree angle. to avoid backflow of material, antibiotic ointment was administered to the eyes to prevent drying and infection while the animals were recovering from the anesthesia. briefly, pupils were dilated with 0.5% tropicamide before the animals were kept in dark overnight. the dark - adopted mice were anesthetized with xylazine / ketamine (10 mg / kg-75 mg / kg body weight, intraperitoneal injection) and were placed on a heating pad to maintain body temperature. the corneal surface was anesthetized with proparacine hydrochloride 1%. a colordome espion erg recording system (diagnosys, lowell, ma) reverse transcription polymerase chain reaction (rt pcr) analysis was performed according to our previous method. briefly, eyeballs were placed in liquid nitrogen immediately after dissection and were stored at 80 c. before the rna samples were prepared, rna samples were prepared with trizol reagent (life technologies, grand island, ny). the first strand cdna was made by random hexamer priming with the superscript first strand synthesis system for rt pcr (life technologies) after brief treatment of rna samples with amplification grade dnase i (life technologies), according to the manufacturer 's instruction. the 411-bp cre mrna was detected with primers (5-agg tgt aga gaa ggc act tag c-3) and (5-cta atc gcc atc ttc cag cag g-3), and the 450 bp -actin mrna was detected with a primer pair (5-gac gag gcg cag agc aag aga gg-3 and 5-ctc ttt gat gtc acg cac gat ttc-3). the rt pcr product was fractionated on 1.2% agarose gel, which was imaged with uvp bioimaging system (upland, ca). immunoblotting for cre with dissected eye cups that contained rpe was performed with an antibody against cre (sigma), according to a previous procedure. -galactosidase (-gal) activity assay, which localized productive cre - mediated recombination in retinal sections, was performed according to our previous procedure. immunostaining for cre - activated--gal on the dissected eye cup was performed with an anti--gal antibody (5 prime-3 prime, boulder, co), according to our previous method. the efficiency of cre - mediated recombination in a particular mouse was calculated by averaging the ratio of cre - positive cells versus total rpe cells from three randomly selected areas (approximately 90 to 120 apart) with approximately 3050 cells in each window. the defined windows in the middle between the optic nerve and one third distance to the edge of the eye cups were deemed the center areas. likewise, those in the middle between the edge of the flatmounts and two thirds distance to optic nerves were defined as peripheral areas. all results were plotted as meanstandard deviation (sd) or standard error (sem). statistical comparison between two samples was performed with the student t test. statistical analysis in a group with more than two samples was performed with one - way anova. to determine the effect of dox induction on cre expression, we performed rt pcr analysis on eye cups (containing the rpe) from inducible rpe - specific cre mice. the cre mrna was detected 7 days after intravitreal dox injection (4 g in 1 l) and was diminished in 2 months in the inducible rpe - specific cre mice (figure 1a). western blot analysis demonstrated that relatively robust cre expression was induced, at a significantly higher level than that from induction by feeding, 15 days after intravitreal dox injection in the eye cups of the inducible rpe - specific cre mice (figure 1b). the level of cre protein was reduced dramatically 2 months after the intravitreal dox injection, and cre was not detectable after another 4 months (figure 1b). a : cre mrna induction was detected with intravitreally delivered doxycycline (dox ; 4 g in 1 l) with reverse transcription (rt)pcr. expression of cre mrna was detected 7 days (7d) after induction and was diminished 60 days (60d) after induction. b : western blot analysis shows relative amounts of cre protein after induction by dox feeding (daily gavage at a dose of 0.4 mg / g bodyweight for 2 days) or intravitreal dox injection. cre expression was robustly elevated 15 days after intravitreal injection, compared with that induced by feeding. cre accumulation was reduced and diminished, 60 days or 180 days after intravitreal dox induction, respectively. whether the intravitreal dox injection elevated cre expression had an effect on the efficiency and reproducibility of productive cre - mediated recombination, which is the key to the success of conditional gene activation or inactivation, we analyzed the expression of -galactosidase (-gal) in double transgenic mice carrying cre and cre - activatable lacz reporter. in ten animals (from three different litters) examined, all demonstrated homogenous cre - activated reporter activity, as judged with the -gal staining assay (figure 2e). to estimate the efficiency of the inducible cre - mediated recombination, we examined the number of -gal - positive cells in rpe flatmounts in cre / lacz double transgenic mice subjected to intravitreal dox injection (4 g in 1 l). this information represented the number of rpe cells that had undergone productive - cre mediated recombination. approximately 60% of the rpe cells were -gal - positive, with no statistical difference between the central (61.64.8%) and peripheral (59.75.4%) regions of the rpe layer (figure 2a d). in some patch areas, -gal - positive cells reached 100%, suggesting almost all rpe cells in these areas had productive cre - mediated recombination. analysis of productive cre - mediated recombination by examining the frequency of -galactosidase (-gal)-expressing rpe cells in f1 mice derived from inducible rpe - specific cre and rosa26 lacz reporter mice. d : representative images and statistical analysis of immunostained rpe flatmounts for cre - activated -gal (green). a : an enlarged confocal image shows the number and frequency of -gal - expressing rpe cells. b c : representative images show the number and frequency of -gal - positive cells in central (b) and peripheral rpe flatmounts (c). d : statistical analysis with the student t test shows that there was no significant difference in the frequency of -gal - positive rpe cells between the central and peripheral regions of the rpe. e : representative image shows homogenous cre - activated -gal activity in the retinal section of the cre/-gal double transgenic mice. productive - cre mediated recombination occurred evenly in approximately 60% of the rpe cells in inducible rpe - specific cre mice after a single intravitreal doxycycline (dox) injection. since our ultimate goal in this line of work is to perform conditional gene targeting in the rpe effectively, it was necessary to determine if there was any loss of retinal integrity in the inducible rpe - specific cre mice after the intravitreal dox injection. the retinal morphology in inducible rpe - specific cre mice appeared normal 12 months after intravitreal dox injection (figure 3a). we then further analyzed the thickness of the photoreceptor outer nuclear layer (onl), a representation of photoreceptor integrity that was mainly supported by healthy rpe cells. no significant difference in onl thickness was identified in these mice (figure 3b), suggesting that the brief cre induction by intravitreal dox injection was not harmful to the retina. to determine whether intravitreal dox injection had a short - term effect on the retinal integrity, we examined retinal function in inducible rpe - specific cre mice with erg. intravitreal dox injection did not alter scotopic erg amplitudes in inducible rpe - specific cre mice 10 days after intravitreal dox injection (figure 4). retinal integrity of inducible rpe - specific cre mice 12 months after intravitreal dox injection (4 g in 1 l). ctrl (control) : age - matched wild - type littermate without dox injection. b : statistical analysis with the student t test for photoreceptor outer nuclear layer (onl) thickness. intravitreal dox injection did not cause changes in retinal morphology in inducible rpe - specific cre mice 12 months after induction. a : representative scotopic electroretinoprahy (erg) amplitudes (flash intensity : 200 cds / m) in inducible rpe - specific cre mice 10 days after intravitreal dox injection (4 g in 1 l). error bar : standard error of the mean (sem) ; p = 0.638 (a - wave) ; p = 0.862 (b - wave). no significant change in retinal function was detected in inducible rpe - specific cre mice 10 days after intravitreal dox injection. to determine the effect of dox induction on cre expression, we performed rt pcr analysis on eye cups (containing the rpe) from inducible rpe - specific cre mice. the cre mrna was detected 7 days after intravitreal dox injection (4 g in 1 l) and was diminished in 2 months in the inducible rpe - specific cre mice (figure 1a). western blot analysis demonstrated that relatively robust cre expression was induced, at a significantly higher level than that from induction by feeding, 15 days after intravitreal dox injection in the eye cups of the inducible rpe - specific cre mice (figure 1b). the level of cre protein was reduced dramatically 2 months after the intravitreal dox injection, and cre was not detectable after another 4 months (figure 1b). a : cre mrna induction was detected with intravitreally delivered doxycycline (dox ; 4 g in 1 l) with reverse transcription (rt)pcr. expression of cre mrna was detected 7 days (7d) after induction and was diminished 60 days (60d) after induction. b : western blot analysis shows relative amounts of cre protein after induction by dox feeding (daily gavage at a dose of 0.4 mg / g bodyweight for 2 days) or intravitreal dox injection. cre expression was robustly elevated 15 days after intravitreal injection, compared with that induced by feeding. cre accumulation was reduced and diminished, 60 days or 180 days after intravitreal dox induction, respectively. to assess whether the intravitreal dox injection elevated cre expression had an effect on the efficiency and reproducibility of productive cre - mediated recombination, which is the key to the success of conditional gene activation or inactivation, we analyzed the expression of -galactosidase (-gal) in double transgenic mice carrying cre and cre - activatable lacz reporter. in ten animals (from three different litters) examined, all demonstrated homogenous cre - activated reporter activity, as judged with the -gal staining assay (figure 2e). to estimate the efficiency of the inducible cre - mediated recombination, we examined the number of -gal - positive cells in rpe flatmounts in cre / lacz double transgenic mice subjected to intravitreal dox injection (4 g in 1 l). this information represented the number of rpe cells that had undergone productive - cre mediated recombination. approximately 60% of the rpe cells were -gal - positive, with no statistical difference between the central (61.64.8%) and peripheral (59.75.4%) regions of the rpe layer (figure 2a d). in some patch areas, -gal - positive cells reached 100%, suggesting almost all rpe cells in these areas had productive cre - mediated recombination. analysis of productive cre - mediated recombination by examining the frequency of -galactosidase (-gal)-expressing rpe cells in f1 mice derived from inducible rpe - specific cre and rosa26 lacz reporter mice. d : representative images and statistical analysis of immunostained rpe flatmounts for cre - activated -gal (green). a : an enlarged confocal image shows the number and frequency of -gal - expressing rpe cells. b c : representative images show the number and frequency of -gal - positive cells in central (b) and peripheral rpe flatmounts (c). d : statistical analysis with the student t test shows that there was no significant difference in the frequency of -gal - positive rpe cells between the central and peripheral regions of the rpe. e : representative image shows homogenous cre - activated -gal activity in the retinal section of the cre/-gal double transgenic mice. productive - cre mediated recombination occurred evenly in approximately 60% of the rpe cells in inducible rpe - specific cre mice after a single intravitreal doxycycline (dox) injection. since our ultimate goal in this line of work is to perform conditional gene targeting in the rpe effectively, it was necessary to determine if there was any loss of retinal integrity in the inducible rpe - specific cre mice after the intravitreal dox injection. the retinal morphology in inducible rpe - specific cre mice appeared normal 12 months after intravitreal dox injection (figure 3a). we then further analyzed the thickness of the photoreceptor outer nuclear layer (onl), a representation of photoreceptor integrity that was mainly supported by healthy rpe cells. no significant difference in onl thickness was identified in these mice (figure 3b), suggesting that the brief cre induction by intravitreal dox injection was not harmful to the retina. to determine whether intravitreal dox injection had a short - term effect on the retinal integrity, we examined retinal function in inducible rpe - specific cre mice with erg. intravitreal dox injection did not alter scotopic erg amplitudes in inducible rpe - specific cre mice 10 days after intravitreal dox injection (figure 4). retinal integrity of inducible rpe - specific cre mice 12 months after intravitreal dox injection (4 g in 1 l). ctrl (control) : age - matched wild - type littermate without dox injection. b : statistical analysis with the student t test for photoreceptor outer nuclear layer (onl) thickness. intravitreal dox injection did not cause changes in retinal morphology in inducible rpe - specific cre mice 12 months after induction. a : representative scotopic electroretinoprahy (erg) amplitudes (flash intensity : 200 cds / m) in inducible rpe - specific cre mice 10 days after intravitreal dox injection (4 g in 1 l). error bar : standard error of the mean (sem) ; p = 0.638 (a - wave) ; p = 0.862 (b - wave). no significant change in retinal function was detected in inducible rpe - specific cre mice 10 days after intravitreal dox injection. although a productive cre - mediated recombination event requires only four cre molecules, which is well below the detection level of conventional methods, it is known that the efficiency of cre - mediated recombination at some chromosomal loci may be lower. this is likely due to the difficulty for cre to access some site - specific recombination loci. in this scenario, associated positional effect may cause variable expression of transgenes among individual animals in a particular line, we observed variable levels of productive cre - mediated recombination, induced by feeding or intraperitoneal (ip) injection, in our inducible rpe - specific cre mice (data not shown). effectiveness in inducing cre expression requires a sufficient amount of the inducer, dox, which is dictated by the limitation of the blood circulation with a half - life of approximately 10 h in mice. since a single ip injection with a high dosage of dox (50 mg / kg bodyweight) resulted in a maximal serum dox concentration of 2 g / ml, it was unclear whether our previous approaches were optimal for inducing cre activity. to improve the inducibility of this cre / lox system, we took advantage of the intravitreal delivery system. intravitreal injection (4 g in 1 l) elevates the average ocular dox concentration to approximate 250-fold of the maximal level in the bloodstream delivered by feeding or ip injection (assuming the diameter of a mouse eye is 3 mm). since feeding or ip injection is dependent on the blood circulation to deliver dox to the eye, it is reasonable to speculate that the relative dox concentration delivered by intravitreal injection is likely much higher than that conferred by feeding or ip injection. indeed, intravitreal dox injection resulted in a significant increase in cre expression, compared with that induced by feeding (figure 1b). as a result, we also observed approximately 60% of the rpe had undergone productive cre - mediated recombination, with patch areas reaching 100% (figure 2b). this result reinforced our assumption that intravitreal dox delivery is advantageous over feeding or ip injection, which provided a higher level of the dox inducer for the inducible cre / lox system in the eye of the inducible rpe - specific cre mice. this higher level of dox is likely to reach the required concentration threshold for the inducible system and, therefore, will reduce the variation in reproducibility of productive cre - mediated recombination. since cre expression rarely reached 100% of the targeted cells in transgenic mice, the efficiency (approximately 60%) of intravitreal dox induced productive cre - mediated recombination in the inducible rpe - specific cre mice will be suitable for most conditional gene activation or inactivation studies in general. in this study, we performed a single intravitreal dox injection (4 g in 1 l) to induce cre expression in inducible rpe - specific cre mice. this procedure did not cause any apparent loss of retinal integrity (figure 3 and figure 4), which is in agreement with a previous result that injecting 4 g dox intravitreally was not harmful to the eye. in our previous characterization, we did not observe any abnormality in retinal integrity 10 months after a brief dox induction through feeding in inducible rpe - specific cre mice. this result also suggests that inserting the cre / rtta transgene cassette did not cause any apparent loss of retinal integrity in this mouse line. cre is a site - specific dna recombinase, and cre overexpression has been shown to cause chromosomal rearrangements in mammals. in the mouse retina, constitutive overexpression of cre may affect rpe integrity. since our ultimate goal in the current work is to establish a genetic system for cre / lox - based conditional gene targeting in the rpe, an inducible approach, which triggers a burst of cre expression transiently (figure 1b), is likely to avoid any potential cre - toxicity. to ascertain whether a single intravitreal dox delivery did not result in the accumulation of a high level of cre for a long time, we analyzed the level of cre protein at various time points after the dox injection. our result suggested that 2 months after intravitreal dox injection the cre level is reduced to that in a dox feeding experiment (figure 1b), which had not caused any loss of retinal integrity in our hands previously. moreover, the fact that the cre protein was not detectable 6 months after the intravitreal dox injection provided more assurance for the successful use of this inducible rpe - specific cre mouse in conditional gene targeting. although tremendous effort has been made by laboratories around the world to generate temporal or / and spatial gene targeting tools for the rpe [11,29 - 31 ], to our knowledge there is no ideal system for rpe - specific gene targeting at this time. we therefore redefined a condition for more effective induction of productive cre - mediated recombination in the inducible rpe - specific cre mice. our work provides a way for more effective use of this mouse line, beyond the knowledge of our previous publication. however, other approaches capable of increasing the efficiency of productive cre - mediated recombination may permit equal or better utilization of this mouse line. although a productive cre - mediated recombination event requires only four cre molecules, which is well below the detection level of conventional methods, it is known that the efficiency of cre - mediated recombination at some chromosomal loci may be lower. this is likely due to the difficulty for cre to access some site - specific recombination loci. in this scenario, associated positional effect may cause variable expression of transgenes among individual animals in a particular line, we observed variable levels of productive cre - mediated recombination, induced by feeding or intraperitoneal (ip) injection, in our inducible rpe - specific cre mice (data not shown). effectiveness in inducing cre expression requires a sufficient amount of the inducer, dox, which is dictated by the limitation of the blood circulation with a half - life of approximately 10 h in mice. since a single ip injection with a high dosage of dox (50 mg / kg bodyweight) resulted in a maximal serum dox concentration of 2 g / ml, it was unclear whether our previous approaches were optimal for inducing cre activity. to improve the inducibility of this cre / lox system, we took advantage of the intravitreal delivery system. intravitreal injection (4 g in 1 l) elevates the average ocular dox concentration to approximate 250-fold of the maximal level in the bloodstream delivered by feeding or ip injection (assuming the diameter of a mouse eye is 3 mm). since feeding or ip injection is dependent on the blood circulation to deliver dox to the eye, it is reasonable to speculate that the relative dox concentration delivered by intravitreal injection is likely much higher than that conferred by feeding or ip injection. indeed, intravitreal dox injection resulted in a significant increase in cre expression, compared with that induced by feeding (figure 1b). as a result, we also observed approximately 60% of the rpe had undergone productive cre - mediated recombination, with patch areas reaching 100% (figure 2b). this result reinforced our assumption that intravitreal dox delivery is advantageous over feeding or ip injection, which provided a higher level of the dox inducer for the inducible cre / lox system in the eye of the inducible rpe - specific cre mice. this higher level of dox is likely to reach the required concentration threshold for the inducible system and, therefore, will reduce the variation in reproducibility of productive cre - mediated recombination. since cre expression rarely reached 100% of the targeted cells in transgenic mice, the efficiency (approximately 60%) of intravitreal dox induced productive cre - mediated recombination in the inducible rpe - specific cre mice will be suitable for most conditional gene activation or inactivation studies in general. in this study, we performed a single intravitreal dox injection (4 g in 1 l) to induce cre expression in inducible rpe - specific cre mice. this procedure did not cause any apparent loss of retinal integrity (figure 3 and figure 4), which is in agreement with a previous result that injecting 4 g dox intravitreally was not harmful to the eye. in our previous characterization, we did not observe any abnormality in retinal integrity 10 months after a brief dox induction through feeding in inducible rpe - specific cre mice. this result also suggests that inserting the cre / rtta transgene cassette did not cause any apparent loss of retinal integrity in this mouse line. cre is a site - specific dna recombinase, and cre overexpression has been shown to cause chromosomal rearrangements in mammals. in the mouse retina, constitutive overexpression of cre may affect rpe integrity. since our ultimate goal in the current work is to establish a genetic system for cre / lox - based conditional gene targeting in the rpe, an inducible approach, which triggers a burst of cre expression transiently (figure 1b), is likely to avoid any potential cre - toxicity. to ascertain whether a single intravitreal dox delivery did not result in the accumulation of a high level of cre for a long time, we analyzed the level of cre protein at various time points after the dox injection. our result suggested that 2 months after intravitreal dox injection the cre level is reduced to that in a dox feeding experiment (figure 1b), which had not caused any loss of retinal integrity in our hands previously. moreover, the fact that the cre protein was not detectable 6 months after the intravitreal dox injection provided more assurance for the successful use of this inducible rpe - specific cre mouse in conditional gene targeting. although tremendous effort has been made by laboratories around the world to generate temporal or / and spatial gene targeting tools for the rpe [11,29 - 31 ], to our knowledge there is no ideal system for rpe - specific gene targeting at this time. we therefore redefined a condition for more effective induction of productive cre - mediated recombination in the inducible rpe - specific cre mice. our work provides a way for more effective use of this mouse line, beyond the knowledge of our previous publication. however, other approaches capable of increasing the efficiency of productive cre - mediated recombination may permit equal or better utilization of this mouse line. | purposeto dissect gene functions in the retinal pigment epithelium (rpe), we previously generated a tetracycline - inducible rpe - specific cre mouse line. although this cre mouse line was useful for several conditional gene targeting studies that were conducted by different laboratories, its potential has not been fully exploited, presumably due to a lack of knowledge or procedure for inducing cre expression appropriately in this mouse line. the goal of the current study is to establish a procedure that will improve the reproducibility of cre - mediated recombination in this mouse line.methodsanalysis of cre expression and function was performed in double transgenic mice derived from inducible rpe - specific cre and cre - activatable rosa26 lacz reporter mice. a tetracycline derivative, doxycycline, was supplied to mice intravitreally to induce cre expression. cre expression and function were examined with reverse transcription pcr, immunoblotting, immunostaining, and in situ enzymatic assay for -galactosidase. retinal integrity was examined with electroretinography and morphometry.resultsintravitreal dox injection elevated cre expression significantly and resulted in productive cre - mediated recombination in approximately 60% of the rpe cells in this mouse line with no apparent change in retinal integrity.conclusionsour results suggest that productive cre - mediated recombination in this mouse line can be induced efficiently with intravitreal dox delivery, with no apparent dox or cre toxicity. therefore, our inducible rpe - specific cre mice are suitable for cre / lox - based gene activation and inactivation in adult rpe, which is critical to the effectiveness and suitability of this cre mouse line in long - term studies requiring conditional gene targeting. |
lupus panniculitis (lp) occurs in 3% of the patients with cutaneous lupus erythematosus. it primarily affects subcutaneous tissues of the face and the proximal areas of the limbs, leading to residual atrophy. lp may lead to major aesthetic sequelae that severely decrease quality of life (even during good long - term control of the autoimmune processes). given the condition 's low prevalence, treatment strategies in lp only 1 case of lipofilling has been reported with a follow - up period limited to 12 months. here, we report on the strikingly good aesthetic outcomes, the long - term quality - of - life benefits, and the safety of lipofilling in 2 patients with lp - associated subcutaneous atrophy. patient 1 (a 13-year - old boy) was referred for an ill - defined subcutaneous inflammatory nodule on the chin. a histopathological examination revealed a marked lymphocytic infiltrate (in superficial and deep tissues) with juxtafollicular involvement, prompting us to diagnose lp. treatment with hydroxychloroquine for 18 months led to the complete disappearance of inflammatory signs but resulted in a cup - shaped lipoatrophy and facial asymmetry. patient 2 (a 32-year - old female) had been monitored over a 6-year period for systemic lupus erythematosus with positive antinuclear and anti - double - stranded dna antibodies. during treatment with hydroxychloroquine, she presented with subcutaneous inflammatory lesions on the thighs and cheeks. a diagnosis of lp was confirmed by histopathological examination of a skin biopsy according to the criteria by peter and su. the initial response to oral prednisone was good, but the patient became steroid dependent. a series of steroid - sparing agents was then administered (including thalidomide, cyclophosphamide, and azathioprine). four weekly intravenous infusions of rituximab (375 mg / m), which were repeated 1 year later, led to complete remission, although atrophic scarring in malar areas persisted. following a request from both patients for correction of the aesthetic sequelae, we chose to use lipofilling. briefly, 10 ml of fat were collected via a 2-mm incision below the umbilicus. next, fat cells were injected into the submental (patient 1) or malar (patient 2) areas using coleman needles. there were no complications, and the positive aesthetic outcome was maintained 3 years after the procedure with no signs of the recurrence of lp (fig 1). the dermatology quality of life index decreased from 16 before lipofilling to 0 six months thereafter (patient 1). lipofilling is a well - established technique widely used in the rejuvenation of the aging face and in iatrogenic lipoatrophy [7, 8 ]. this technique was also used successfully in some cases of linear scleroderma en coup de sabre. most of the common side effects of lipofilling are not severe. nevertheless, it is critical for surgeons to have a firm knowledge of the vascular anatomy because some cases of irreversible blindness have been reported. the good response of patient 2 to rituximab suggests that the efficacy of rituximab as a salvage therapy for treatment - refractory lp should be assessed in prospective trials. along with a previous report, the 2 present cases suggest that lipofilling is effective for treating the atrophic scars that result from permanent alterations of the subcutaneous fatty tissue in lp. moreover, the short- and long - term absence of any signs of lupus relapse or postsurgical complications is encouraging. the lipofilling procedure appears to be very safe when performed in patients with stable disease and thus paves the way for prospective, larger - scale studies in patients with completely controlled autoimmune disorder. these long - term, controlled trials will be able to accurately assess the benefit - risk ratio of lipofilling in lp and other immune - related subcutaneous scarring conditions. | lupus panniculitis is a rare manifestation of cutaneous lupus erythematosus, which may lead to major aesthetic sequelae with a severe impact on patients quality of life. we report 2 cases supporting the short- and long - term efficacy and safety of lipofilling in the treatment of lupus panniculitis - induced atrophy. these observations pave the way for prospective, larger - scale studies in patients with scarring lupus panniculitis, provided that the autoimmune pathogenic process is in complete, stable remission. |
synovial sarcoma (ss) most commonly affects the para - articular regions of the extremities of young adults,. occasionally, however, ss has been identified in less common locations, including the head and neck, heart, lungs and prostate. herein, we describe a case of primary renal ss, an exceedingly rare diagnosis, in a 52-year - old woman. the diagnosis was established by histology and immunostaining. to our knowledge, based on a review of the english literature, this marks the first case report describing the magnetic resonance (mr) features of synovial sarcoma of the kidney. a 52-year - old female presenting with a history of right flank pain underwent computed tomography (ct) examination which revealed an amorphous partially cystic right lower pole renal mass with heterogeneous enhancement. further workup with magnetic resonance imaging revealed a 6.0 5.9 6.1 cm mass in the lower pole of the right kidney. three - plane localizer, coronal single shot fast spin echo (ssfse) t2, axial in and out of phase, axial fat suppressed t2 weighted, axial 5-min and 10-min delayed, and axial 3d liver acquisition with volume acceleration (lava) sequences were acquired. on t1 weighted imaging (fig. 1a), the mass was heterogeneous, with the posterior region having similar intensity to skeletal muscle and the anterior region with higher signal intensity. axial t2 weighted imaging revealed marked heterogeneity with areas of high, intermediate, and low signal, known as the triple sign (fig. 1b). the mass contained multiple areas of high t2 signal emanating from the center of the mass, with surrounding intermediate and low t2 signal intensity regions (fig. figure 1axial in - phase t1 weighted gradient recalled echo (gre) image (a), showing a heterogeneous mass lesion involving the medial portion of the lower pole of the right kidney (arrow), with a posterior isointense area and an anterior area of higher signal intensity, relative to skeletal muscle. axial fat suppressed t2 (b) and coronal ssfse t2 images (e) show marked heterogeneity and the triple sign ; : areas of low, intermediate and high signal intensity (arrows). axial post - contrast gre image (c and d) shows enhancement of the t2 hypo - intense areas (arrow). axial in - phase t1 weighted gradient recalled echo (gre) image (a), showing a heterogeneous mass lesion involving the medial portion of the lower pole of the right kidney (arrow), with a posterior isointense area and an anterior area of higher signal intensity, relative to skeletal muscle. axial fat suppressed t2 (b) and coronal ssfse t2 images (e) show marked heterogeneity and the triple sign ; : areas of low, intermediate and high signal intensity (arrows). axial post - contrast gre image (c and d) shows enhancement of the t2 hypo - intense areas (arrow). the patient underwent right nephrectomy with a pathological diagnosis of high - grade, poorly differentiated spindle - cell synovial sarcoma. immunohistochemical staining showed focal positivity for cytokeratin cam 5.2 and negativity for s100, ck7, and epithelial membrane antigen (ema). the most common sarcoma of the kidney is leiomyosarcoma, accounting for 4060% of reported cases. the first set of 15 cases reported was in 2000, by argani, with few other cases reported in the english literature. there have been no reports to date describing the mr imaging findings of primary ss of the kidney. there is a slight male predominance, with a male - to - female ratio of 1.2. furthermore, there seems to be a predilection for the right kidney, with a right - to - left ratio of 2.2. the typical presentation of ss of the kidney is non - specific, and resembles that of other primary tumors of the kidney. patients may complain of localized flank or back pain, hematuria, or a palpable abdominal mass. the imaging modality of choice is magnetic resonance imaging (mri), given its high sensitivity for soft tissue abnormalities. although diagnostic for some soft tissue tumors, mr signal intensity characteristics are usually non - specific for ss. most synovial sarcomas tend to be large masses, averaging 8 cm in size. the most common mr finding is an oval, well - defined nodular mass. on t1 weighted imaging, small lesions are typically homogenous, with a signal intensity similar to that of skeletal muscle, while lesions larger than 5 cm often exhibit intermediate intensity signal and are heterogeneous secondary to hemorrhagic and necrotic areas. on t2 weighted imaging,, lesions are hyperintense, usually with intratumoral hemorrhage (73%) and cystic components (77%) ; 3557% exhibit the triple sign, showing areas that are hyper-, iso-, and hypo - intense relative to skeletal muscle, representing hemorrhage / necrosis, septa / cellular elements, and calcified / fibrotic areas ; however, this is also seen in other soft tissue tumors, namely malignant fibrous histiocytoma,. mr imaging after intravenous contrast injection shows heterogeneous enhancement. histologically, ss consists of round spindle cells with minimal cytoplasm and active mitotic figures. these tumors are further divided into two subtypes, monophasic and biphasic, depending on the absence or presence of a well - develop glandular epithelium. although, synovial sarcoma in any location is regarded as slow growing, it should be considered a systemic disease. indeed, 25% have pulmonary metastases at the time of diagnosis. despite wide surgical resection, local recurrence and metastatic disease however, ss, more than other sarcomas, are more often treated with neoadjuvant chemotherapy followed by surgical treatment. these tumors demonstrate moderate chemo sensitivity, with 50% response rates to ifosfamide - based or doxorubicin - based regimens. studies of extremity ss have shown that ifosfamide - based chemotherapy reduces tumor volume by greater than 50% and improves disease - specific survival. in contrast, renal cell carcinoma is generally considered chemoresistant, although newer drug therapies such as sorafenib and sunitinib have led to some optimism,. multiple studies of ss of the extremity have shown a strong association between tumor size, volume, and histological grade to disease recurrence and patient mortality. high grade ss is favored in tumors that exhibit cystic components, hemorrhage, and fluid levels, as well as the triple sign. | abstractprimary renal synovial sarcoma (ss) was first described in 2000 by argani, with only a few subsequent cases being reported in the english literature. herein, we describe a case of a 52-year - old woman who presented with right flank pain. magnetic resonance imaging revealed a 6-cm mass in the lower pole of the right kidney. t1 and t2 weighted imaging revealed a heterogeneous mass with triple sign. there was post - contrast enhancement. imaging, histology and immunostaining together made the diagnosis of ss of the kidney. |
first described pleuropulmonary blastoma (ppb) as a distinctive tumor in young children, occurring almost exclusively in the first decade of life. since then, individual cases as well as series of similar tumors in children have been reported with the diagnosis of ppb. international ppb registry is maintaining a record of patients with ppb for last 20 years having published [n = 50 ] as well as unpublished [n = 128 ] registry series. it has a rare occurrence beyond the first decade of life and only a few isolated cases in adults have been reported. these tumors usually require a multimodality treatment approach for improving the survival including surgery, chemotherapy and radiation therapy. we herein report a short series of two cases of ppb in adolescence, with one of them having a relatively long survival, an unusual presentation beyond the first decade of life. a 14-year - old adolescent male presented with complaints of nonproductive cough for three months associated with dyspnea on exertion in july 2009. computed tomography (ct) of the chest showed an approximately 6 5 3 cm sized heterogenous mass lesion in the right lower lobe [figure 1a ]. endobronchial biopsy was done, which showed nests of malignant cells with hyperchromatic, elongated nuclei and moderate cytoplasm. these cells were positive for vimentin on immunohistochemistry. a possibility of ppb was suggested., tumor was seen occupying approximately 90% of lower lobe and was greyish white in color with areas of cystic changes. on microscopy, plump to spindle - shaped cells arranged in short fascicles as well as storiform pattern were seen. the cells were showing moderate nuclear atypia, elongated nucleus, some showing nucleoli and moderate amount of cytoplasm with brisk mitoses. in between, there were vague nests of primitive blastemal cells. based on these features, a diagnosis of ppb preoperative (a) axial computed tomography (ct) image showing a large heterogenously enhancing soft tissue mass lesion in the right hemithorax. postoperative and postchemotherapy (b) ct image showing a heterogenous paravertebral soft tissue on the right side the patient was referred for radiotherapy, where he received a cumulative dose of 50 gray (gy) in 25 fractions. the follow - up ct showed a heterogenous soft tissue in the right paravertebral location abutting the mediastinal pleura [figure 1b ]. in view of residual lesion, the patient received six cycles of chemotherapy (250 mg paclitaxel and 70 mg cisplatin) at 3-week intervals. the sixth cycle was completed in march 2010, and no residual lesion was seen in the follow - up ct. however, in september 2011, the patient started complaining of vague abdominal pain on and off. fine - needle aspiration (fna) was done under ultrasound guidance, which showed highly cellular smears and sheets of undifferentiated cells having high nuclear / cytoplasmic ratio. on immunocytochemistry, cells were positive for vimentin and neuron - specific enolase (nse) showed strong focal positivity. in addition, nuclear positivity for wilms tumor (wt-1) was seen in about 50% of cells. the patient was referred to the urology department for the surgical consideration, however, was lost to follow up. an 18-year - old adolescent male patient was referred recently to our institute from a hospital with complaints of abdominal pain with recurrent fever for the last 4 months. a clinical possibility of a ruptured liver abscess in the right pleural cavity based on sonographic findings was documented in the records. the ct showed a large solid - cystic mass lesion with thick enhancing septae filling almost the entire right hemithorax and extending inferiorly, indenting upon the right hemidiaphragm and displacing the liver inferiorly. the mass was having loss of fat planes at places with the mediastinal structures, including trachea, heart, right innominate artery, and was compressing upon right main bronchus. overlying ribs and soft tissues were normal with no extrathoracic extension [figure 2 ]. axial ct image showing a large heterogenous solid - cystic mass lesion occupying the entire right hemithorax. no definite rib erosion and extrathoracic extension is seen a percutaneous ultrasound (us)-guided fna was performed. the aspirate was particulate and the smears were air - dried as well as alcohol - fixed and stained with may - grnwald giemsa and hematoxylin and eosin, respectively. a cell block was also prepared. on microscopy, the smears were cellular, and showed sheets of small variably shaped poorly differentiated cells. the cells were predominantly oval to spindle - shaped and a prominent perivascular arrangement was noted. the tumor cells had high nuclear : cytoplasmic ratio, finely granular chromatin, inconspicuous nucleoli, and scanty eosinophilic cytoplasm with indistinct cell borders. immunochemically, the tumor cells showed strong positivity for vimentin and focal positivity for smooth muscle actin [figure 4 ]. aspiration cytology of pleuropulmonary blastoma : (a) cellular smear with prominent perivascular pattern (hematoxylin and eosin 200) (b) small ovoid to spindled cells with occasional cell showing cytoplasmic vacuolation, inset : small cell with cytoplasmic vacuolation [may - grnwald giemsa (mgg) 400 ] (c and d) cell block showing oval to spindle - shaped cells with scanty eosinophilic cytoplasm with indistinct borders (mgg 400) case 2. immunohistochemistry slides showing strong positivity of tumor cells for vimentin (a - c) and weak focal positivity for smooth muscle actin (d) the patient developed progressive respiratory dysfunction and died approximately seven days after the diagnosis of tumor. a 14-year - old adolescent male presented with complaints of nonproductive cough for three months associated with dyspnea on exertion in july 2009. computed tomography (ct) of the chest showed an approximately 6 5 3 cm sized heterogenous mass lesion in the right lower lobe [figure 1a ]. endobronchial biopsy was done, which showed nests of malignant cells with hyperchromatic, elongated nuclei and moderate cytoplasm. these cells were positive for vimentin on immunohistochemistry. a possibility of ppb was suggested., tumor was seen occupying approximately 90% of lower lobe and was greyish white in color with areas of cystic changes. on microscopy, plump to spindle - shaped cells arranged in short fascicles as well as storiform pattern were seen. the cells were showing moderate nuclear atypia, elongated nucleus, some showing nucleoli and moderate amount of cytoplasm with brisk mitoses. in between, there were vague nests of primitive blastemal cells. based on these features, a diagnosis of ppb preoperative (a) axial computed tomography (ct) image showing a large heterogenously enhancing soft tissue mass lesion in the right hemithorax. postoperative and postchemotherapy (b) ct image showing a heterogenous paravertebral soft tissue on the right side the patient was referred for radiotherapy, where he received a cumulative dose of 50 gray (gy) in 25 fractions. the follow - up ct showed a heterogenous soft tissue in the right paravertebral location abutting the mediastinal pleura [figure 1b ]. in view of residual lesion, the patient received six cycles of chemotherapy (250 mg paclitaxel and 70 mg cisplatin) at 3-week intervals. the sixth cycle was completed in march 2010, and no residual lesion was seen in the follow - up ct. however, in september 2011, the patient started complaining of vague abdominal pain on and off. fine - needle aspiration (fna) was done under ultrasound guidance, which showed highly cellular smears and sheets of undifferentiated cells having high nuclear / cytoplasmic ratio. on immunocytochemistry, cells were positive for vimentin and neuron - specific enolase (nse) showed strong focal positivity. in addition, nuclear positivity for wilms tumor (wt-1) was seen in about 50% of cells. the patient was referred to the urology department for the surgical consideration, however, was lost to follow up. an 18-year - old adolescent male patient was referred recently to our institute from a hospital with complaints of abdominal pain with recurrent fever for the last 4 months. a clinical possibility of a ruptured liver abscess in the right pleural cavity based on sonographic findings was documented in the records. the ct showed a large solid - cystic mass lesion with thick enhancing septae filling almost the entire right hemithorax and extending inferiorly, indenting upon the right hemidiaphragm and displacing the liver inferiorly. the mass was having loss of fat planes at places with the mediastinal structures, including trachea, heart, right innominate artery, and was compressing upon right main bronchus. overlying ribs and soft tissues were normal with no extrathoracic extension [figure 2 ]. axial ct image showing a large heterogenous solid - cystic mass lesion occupying the entire right hemithorax. no definite rib erosion and extrathoracic extension is seen a percutaneous ultrasound (us)-guided fna was performed. the aspirate was particulate and the smears were air - dried as well as alcohol - fixed and stained with may - grnwald giemsa and hematoxylin and eosin, respectively. a cell block was also prepared. on microscopy, the smears were cellular, and showed sheets of small variably shaped poorly differentiated cells. the cells were predominantly oval to spindle - shaped and a prominent perivascular arrangement was noted. the tumor cells had high nuclear : cytoplasmic ratio, finely granular chromatin, inconspicuous nucleoli, and scanty eosinophilic cytoplasm with indistinct cell borders. immunochemically, the tumor cells showed strong positivity for vimentin and focal positivity for smooth muscle actin [figure 4 ]. aspiration cytology of pleuropulmonary blastoma : (a) cellular smear with prominent perivascular pattern (hematoxylin and eosin 200) (b) small ovoid to spindled cells with occasional cell showing cytoplasmic vacuolation, inset : small cell with cytoplasmic vacuolation [may - grnwald giemsa (mgg) 400 ] (c and d) cell block showing oval to spindle - shaped cells with scanty eosinophilic cytoplasm with indistinct borders (mgg 400) case 2. immunohistochemistry slides showing strong positivity of tumor cells for vimentin (a - c) and weak focal positivity for smooth muscle actin (d) the patient developed progressive respiratory dysfunction and died approximately seven days after the diagnosis of tumor. ppb, which is a rare aggressive malignant primary neoplasm of the pleuropulmonary mesenchyme occurring in early childhood associated with poor prognosis.. only a few isolated cases of ppbs have been reported in literature in adult population. table 1 summarizes the key findings of previously published cases in literature in adult population. there are three pathological types (type i, ii, and iii) of ppb based on gross and microscopic features. the median ages at diagnosis are 10, 34, and 44 months for types i, ii, and iii, respectively. clinicoradiological details of published cases of ppb in adult population the tumor may arise from the lung or it may be extrapulmonary, involving the mediastinum or parietal pleura. our case showed the tumor filling nearly the entire right hemithorax in the second case, whereas it was central in location in the first case. it is to be kept in mind that type i ppb can not be differentiated from other benign cystic lung lesions on imaging studies, which include congenital lung cysts such as congenital adenomatoid malformations. type ii and iii ppb usually present as a large heterogenous masses, which may invade mediastinum, vessels, and diaphragm. type ii ppb is a solid - cystic tumor, whereas type iii ppb is solid tumor without epithelial - lined cystic spaces. in view of presence of solid - enhancing areas as well as nonenhancing cystic areas, the tumors in our cases were type ii ppb radiologically. type ii and iii ppb tumors can metastasize to central nervous system, bones, and liver. microscopically, there is presence of primitive blastema and a malignant mesenchymal stroma that often demonstrates heterologous elements such as cartilage differentiation. blastema components may show numerous mitoses and foci of necrosis, as seen in the resected specimen in the first case. cystic component is lined by benign respiratory - type epithelium. due to histologic variability in the solid component of the ppb however, based on the some characteristic features such as cystic architecture, blastemal tissue, and other mesenchymal components including cartilage, ppb can be designated as a distinct entity. the malignant mesenchymal cells are usually immunoreactive for vimentin, muscle - specific actin and desmin. the cells were vimentin positive in both of our cases, whereas actin positivity was seen in the second case only. clinically, the tumor equally affects both genders. in our series, both patients are males. shortness of breath in the absence of other respiratory tract symptoms is the main symptom in most cases of ppb reported. a suspected pulmonary infection, including an empyema, is the most frequent clinical impression in these patients. our first patient presented with respiratory complaints of nonproductive cough and dyspnea. however, the second patient presented with abdominal pain with recurrent fever for last 4 months and hence a pulmonary infection was not a clinical consideration. chemotherapy is based on the institutional practice, which includes either specific chemotherapeutic agents or their combination. the various agents that have been used include vincristine, actinomycin d, cyclosphosphamide, doxorubicin, ifosfamide, etoposide, cisplatin, carboplatin, epirubicin, methotrexate, and 5-fluorouracil. however, no specific association has been shown between the survival and the chemotherapy used. our first patient received cisplatin - paclitaxel combination and showed a complete response (100% decrease) according to response evaluation criteria in solid tumors (recist) criteria. types ii and iii ppbs are aggressive tumors and even after a multimodality therapy have a survival rate of 62% at 2 years and 42% at 5 years. our first patient underwent multimodality treatment and had an overall survival of 27 months (from the diagnosis to the last contact). however, the second patient died within seven days due to progressive respiratory dysfunction even before the treatment could be started. ppbs are familial childhood cancer with a high incidence of childhood cancers in close relatives (25%). these include ppb, malignant germ cell tumor, medulloblastoma, thyroid neoplasia, and others. the patients with ppb are also at a higher risk of developing childhood cancers, as seen in the first case in which the patient developed renal wilms tumor during the follow - up period. to conclude, ppbs are primitive dysodontogenic neoplasms of childhood. | pleuropulmonary blastoma (ppb) is a unique dysontogenetic and a primitive neoplasm occuring almost exclusively in the first decade of life, as a pulmonary- and/or pleural - based tumor with cystic, solid, or combined cystic and solid features. it is characterized histologically by a primitive, variably mixed blastematous and sarcomatous tissues. these tumors are usually associated with a poor prognosis. however, with a multimodality treatment approach, the survival of the patient can be prolonged. we herein report two cases of ppb in adolescence, a rare presentation beyond first decade of life with a short review of literature. |
intraoral and extraoral radiographs are integral part of diagnosis of dental disease and treatment planning. while in forensic odontology, radiographs are an implicit part of practice used mainly in identification and age estimation. the tenet of identification by dental means is the comparison of antemortem records with postmortem records. radiographs are nonabstract and observable when antemortem and postmortem data are presented to the end users of forensic odontological reports. indeed, a picture is worth a thousand words, attributed to french leader napoleon bonaparte is an apt allegory here. however, radiographs are obtained through a multi - step procedure, and as outlined later in the text, operator error can be introduced at different stages. the main errors that will be presented and discussed are those that lead to difficulties or mistakes in orientation of the radiographs and the possible ramifications arising from that. focus of this paper is as follows : the inversion of the right and left marker orthopantomographs (opg), causing the image to be laterally invertedthe reverse placement of phosphor storage plate (psp) receptor for intraoral dental radiograph resulting in confusion of the right and left.both of these errors can result in potential problems in practice as illustrated by the case examples presented here. the inversion of the right and left marker orthopantomographs (opg), causing the image to be laterally inverted the reverse placement of phosphor storage plate (psp) receptor for intraoral dental radiograph resulting in confusion of the right and left. both of these errors can result in potential problems in practice as illustrated by the case examples presented here. an adult male was referred for an extraction of lower left erupted carious third molar under local anesthetic. it was noted that on opg, when placed in the correct orientation for viewing according to the indicator, the carious tooth to be extracted was the right lower third molar. consultation before the procedure and confirmation with previously obtained periapical radiographs confirmed that opg was laterally inversed [figures 1 and 2 ] as illustrated above, the indicator in the digital intraoral radiograph can help with orientating an x - ray. this is the case provided that the correct side of the receptor has been placed toward the x - ray tube. when the psp is used with the correct side facing the x - ray source, and if placed with the indicator to the distal, a lower left radiograph should have the psp = phosphor storage plate the above is a set of bitewings taken with the correct side facing the tube and the indicator in the correct orientation. in the right bitewing, the indicator is on the top distal corner while in the left bitewing, the indicator is the left distal corner. (note : the common operator error cone cutting) this is the same right bitewing taken with reversed placement. the exposure is done with the correct side of the receptor not facing the x - ray source. note the only clue is the indicator appearing on the lower right distal corner of the image ; the contrast and clarity of the image is not affected this is the same radiograph as the one in figure 4. it was laterally rotated to correct the alignment of the indicator, resulting in an inversion of what was a right bitewing into a left bitewing bitewings or periapical radiographs are used for intraoral views, and opg are used for extraoral views. they can also be classified by method of acquisition into either digital or analog radiographs. regardless of the method of acquisition, radiographs are obtained by exposure of ionizing radiation to a receptor. the receptors can be analog radiographic films or digital charge - couple devices (ccd) plates and psps. the exposed receptor containing the latent image analog dental radiographic films require a chemical process to develop the latent image, whereas digital receptors require a computer to process the stored information into a visual image on a computer monitor. all of these receptors, analog and digital, have only one recommended surface of exposure toward the source of radiation. to help with film placement, the analog dental intraoral radiograph has an embossed dot on the film that serves as an indicator for the surface for exposure. as for digital and film opg, l for left is indicated on the receptor and these are transferred to the final radiographic image. in digital intraoral radiographs, the indicator may be marked on ccd plates and phosphor plates. the embossed dot on an exposed dental intraoral film and the indicator on digital receptors in the resultant images allow orientation of the radiographs when viewing, giving a guide to the site of exposure. there may be protocols as to how the x - ray should be placed. for example, in bitewings, it may be recommended that the embossed dot on an analog film be placed in the distal portion of the quadrants, and in a periapical view, the embossed dot might be placed in the occlusal area.. this may not always be the case in practice ; nevertheless, an experienced clinician is usually able to identify the region the radiograph is obtained from based on characteristics of the different teeth and surrounding structures together with the position of the indicator when the receptor has been placed with the recommended surface toward the radiation source in digital radiographs. however, this may become a problem when the radiograph is viewed at a later date or by a different operator. the lack of an attached wire, unlike ccd receptors, makes their use similar to plain film x - rays and thus easier to use as most clinicians are familiar with analog films. however, the recommended side of exposure in some older model is not as obvious compared with ccd receptors for the same reason. reverse placement of the receptor has been reported. more recent receptors have an indicator incorporated to help orientate films. however, at least in one older model, when the wrong side of the receptor is exposed, there is no visual clue to indicate the reversed placement. when this happens, a clinician might confuse the right and left side, especially when there are no, or few restorations, or lack of distinctive characteristics. unlike analog periapical films where the characteristic herring bone appearance, due to the lead lining incorported on the reverse side of the film, provides a clue to the fact the exposure has been done on the wrong side of the film, is is not apparent in psp receptors. certain types of digital intraoral radiographs do not provide any indicators equivalent to the embossed dot on analogous films. this is further complicated by the fact that most native or included software viewers for dental radiographs allow rotation and lateral inversion of the image. inverted or reversed placement of the digital psp opg system receptor has been reported to cause confusion of the true left and right. backward placement of opg cassette may have caused the lateral inversion of the opg films in the case examples. in clinical practice, failure to detect this error can result in initiation of procedures on the wrong tooth or site, especially when this error is not detected in patients with few distinctive characteristics to help orientate the radiograph. for example, in bitewings of fully dentate individuals with the absence of existing restorations. in addition, dental records are legal records ; when legal disputes arise, inaccurate records can be confusing and problematic. some may argue that in routine practice, if the clinician who takes the x - ray is the end user, the clinician should be able to orientate the x - ray to allow meaningful analysis and correct interpretation. however, if the x - ray is used for a referral, this may cause confusion as to the area of interest and result in a procedure being carried out on the wrong tooth or site. in forensic dentistry non - radiographic dental records alone have been shown to be a valuable source as the different permutations and combinations of patterns of missing and filled teeth may allow identification to be done with a certain degree of confidence. however, such records have also been shown to contain mischarting, incorrect reports, and even fraud. forrest and wu state that radiographs are a direct representation of a physical item and are an objective method of recording information, whereas clinical notes are less reliable especially when recording may not even be done by the clinician. wood and kogon mentioned that few odontologists would be confident enough to depend solely on non - radiographic records and that x - rays are important component in identification. radiographs are also useful when few restorations are present, where other anatomical features may be used such as root morphology, canal, trabecular bone pattern, and sinus radiographic outlines. when dental records are required to be submitted as antemortem records (and this can occur months or years since last visit or treatment), the clinician responsible for submitting the records may have forgotten about the error or may overlook the mistake. it is important to submit original and full records even though some records may seem irrelevant. full records afford the opportunity and possibility of this error being noted by the dentist who receives the antemortem record for transcription into antemortem chart. otherwise, errors of this nature can result in exclusion due to visually obvious non - matching of x - rays. furthermore, partial recovery of remains or inexperienced forensic odontologists may lead to exclusion, particularly in mass fatality incidents where multiple reconciliations are required. in a disaster victim identification (dvi) dvi is a complex process, especially when different nationalities are involved, and where different disciplines are required to work together for the process to work. this usually involves multidisciplinary collaboration and numerous people working within each discipline to collate information under the challenging physical and political backdrop of the inherent chaos that accompanies the event. within the forensic odontology discipline, there will usually be teams working in each section with rotation of personnel in a large - scaled dvi. the complexity of a large - scale operation with information (antemortem and postmortem data) coming from different sources and often at different points of time throughout the process provides the potential for complications. often, multiple antemortem dental records from one potential victim can come from different practitioners and may be only made available at different points during the progress of the dvi process. dental records can be handwritten or computer generated ; the task of collating and consolidating records and x - rays for each possible victim is an onerous one. in addition, there can be a large amount of postmortem information by virtue of the sheer number of victims involved. for example, in the thai tsunami, as many as 200 dental radiographs were obtained in a single day. this, together with many other challenges that are inherent in a large - scale dvi process, e.g., physical and mental fatigue or when records are from international sources with different languages and systems of recording, can further complicate the process. one of the reasons why original x - rays are important is that original analog x - rays allow unambiguous identification of the correct site. this applies to films that have been scanned or photocopied, and this important information may not be transmitted correctly. with scanned digital radiographs, mistakes have been made where the radiographs have been scanned in a reversed order. identification by comparison of dental features is a primary scientific method that is stand alone identification in dvi interpol guidelines. in routine practice, a majority of cases are confirmatory, where possible identification has already been made and a dental identification is needed to confirm other evidential or circumstantial finding. the task of matching antemortem and postmortem radiographs is one where judgment is required as differences may be explainable. the australian society of forensic odontology recommends the inclusion of statements about the consistencies and lack of irreconcilable discrepancies in reports of cases of established identification. the occurrence of errors in the interpretation of antemortem and postmortem radiographic or digital images will lead to incorrect identifications. forensic odontologists do not have complete control of the antemortem evidence submitted ; it is not possible to check the whole trail and history of the evidence to ensure that evidence submitted is true and correct. quality checks can be done, e.g. through direct communication with the dentist who submitted the evidence to confirm records but this can only go so far. in dvi situations, original records may not be submitted and communication with the treating dentist can be difficult especially where the records are from overseas. it is important to include in statements and reports, descriptions of the evidence as they were received. in other areas of clinical practice, being aware of the possibility of such error can save some difficult and troublesome medicolegal problems at a later stage. | digital dental radiography, intraoral and extraoral, is becoming more popular in dental practice. it offers convenience, such as lower exposure to radiation, ease of storing of images, and elimination of chemical processing. however, it also has disadvantages and drawbacks. one of these is the potential for confusion of the orientation of the image. this paper outlines one example of this, namely, the lateral inversion of the image. this source of confusion is partly inherent in the older model of phosphor storage plates (psps), as they allow both sides to be exposed without clue to the fact that the image is acquired on the wrong side. the native software allows digital manipulation of the x - ray image, permitting both rotation and inversion. attempts to orientate the x - ray according to the indicator incorporated on the plate can then sometimes lead to inadvertent lateral inversion of the image. this article discusses the implications of such mistakes in dental digital radiography to forensic odontology and general dental practice. |
the aim of regenerative medicine is currently underway to use the body s own cells to repair diseased or damaged tissue. the use of stem cells from different tissues for regenerative medicine applications has increased over the past few years. amniotic fluid (af) represents rich sources of stem cells population deriving from either the fetus or the surrounding amniotic membrane that can be used for clinical therapeutic applications in the patients who develop organ failure that have resistance to current therapies. the af was first studied at the beginning of the 20th century (1). initial studies have been performed in order to detect fetal abnormalities during development the fetus (2). isolation and identification of amniotic fluid- stem cells (afscs) dating back to the early 1990 s (3). first, af is easily collected during the first trimester of pregnancy by scheduled amniocenteses for fetal karyotyping, prenatal diagnosis and detection a variety of genetic diseases (4). finally, these cells could be stored in cell banks and used in disease research, drug screening and genetic disorders. human af is a protective and nourishing watery liquid that providing mechanical support during embryogenesis and is constituted of about 98% water (4). other ingredients include electrolytes, pigments, sugars, fats, amino acids, proteins, carbohydrates, enzymes, growth factors and cells (5). after formation of the amniotic cavity, 7~8 days after fertilization, this fluid starts to gather immediately. af volume increases progressively and is then completely surrounding the embryo after 4 weeks of pregnancy. the average volume is 270 ml at week 16 which increases to 400 ml at week 20 of pregnancy and 800 ml at birth (5). at the beginning of pregnancy, after keratinization of the fetal skin, which usually occurs at week 24 of pregnancy, amniotic osmolarity decreases relatively to maternal or fetal plasma, mainly due to the inflow of fetal urine (7). additional investigations have been recently focused on the cellular and molecular properties of amniotic derived cells and their potential use in pre - clinical models and in cell therapies (4). the first one is based on single - stage method (8). in this method af collected from second - trimester amniocentesis is centrifuged. the number of cells is counted by hemocytometer and mixed with an equal volume of culture medium, usually dulbecco s modified eagle medium (dmem) supplemented with fetal bovine serum (fbs) and the cells allowed to adhere to a plastic culture plate at 10 cells / cm and incubated overnight at 37c under 5% co2 (9). culture medium is changed after 3~5 days to remove non adherent cells and twice weekly thereafter. the primary cells are cultured for 4~5 days until they reached confluence and are defined as passage 0. in this step the heterogeneous morphological cell population appears and after several sub - culturing the fibroblastic like cells was dominated. the cells typically reach confluence in 4 to 6 days and the remaining cells are cryopreserved in cryopreservation media (10% dimethylsulfoxide, 90% fbs), frozen at 80c for 24 h, and stored in liquid nitrogen the next day. ditadi was the first researcher to show that the c - kit population cells extracted from the af do have hematopoietic potential (10). at this time, atala. (11) isolated cd117 positive cells by c - kit (a rabbit polyclonal antibody to cd117) and cd133 positive cells by cd133 magnetic beads, respectively. their study showed these cells can be easily expanded in cultures and sub - population of cd133 positive exhibited similar characteristics of mesenchymal progenitors cells (11). following these studies, arnhold. they indicated that the percentage of cd117 positive cells was 3.21.03% of the whole cell population and demonstrated that these cells could differentiate to adipogenic, osteogenic, myogenic and neurogenic lineage (12). the third one is the two - stage culture protocol established by tsai., using nonadhering af cells of the primary amniocytes culture to isolate afscs. in this protocol, nonadhering af cells are collected from supernatant of afcs that cultured in serum - free changes medium (first stage). then collected cells are plated for afscs culturing after the completion of fetal chromosome analysis (second stage) (13). major advantage of this culture protocol comparing to the other two is that instead of the adhering cells derived in af it isolates from the nonadhering cells, which is being left in the incubator without any added nutrition for 7~10 days (13). as mentioned two - stage method is more superior compared to other methods, the use of this method have been proposed. the afscs are mainly composed of three heterogeneous groups of adherent cells, calcified based on their growth, morphological and biochemical characteristics that derived from the three germ layers (14). epithelioid (e - type) cells that are cuboidal to columnar, derived from the fetal skin and urine, af (af - type) cells are originating from fetal membranes, and fibroblastic (f - type) cells are generated mainly from fibrous connective tissue. some studies have reported that afscs can be easily obtained from a small amount of af (4, 15). like other mesenchymal stem cells (mscs), the afscs expressed cd73, cd90, cd105, cd29, cd166, cd49e, cd58, cd44 and hla - abc antigens and are negative for the hematopoietic markers such as cd14, cd34, cd45 and cd133, the endothelial marker such as cd31, and the hla - dr antigen (fig. 1) (4). these cells are able to differentiate along adipogenic, osteogenic, myogenic, endothelial, neurogenic and hepatic pathways (table 1) (1619). additionally, the majority of these cells expressed the pluripotency markers such as the octamer binding protein 3/4 (oct-3/4), the homebox transcription factor nanog, and the stage - specific embryonic antigen 4 (ssea-4) (20, 21). similar to mscs, afscs express mhc ii at a very low level. unlike human mscs that are telomerase negative, low to moderate levels of the enzyme have been described in afscs (5, 22). telomeres consist of ttaggg repeats protect the ends of chromosomes from end - to - end fusion, recombination and deterioration (5). the presence of telomerase activity in both cultured and uncultured cells was found in 1999 (24). another interesting finding have shown the presence of a population of oct-4-positive cells in af (25). afscs also express vimentin and alkaline phosphatase, which are markers of pluripotent stem cells (5, 26). like other mscs, afscs are attractive candidates for clinical applications, which were reviewed in table 2. for instance some reports indicated that the af can be a reliable and practical source of cells for the engineering of select fetal tissue constructs (27, 28). another clinical application of afscs is the use as produce mineralized bioengineered constructs in vivo, functional repair of bone defects and bone engineering (2, 2931), neural tissue regeneration and nerve myelination (3235), lung epithelial regeneration (36, 37), cardiac regeneration (38, 39) and kidney regeneration (21). like other mscs, afscs have advantages such as rapid cell proliferation, low or negligible immunogenicity. thus far they have been used in pre - clinical settings to treat a variety of diseases such as osteogenesis imperfecta, congenital diaphragmatic hernia, parkinson s disease and cancer with encouraging results. finally, their usefulness for afscs is very likely to expand their future clinical use even further. human afscs could be isolated by several methods including immunoselection method based on surface antigens, single - stage and two - stage methods. for example this method does nt interfere with the normal culture process for fetal karyotyping and also illustrated their ability to successfully differentiate into osteocyte, adipocyte and etc in vitro. as two - stage method is more superior compared to other methods, | amniotic fluid represents rich sources of stem cells that can be used in treatments for a wide range of diseases. amniotic fluid- stem cells have properties intermediate between embryonic and adult mesenchymal stem cells which make them particularly attractive for cellular regeneration and tissue engineering. furthermore, scientists are interested in these cells because they come from the amniotic fluid that is routinely discarded after birth. in this review we give a brief introduction of amniotic fluid followed by a description of the cells present within this fluid and aim to summarize the all existing isolation methods, culturing, characterization and application of these cells. finally, we elaborate on the differentiation and potential for these cells to promote regeneration of various tissue defects, including fetal tissue, the nervous system, heart, lungs, kidneys, bones, and cartilage in the form of table. |
sepsis is a common clinical condition with a significant impact on healthcare resources and expense. according to world health organization estimates, sepsis accounts for 60 - 80% of lost lives per year in childhood. it accounts for approximately 20% of all admissions to the intensive care unit and is remain the leading cause of morbidity and mortality in the pediatric intensive care unit (picu). data from the surviving sepsis campaign showed a mortality of 34.8% and the number is higher in developing country. recent data from italian picu (n = 22) reported mortality as high as 50% in children with septic shock. the growing number of patients with severe sepsis and septic shock with significant mortality requires changes in the current management protocols. in 2002, the american college of critical care medicine first published clinical practice parameters for hemodynamic support of pediatric and neonatal with septic shock. han., later reported that early recognition and aggressive resuscitation of pediatric - neonatal septic shock by community physicians can save lives. previous reports have shown a reduction of incidence with using bundled care such as catheter - related infection or morbidity and mortality in mechanically ventilated patients by changing clinical practice. the current guidelines recommend stepwise use of fluid resuscitation and inotropic support to attain optimal blood pressure and achieve adequate tissue perfusion. we believe that if the bundle guidelines are well - organized, and performed reliably and timely, we can achieve the desired outcome of reducing sepsis - related death at our institution by at least 5 - 10%. thus, the purpose of our study was to determine the clinical outcomes after using surviving sepsis campaign guideline also assess the prognostic value of initial nt - probnp and plasma procalcitonin in children with severe sepsis and septic shock. we prospectively employed a before and after study in the picu of university referral hospital, bangkok, thailand. the study compares time periods before (2007 - 2009) implementation of the interventions, and the period of intervention from april, 2010 to may, 2011 with wash - out period from january, 2010-march, 2010. the educational program was done through a protocol based on surviving sepsis campaign guideline including lectures by er personnel, healthcare workers at pediatric ward, pediatric residents and fellows. the protocol addressed the importance of early diagnosis and therapeutic interventions, quality indicators and the data collection (see details of protocol in appendix 1). patients aged between 1 month through 15 years with diagnosis of severe sepsis or septic shock who were admitted to the pediatric ward prior to picu admission or directly to picu were recruited to our study. clinical data collected included age, sex, admission date, the time of diagnosis before picu admission, location, time of antibiotics given, length of stay and prism score. once a patient met the inclusion criteria, the acute interventions including fluid resuscitation to achieve adequate tissue perfusion, administration of antibiotic, hydrocortisone if clinically indicated, early use of inotrope or vasopressor and post acute intervention bundle care to be completed within 24 h. blood was collected for a basic laboratory work up including blood culture prior to antibiotic administration. the picu physicians, fellows, and nurses were notified simultaneously of the cases enrolled. a modified early goal directed in children with severe sepsis and septic shock for 6 hrs and 24 hrs bundle. data are presented with mean standard deviation or median depending of the normality of their distribution. if data were normally distributed, the comparisons were performed using student 's t - test, otherwise, wilcox on rank - sum (mann - whitney) test was used. receiver - operating characteristic (roc) curves were constructed to illustrate a cut off value of pct and nt - probnp levels. all statistical analyses were performed using the software spss, version 16 (chicago, il, usa). data are presented with mean standard deviation or median depending of the normality of their distribution. if data were normally distributed, the comparisons were performed using student 's t - test, otherwise, wilcox on rank - sum (mann - whitney) test was used. receiver - operating characteristic (roc) curves were constructed to illustrate a cut off value of pct and nt - probnp levels. all statistical analyses were performed using the software spss, version 16 (chicago, il, usa). data are presented with mean standard deviation or median depending of the normality of their distribution. if data were normally distributed, the comparisons were performed using student 's t - test, otherwise, wilcox on rank - sum (mann - whitney) test was used. receiver - operating characteristic (roc) curves were constructed to illustrate a cut off value of pct and nt - probnp levels. all statistical analyses were performed using the software spss, version 16 (chicago, il, usa). their age was at 5.4 4.9 years (range : 1 month-15 years). thirty patients (63.8%) had underlying diseases in addition to sepsis, leukemia (25.5%) was the most common. chronic liver disease was found in 6 (12.7%), neuromuscular disease in 4 (8.5%), and immunodeficiency in 3 (6.4%). there were 31 (66%) patients who developed respiratory failure and required mechanical ventilation. mean initial superior vena cava oxygen saturation (iscvo2) at admission to picu was 73.4% 6.2% (range : 52 - 87%) as shown in table 1. initial oxygen index was 9.9 + 8.6 and pao2 /fio2 ratio was 210.1 + 95.1. initial positive inspiratory pressure (pip) for those mechanical ventilated was at 23.7 + 6.90 (cmh2 o). maximum pip was significantly lower in survivors compared with nonsurvivors (21.9 7.2, 28.4 2.7 cm h2 o, p = 0.02). initial arterial ph was significantly lower in the non survival group compared to the survival group (7.3 0.1 and 7.4 0.1, p = 0.04) [table 2 ]. baseline demographic data of subjects enrolled in the study compared between preinterventional period (2007 - 2009) and interventional period (2010 - 2011). (data represent as mean standard deviation) baseline clinical characteristic and laboratory parameters admitted to pediatric intensive care unit compared between survivor and nonsurvivor group. (significant p 70% after initial fluids resuscitation and the group that had scvo2 11,200 ng / l, sensitivity 85%, specificity 93%) and initial procalcitonin (levels > 1.42 / l, sensitivity 85%, specificity 49%) the overall compliance of our sepsis bundle was at 70% by measuring from each item. to achieve optimum oxygen delivery by keeping hb at 10 g / dl, 17 patients (36%) a total of 42 patients (87.2%) were given antibiotics within an hour of severe sepsis or septic shock diagnosis. all patients were transferred out of er to picu within 1.1 0.2 h. a total of 0.9% nss was selected as a first choice in 95% of initial fluid resuscitation after enrollment. 18.8 3.4 cc / kg was given in the first 15 min, 43.1 13.1 cc / kg in the 1 hour, and 68.3 19.22 cc / kg in the first 6 h of fluid resuscitation. inotropic or vasopressor agents were started via peripheral line after fluid resuscitation within 33.8 5.5 min to achieve optimum cardiac output (by using update pediatric hemodynamic parameters guideline). dopamine was used as a first - line inotrope in 95.7% after fluid refractory shock was suspected. central venous line (internal jugular 38.3%, subclavian 23.4%, and femoral 38.3%) were inserted in children with fluid refractory septic shock within 2 0.3 h after initiation of fluid resuscitation to achieve a cvp goal of 8 - 12 mmhg. central venous oxygen saturation (scvo2) level of at least 70% was achieved in 89.4% of patients in the first 6 h. levels were significantly different between survivors and non - survivors [figure 1, table 2, p 70% after initial fluids resuscitation and the group that had scvo2 11,200 ng / l, sensitivity 85%, specificity 93%) and initial procalcitonin (levels > 1.42 / l, sensitivity 85%, specificity 49%) the overall compliance of our sepsis bundle was at 70% by measuring from each item. to achieve optimum oxygen delivery by keeping hb at 10 g / dl, 17 patients (36%) a total of 42 patients (87.2%) were given antibiotics within an hour of severe sepsis or septic shock diagnosis. a total of 0.9% nss was selected as a first choice in 95% of initial fluid resuscitation after enrollment. 18.8 3.4 cc / kg was given in the first 15 min, 43.1 13.1 cc / kg in the 1 hour, and 68.3 19.22 cc / kg in the first 6 h of fluid resuscitation. inotropic or vasopressor agents were started via peripheral line after fluid resuscitation within 33.8 5.5 min to achieve optimum cardiac output (by using update pediatric hemodynamic parameters guideline). dopamine was used as a first - line inotrope in 95.7% after fluid refractory shock was suspected. central venous line (internal jugular 38.3%, subclavian 23.4%, and femoral 38.3%) were inserted in children with fluid refractory septic shock within 2 0.3 h after initiation of fluid resuscitation to achieve a cvp goal of 8 - 12 mmhg. central venous oxygen saturation (scvo2) level of at least 70% was achieved in 89.4% of patients in the first 6 h. levels were significantly different between survivors and non - survivors [figure 1, table 2, p 70% after initial fluids resuscitation and the group that had scvo2 11,200 ng / l, sensitivity 85%, specificity 93%) and initial procalcitonin (levels > 1.42 / l, sensitivity 85%, specificity 49%) the mortality rate of severe sepsis and septic shock in our picu was previously quite high similar to other developing countries. sepsis is associated with imbalance between oxygen delivery and demand. the treatment strategy to correct this imbalance rivers., firstly introduced early goal - directed therapy management algorithm in adults sepsis since admission to emergency department. early resuscitation in severe sepsis and septic shock can modulate inflammation and result in significant reduction morbidity and mortality as well as health care resource consumption. han., previously reported that early reversal of pediatric - neonatal septic shock by community physicians is associated with improved outcome. it showed the importance of early and aggressive hemodynamic resuscitation in children with septic shock. additionally, previous analysis of utilized bundled care for adult septic shock demonstrated consistent and significant improvement in survival. this study also shows that implementing a sepsis bundle in our hospital could significantly improve outcome, given the fact that our previous baseline clinical data for the preintervention period (3 years prior) is comparable with 14 months of interventional period [tables 1 and 3 ]. early recognition of sepsis, hemodynamic optimization with fluid resuscitation, and rapid vasopressor used may contribute to our success in reducing sepsis mortality. every hour of delay in appropriate antibiotic administration was associated with a significant increase in mortality. this study demonstrated that 87.2% (n = 41) of our subjects received antibiotics within 1 h of enrollment. the compliance of the sepsis bundle played a significant part of improving outcome. prior to the implementation of a written sepsis protocol and educational program, our compliance of the bundle was at 30% at the baseline and continues to increase over time. this study achieved a high compliance rate (70%) of the sepsis bundle at the end of the study which was much better compared to most of the previous published data that ranged between 30% and 55%. additionally, recent studies in adult sepsis from most of the asian countries had reported even lower compliance rate (7.6 - 54%). we utilized a modified model of rapid response team (trained fellow, pediatric residents, and nurses) with rapid transfer of critically ill sepsis patients from their initial location (er, medical, and surgical ward) to the picu or wards for continued egdt. multiple clinical staff models may be required to achieve a consistent level of quality care for the treatment of sepsis. this team model plays a vital part in achieving sepsis bundle compliance which resulted in early therapeutic interventions. logistic issues regarding early hemodynamic optimization limit its generalizability because of inadequate resources even in industrialized country. recent multicenter study reported overall in - hospital mortality rate reduction from 23.1% to 18.8% without including scvo2 > 70% as a directed goal therapy. in contrast, another multicenter study showed that the only intervention from the sepsis bundle that impacts on mortality was the achievement of scvo2 > 70%. nevertheless, we found in our study that targeted scvo2 (> 70%) at 6 has a better outcome (89.4% in first 6 h, or : 0.8 (0.7 - 0.9), 95% ci, p = 0.02). although a normal scvo2 does not exclude tissue hypoxia, a low scvo2 is an important sign of inadequate tissue perfusion. in addition, children are more likely to have cardiac dysfunction than adults in severe sepsis or septic shock, with 58% having low cardiac output with high systemic vascular resistance and 22% having both low cardiac output and low systemic vascular resistance. thus, a biomarker that could be linked to cardiac dysfunction in children with septic shock would be helpful. plasma nt - pbnp is a biomarker, synthesized and secreted by myocytes and fibroblasts in the atria and ventricle that has been associated with cardiac dysfunction. our study reveals significant correlation between initial procalcitonin level and plasma nt - probnp levels drawn at initial resuscitation. this may indicate the severity of infection related with higher level of nt - probnp. we observed that initial plasma nt - pbnp levels were significantly elevated in all septic patients and were significantly higher in non survivors compared with survivors. moreover, initial plasma nt - probnp levels were also found to be an independent prognostic marker of hospital mortality and were a better predict picu mortality than initial procalcitonin. a recent article reported that bnp level at intensive care unit entry correlates with mortality and could have a role in guiding fluid therapy in septic patients. they reported that the volume of positive fluid balance was independently associated with rise in bnp level. although early goal - directed therapy requires volume resuscitation, negative fluid balance should be targeted after hemodynamic stabilization. further studies on the role of this marker in relation to cardiac dysfunction in sepsis and guiding fluid management are required. applying the sepsis management bundle in a single center tertiary care setting resulted in reduced mortality for children presenting with severe sepsis and septic shock. the sepsis bundle is a quality improvement program that should be implemented in all hospital settings and effort should be made to continue improving compliance. it also requires a coordinated effort among healthcare team such as emergency room physicians, ward physicians, nurses, and intensivists. however, the importance of each component in the bundle should be studied perhaps the bundle could be modified to suit individual institution capabilities. last, continued education for all healthcare providers involved in sepsis care is mandatory for sustained improvement in patient outcomes. | background and objective : the surviving sepsis campaign treatment guideline (ssc) implementation is associated with improved outcome in adults with severe sepsis. the effect on outcome of pediatric sepsis is less clear.purpose:to determine the clinical outcomes of ssc implementation and to investigate the prognostic value of initial plasma nt - probnp and procalcitonin in children.materials and methods : infants and children (aged 1month/0 - 15 years with severe sepsis or septic shock) were prospectively enrolled and treated according to the guidelines. initial blood drawn was saved for nt - pro - bnp, procalcitonin measurements and clinical data were also recorded.results:a total of 47 subjects were recruited. since the application of the ssc, our mortality rate had significantly decreased from 42 - 19% (p = 0.003) as compared to the data in the previous 3 years. clinical factors that significantly increased the mortality rate were : initial central venous oxygen saturation < 7 0% after fluid resuscitation [odds ratio (or) = 23.3 ; 95% confidence interval (ci) 3.7 - 143 ; p = 0.001 ], and initial albumin level (3 g / dl, or = 6.7 ; 95% ci 1.2 - 37.5, p = 0.03). there was asignificant difference between the initial nt - probnp levels between survivors and non survivors, (6280.3 9597 ng / l, p < 0.001), but not for procalcitonin (12.7 24.8, 29.3 46 g / l, p = 0.1), respectively. an initial nt - probnp level of more than 11,200 pg / ml predicted pediatric intensive care unit (picu) mortality with a sensitivity of 85.7% and a specificity of 90%.conclusions : a modified ssc for severe sepsis and septic shock significantly reduced the mortality rate in our picu. high initial nt - probnp level was associated with mortality. |
this series of articles provides regular surveillance of new technologies which may impact on critical care. several countries have developed national horizon scanning systems to identify and monitor new health technologies. there is variation in how these centres gather information, but a consistent set of high priority sources has been identified. for the purposes of this article, the outputs of major health technology assessment centres, national regulatory authorities, and recognized scientific news sources (table 1) were systematically searched for developments relevant to acute and critical care this was combined with a manual medical literature search, along key editorial themes subjectively selected for this issue. point - of - care testing is a major emerging theme throughout the health sector, encompassing both new diagnoses and monitoring of known diseases and their treatment. areas of research range from the potentially lucrative markets for outpatient, ' office'-based and patient self - testing, through to in - hospital diagnostics, which include both rapid access analysis of traditionally laboratory bound diagnostics and direct patient imaging. both aspects are particularly relevant to critical care clinicians, who rely on time sensitive diagnosis and treatment in a hyper - acute setting. an example of bedside imaging in cardiac assessment has already been cited in the first article of the present series. sample analysis, meanwhile, is rapidly developing to encompass bedside biochemical markers, physiological homeostasis monitoring, and novel ultra - rapid forms of infectious disease diagnosis. b - type natriuretic peptide can be a rapid and effective marker of ventricular strain and heart failure, and can now be measured using a point - of - care diagnostic panel (triage bnp test ; biosite inc., san diego, ca, usa). similar current and forthcoming technologies include rapid access d - dimer assays for diagnosis of pulmonary embolism as part of a structured point - of - care algorithm and unpublished early developments in stroke diagnostics. validation and clinical trials of these technologies have taken place primarily in the emergency department setting, but heart failure, cerebrovascular accident and pulmonary embolism are all of added significance in the intensive care unit (icu) as both primary and acquired conditions. rapid bedside diagnosis of such conditions with minimal need for intrahospital transport may be of great potential benefit to intensivists. the importance of tight glucose control in sepsis is becoming well established, although work continues on refining the target range, with a study of 4,000 patients now in progress (normoglycaemia in intensive care study, anzics, commencing 2004). the first major prospective study of tight glucose control in sepsis introduced a novel algorithm requiring frequent measurements, which raised concerns over patient safety and resource utilization in general icus. point - of - care ' stick ' glucose testing is already prevalent, but technology now exists for continuous in vivo glucose monitoring, which, although intended for ambulatory use, could improve accuracy in the acute setting. a subcutaneous interstitial glucose sensor system (continuous glucose monitoring system ; medtronic minimed, inc., northridge, ca, usa) was tested against clamp controlled hypoglycaemic and hyperglycaemic excursions in volunteers ; it was shown to be closely correlated with reference analyzer results (r= 0.91 ; p < 0.001) and highly responsive (half - time 4.0 1.0 min). similarly, another device (glucoday ; a. menarini diagnostics, florence, italy), utilizing a 15100 l micropump and a biosensor coupled with microdialysis to give a claimed response time of 2 min, will reach european markets this year. such devices may be incorporated into manual algorithms, or they may potentially open the way to automated closed - loop glucose control. microbiological diagnosis within clinical laboratories has been advancing apace. polymerase chain reaction technology is well established, but progressive refinements have made possible the rapid and near real - time diagnosis of current, novel, or newly relevant pathogens, including hiv and sars (severe acute respiratory syndrome). techniques initially aimed at viruses because of their manageable size can now also be applied to bacteria and can be used for broad, simultaneous screening of multiple pathogens (pneumoplex, prodesse, milwaukee, wi, usa). further refinements in microarrays and microfluidics are anticipated to bring handhand and point - of - care systems into use in the near future. point - of - care and rapid laboratory based technologies will soon be able to elicit not only pathogen identity but also patterns of drug resistance. developments include the use of adenylate kinase assay for accelerated laboratory based identification of drug - resistant bacteria, including methicillin - resistant staphylococcus aureus and vancomycin - resistant enterococci (baclite, acolyte biomedica, salisbury, uk ;). point - of - care testing within emergency and critical care areas is likely to develop rapidly in the next 5 years, but it will bring complications relating to quality control, medicolegal liability, certificated training for icu and other nonlaboratory staff, increased cost, and territoriality issues. finally, other bedside technologies that have recently been assessed include the use of handheld ultrasound devices to detect occult pneumothoraces, which have been shown to have a higher sensitivity than chest radiography (48.8% versus 20.9%) against a computed tomography standard. preliminary investigations suggest that handheld infrared pupillometry may be of clinical use in detecting midline cerebral shift in head injury patients. more procedure orientated assistance may become available from near - infrared technology, which has been piloted in a computerized bedside visualization device to aid venous cannulation. a study of icu patients in calgary demonstrated crude death rates of 45% among patients with icu - acquired bsi, as compared with 21% in those without (p < 0.0001). s aureus was isolated in 18% of cases in the study cited above. in this context, the development of an antistaphylococcal vaccine (staphvax ; nabi pharmaceuticals, boca raton, fl, usa) represents a promising new health technology. staphvax is currently in phase iii trials for end - stage renal disease, but phase ii trials are under way in postoperative and long hospital stay patients. health technology assessment encompasses the best use of current health care devices as well as emerging technologies. medical devices represent a prime infection hazard, and us centers for disease control and prevention guidelines cover the safe use of intravascular devices to minimize acquired bsi. however more recent work demonstrates that the incidence of catheter - related bsi may be significantly reduced by adding a further device needle - free, disinfectable connectors instead of three - way stopcocks to the existing recommendations (0.7 infections/1000 days versus 5.0 infections/1000 days of catheter use ; p < 0.03). clinical management of sepsis is normally outside the remit of this section of the journal. however, it is noteworthy that new mechanical technology has been applied to the direct treatment of sepsis rather than to cardiovascular or tissue perfusion monitoring. a recent european multicentre open randomized phase ii trial investigated the use of the endotoxin adsorber system en500 (fresenius, bad homburg, germany) in 145 patients with severe sepsis or septic shock due to suspected gram - negative infection. the study demonstrated a trend toward reduced icu stay and more rapid reduction in lipopolysaccharide levels, but it failed to show any difference in outcome. isabel is a web - based, diagnostic decision support tool intended to provide diagnosis reminders and minimize missed diagnosis of critical disease processes. it is currently in use in several uk and overseas hospitals, with development supported by uk department of health funding followed by a commercial launch. the methodology is novel ; a commercial artificial intelligence inference engine (autonomy, cambridge, uk) is used to extract and structure information from standard paediatric textbooks, and to generate diagnostic reminders from this knowledge base in response to unstructured free text clinical information. the software has been under development for some time and was reviewed in this journal in 2002, but it is now being modified to encompass adult critical illness. a review of decision support systems by the uk national institute of clinical excellence is pending. the isabel project was initially set up on a charitable basis by the parents of a child who survived a prolonged stay in paediatric intensive care after a missed diagnosis of necrotizing fasciitis. although the system is as yet little known among adult intensivists, its technology is innovative and its proposed status as an ' online second opinion ' may give it, together with similar expert systems, a powerful consumerist resonance with patients, carers and managers. the uk national institute of clinical excellence findings should be monitored with interest by critical care providers. more broadly, the uk health service is currently in the grip of a globally unprecedented large - scale national project for it (npfit). structured as a series of private finance initiatives, this ambitious programme will ultimately see in a host of regionally standardized patient information systems, image storage, and networked monitoring and audit systems, linked to a national electronic patient record ' spine '. broader concern is growing about catastrophic and unpredictable ' emergent behaviour ' in massively interconnected information technology (it) systems, which are rapidly becoming too complex to test or accurately model. emergent behaviour in complex systems has already been explored in popular fictional media, in which predicted outcomes are spectacular but somewhat discouraging ; however, even without quite such an apocalyptic scenario, we may well see a rising incidence of total system failures due to unpredictable nonlinear behaviour that is, major collapses triggered by small unforeseen causes. in the light of recent north american power outages and destructive computer failures in the uk social service and tax systems, emergent behavour must now be considered a clear and present threat to our increasingly networked health services and their supporting infrastructure. levels of concern are such that the uk government is funding a 10 million research programme into it complexity and catastrophic failures. they therefore require the successful convergence of multiple hospital systems, which makes them uniquely vulnerable to the consequences of system failures, whether in diagnostics, supplies, information flow, or indeed electrical power. second, the currently stated uk npfit vision is that all icu subsystems, including networked monitoring, telemetry and audit systems, will eventually be integrated into npfit, with control over equipment selection and data collection handed to the regional private sector consortia and to national audit bodies. clinician engagement and choice may not feature highly on the agenda, and there are clear concerns over the future of independent research and audit. finally, clinicians from other countries would be well advised to follow such developments because the uk is not unique in its desire to radically modernize and standardize health it, starting with a drive toward electronic patient records. in april 2004, president bush issued an executive order calling for us national implementation of electronic medical records within 10 years, from a current baseline of 19% implementation. in a series of presidential speeches he went on therefore, this represents another area in which political and technological developments outside the icu may have a direct impact on clinical practice and patient safety, and intensivists are strongly recommended to consult early and engage with those driving their local and national health economy. very few of these are designed or marketed to be specific to intensive care, and few are traditional ' devices ' that can be physically handled or attached to a patient. however, critical care is a distillation of acute hospital practice, and any health care technology that has an impact on diagnosis, monitoring, and management of acute conditions will be of heightened importance in the clinical pressure cooker of intensive care. point - of - care testing, accelerated microbiological diagnostics, decision support systems and networked it systems are all key developments that will exert an impact on future critical care practice. bsi = bloodstream infection ; icu = intensive care unit ; it = information technology. | this commentary represents a selective survey of developments relevant to critical care. selected themes include advances in point - of - care diagnostic testing, glucose control, novel microbiological diagnostics and infection control measures, and developments in information technology that have implications for intensive care. the latter encompasses an early example of an artificially intelligent clinical decision support mechanism, the introduction of a national health care information technology programme (uk npfit) and its implications, and exotic threats to patient safety due to emergent behaviour in complex information systems. |
metastatic cancer is usually found in the bone, which is the third most common site of cancer metastasis. metastatic bone cancer often occurs in the axial skeleton such as pelvis, ribs, skull, and vertebrae. metastatic cancer of the limbs is frequently found at the proximal end of long bones such as the proximal end of the humerus and femurs. metastatic cancer of the limbs inevitably causes pain, limb dysfunction and formation of a bump, or even results in pathological fracture. these complications significantly influence the quality of life (qol) and affect the performance of adjunctive therapies, including chemotherapy and radiotherapy, which may further influence the survival time and the prognosis of these patients [46 ]. non - surgical treatment is a major strategy for the treatment of multiple metastatic bone cancers of limbs, and, in addition to radiation (external beam radiation, radiopharmaceuticals), adjunctive therapies include bisphosphonates and chemotherapeutic agents, ablative techniques (radiofrequency ablation [rfa ] and cryoablation), inhibitors of rank - rankl interaction (eg, denosumab), hormonal therapies, and interventional techniques (eg, kyphoplasty). for patients developing pathological fracture at the limbs, palliative surgical treatment is frequently adopted in which the lesions are removed completely, followed by filling of bone cement and fixation with plates and screws or intramedullary nails [911 ]. these treatments may relieve the pain, but are less beneficial for the recovery of limb function, and weight - bearing walking is usually not possible using a crutch or a walker, so these patients often have poor qol [1214 ]. for patients with solitary metastatic bone cancer of the limbs, palliative surgical treatment is often clinically preferred. in the present study, solitary metastatic bone cancer of the limbs was treated according to the principles for the treatment of primary bone cancer. en bloc resection was performed, followed by limb - salvage reconstruction of bone defects and limb function. to analyze the effectiveness of this strategy, the pain, qol, limb function, tumor - free survival and operative complications were evaluated. from 2007 to 2010, a total of 27 patients with solitary metastatic bone cancer were treated in our department, including 15 males and 12 females, with a mean age of 588.58 years (range : 4375 years). follow - up was carried out for 16.157.47 months (range : 631) (t=1.073, p=0.294). the primary cancers included lung cancer in 7 patients, renal carcinoma in 6, breast cancer in 5, prostate cancer in 2, endometrial carcinoma in 1, thyroid cancer in 1, colon cancer in 1, rectal cancer in 1, liver cancer in 1, glioma in 1, and gastric cancer in 1 patient. metastatic bone cancer was detected 8~123 months (mean : 42.2033.61 months) after diagnosis of primary cancers in 20 patients, but metastatic bone cancer as the first sign facilitating the diagnosis of primary cancers was detected in 7 patients. the cancer metastasis sites included the proximal end of the femur in 11 patients, proximal end of the humerus in 6, middle part of the humerus in 3, scapula in 2, middle part of the femur in 2, distal end of the femur in 1, middle part of the ulna in 1, and proximal end of the tibia in 1 patient. the complaints included simple pain in 10 patients, pain and bump in 6, and pathological fracture in 7 patients. four patients had no complaints and metastatic bone cancer was identified by routine ect or pet. limb - salvage surgery was performed in all patients according to the principles for the treatment of primary bone cancer. artificial prosthetic replacement was performed in 21 patients, simple resection in 2, reconstruction with allogeneic bones in 2, inactivated bone replantation in 1, and removal of hemi - cortex of the bone, filling of bone cerement, and internal fixation in 1 patient. in 7 patients, primary cancer and metastatic bone cancer were identified simultaneously and then a one - stage operation was carried out for resection of primary cancer and metastatic cancer simultaneously. according to the enneking s staging system, wide resection was done in 19 patients and marginal resection in 8 patients. in 21 patients, post - operative systemic chemotherapy and/or local radiotherapy were performed, of whom 7 patients receiving marginal resection underwent local radiotherapy post - operatively. qol and pain were evaluated before and after surgery in 21 patients. pain was assessed with the 10-point scale before and 1 month after surgery. the qol was assessed with the sf-36 scale before and 3 months after surgery. post - operative limb function was evaluated with the american musculoskeletal tumor society (msts) system 3 months after surgery. quantitative data with normal distribution were expressed as mean standard deviation to describe the central tendency or discrete tendency. the scores of pain, qol, and limb function were compared with the t test. tumor - free survival was defined as the time from the end of surgery to the presence of new lesions. from 2007 to 2010, a total of 27 patients with solitary metastatic bone cancer were treated in our department, including 15 males and 12 females, with a mean age of 588.58 years (range : 4375 years). follow - up was carried out for 16.157.47 months (range : 631) (t=1.073, p=0.294). the primary cancers included lung cancer in 7 patients, renal carcinoma in 6, breast cancer in 5, prostate cancer in 2, endometrial carcinoma in 1, thyroid cancer in 1, colon cancer in 1, rectal cancer in 1, liver cancer in 1, glioma in 1, and gastric cancer in 1 patient. metastatic bone cancer was detected 8~123 months (mean : 42.2033.61 months) after diagnosis of primary cancers in 20 patients, but metastatic bone cancer as the first sign facilitating the diagnosis of primary cancers was detected in 7 patients. the cancer metastasis sites included the proximal end of the femur in 11 patients, proximal end of the humerus in 6, middle part of the humerus in 3, scapula in 2, middle part of the femur in 2, distal end of the femur in 1, middle part of the ulna in 1, and proximal end of the tibia in 1 patient. the complaints included simple pain in 10 patients, pain and bump in 6, and pathological fracture in 7 patients. four patients had no complaints and metastatic bone cancer was identified by routine ect or pet. limb - salvage surgery was performed in all patients according to the principles for the treatment of primary bone cancer. artificial prosthetic replacement was performed in 21 patients, simple resection in 2, reconstruction with allogeneic bones in 2, inactivated bone replantation in 1, and removal of hemi - cortex of the bone, filling of bone cerement, and internal fixation in 1 patient. in 7 patients, primary cancer and metastatic bone cancer were identified simultaneously and then a one - stage operation was carried out for resection of primary cancer and metastatic cancer simultaneously. according to the enneking s staging system, wide resection was done in 19 patients and marginal resection in 8 patients. in 21 patients, post - operative systemic chemotherapy and/or local radiotherapy were performed, of whom 7 patients receiving marginal resection underwent local radiotherapy post - operatively. the qol was assessed with the sf-36 scale before and 3 months after surgery. post - operative limb function was evaluated with the american musculoskeletal tumor society (msts) system 3 months after surgery. quantitative data with normal distribution were expressed as mean standard deviation to describe the central tendency or discrete tendency. the scores of pain, qol, and limb function were compared with the t test. tumor - free survival was defined as the time from the end of surgery to the presence of new lesions. all procedures were performed successfully, and no patient died during the follow - up period. post - operative prosthesis dislocation occurred in 1 patient and closed reduction was successfully carried out. deep venous thrombosis was found in 3 patients of whom inferior vena cava filter was placed in 2 and systemic anti - coagulation therapy done in 1 patient. pulmonary infection was controlled following anti - infection therapy in 1 patient. as shown in table 2, the score of pain was 6.853.11 and 1.260.81 before and 1 month after surgery, respectively, indicating that the post - operative pain was markedly improved (t=9.978, p<0.001). the qol score was 38.3013.05 and 65.7810.65 before and 3 months after surgery, respectively, revealing the quality of life was dramatically improved following surgery (t=18.550, p<0.001). the post - operative score of limb function ranged from 17 to 30 (mean : 233) (t=1.450, p=0.016). activities of daily living recovered in all patients. for patients with metastatic bone cancer in lower limbs, they could walk with or without a walker. regarding cancer control, as shown in figure 1, log rank testing revealed there was no significant difference in the tumor - free survival between patients receiving wide resection and marginal resection (=0.121, p=0.728). he was admitted due to pain and presence of pain and bump at the right hip for 2 months. on examination, a mass was found in the right lung and subsequent pathological examination revealed lung cancer. radical resection for lung cancer and marginal resection of the bone cancer were performed (figure 2). case 2 : male, 56 years old, prostate cancer concomitant with pathological fracture at proximal end of bilateral femurs. en bloc resection of the bone cancer and bilateral prosthetic replacement were performed (figure 3). pathological fracture at the left humerus was found and subsequent pathological examination confirmed renal carcinoma. left radical nephrectomy and en bloc resection of cancer at the left humerus were performed, followed by artificial prosthetic replacement (figure 4). case 17 : male, 62 year old, swelling and pain at left forearm 2 years after resection of rectal cancer. implantation of inactivated bone and internal fixation with plates were performed after marginal resection of the bone cancer (figure 5). all procedures were performed successfully, and no patient died during the follow - up period. post - operative prosthesis dislocation occurred in 1 patient and closed reduction was successfully carried out. deep venous thrombosis was found in 3 patients of whom inferior vena cava filter was placed in 2 and systemic anti - coagulation therapy done in 1 patient. as shown in table 2, the score of pain was 6.853.11 and 1.260.81 before and 1 month after surgery, respectively, indicating that the post - operative pain was markedly improved (t=9.978, p<0.001). the qol score was 38.3013.05 and 65.7810.65 before and 3 months after surgery, respectively, revealing the quality of life was dramatically improved following surgery (t=18.550, p<0.001). the post - operative score of limb function ranged from 17 to 30 (mean : 233) (t=1.450, p=0.016). activities of daily living recovered in all patients. for patients with metastatic bone cancer in lower limbs regarding cancer control, no local recurrence occurred in these 27 patients during the follow - up period. as shown in figure 1, log rank testing revealed there was no significant difference in the tumor - free survival between patients receiving wide resection and marginal resection (=0.121, p=0.728). he was admitted due to pain and presence of pain and bump at the right hip for 2 months. on examination, a mass was found in the right lung and subsequent pathological examination revealed lung cancer. radical resection for lung cancer and marginal resection of the bone cancer were performed (figure 2). case 2 : male, 56 years old, prostate cancer concomitant with pathological fracture at proximal end of bilateral femurs. en bloc resection of the bone cancer and bilateral prosthetic replacement were performed (figure 3). pathological fracture at the left humerus was found and subsequent pathological examination confirmed renal carcinoma. left radical nephrectomy and en bloc resection of cancer at the left humerus were performed, followed by artificial prosthetic replacement (figure 4). case 17 : male, 62 year old, swelling and pain at left forearm 2 years after resection of rectal cancer. implantation of inactivated bone and internal fixation with plates were performed after marginal resection of the bone cancer (figure 5). the bone is the third most common site of cancer metastasis, following lung and liver. in addition to the limbs, the spine is also a common site of cancer metastasis. in the present study, we primarily focus on the surgical treatment of metastatic bone cancer of the limbs. comprehensive therapy is preferred for the metastatic bone cancer, including surgical intervention, chemotherapy, radiotherapy and other conservative therapies. metastatic bone cancer is a systemic disease, thus surgical therapy is usually performed on the basis of other adjunctive therapies. the majority of metastatic bone cancers are predominantly derived from lung cancer and breast cancer, followed by renal carcinoma, thyroid cancer and prostate cancer [1820 ]. metastatic bone cancer from lung cancer / renal carcinoma is usually characterized by osteolytic lesions, thus patients with this disease are susceptible to pathological fracture, and surgical intervention is required. metastatic bone cancer from breast cancer / prostate cancer is usually osteogenic, especially in patients with prostate cancer. when compared to patients with osteolytic lesions, those with osteogenic lesions seldom develop pathological fracture ; conservative therapy is often performed and surgical intervention less often required. in the present study, 1 patient with prostate cancer developed metastatic bone cancer and subsequent pathological fracture at the proximal end of bilateral femurs. thus, for metastatic bone cancer patients with osteogenic lesions, the risk for pathological fracture should also be paid attention to, especially in those with intertrochanteric lesions. patients with a mirels score of 9 are regarded to have high risk for fracture, and active prophylactic surgery is required. for patients developing pathological fracture, determination of surgical intervention is done based on the general conditions : tolerance to surgery, time from identification of primary disease to metastatic bone cancer, disease progression, expected survival time, extent of post - operative qol improvement, and post - operative limb function. generally, surgical intervention should be considered for patients with favorable general condition, able to tolerate surgery, having no progression of primary disease, with expected survival time of longer than 6 months, and having poor qol due to pathological fracture. once surgical intervention is determined for the treatment of pathological fracture, the requirements are similar to those in the treatment of primary bone cancer. the reconstruction should be beneficial for the rapid recovery of limb function or partial load - bearing. usually, artificial prosthetic replacement is performed in patients with lesions near the joint ; implantation of allogeneic bone or inactivated cancer - bearing bone, followed by internal fixation with plates, is recommended for patients with lesions at the bone shaft or non - weight - bearing site. in the present study, lesions were mainly found at the site near the joint, and thus artificial prosthetic replacement was the major strategy for the treatment ; the post - operative limb function was markedly improved. for pathological fracture, surgical treatment aims to relieve the pain and improve the qol and limb function. in the present study results demonstrate that post - operative pain, qol, and limb function were dramatically improved. however, for patients with multiple bone metastases, surgical intervention is unable to achieve wide resection or resection of foci, and is usually highly invasive. under this condition, patients usually have high surgical risk. wide resection was performed in the majority of patients and a minority of patients received marginal resection. post - operative local radiotherapy was also carried out as an adjunctive therapy, achieving favorable control of local tumors. in addition, statistical analysis showed there was no significant difference in the tumor - free survival time between patients receiving wide vs. marginal resection. this demonstrates that only systemic treatment is an effective strategy to improve the survival of cancer patients. nevertheless, the wide or marginal resection of the cancer plays an important role in the control of local cancer and pain, and improvement of qol and limb function. in our study, metastatic bone cancer was the first sign in 7 patients and further examinations identified primary diseases. of these patients, 6 received 1-stage resection of primary cancer and metastatic bone cancer simultaneously. the remaining patient had prostate cancer and pathological fracture at the proximal end of the bilateral femurs. bilateral lesions were resectable and treatment was similar to that in patients with solitary metastatic bone cancer. one - stage resection of lesions at bilateral femurs was performed, followed by joint replacement, and endocrine therapy was done as a treatment for primary prostate cancer. for patients with resectable primary and metastatic lesions, post - operative chemotherapy and/or radiotherapy is recommended, which may significantly delay disease progression in the tumor - free survival time, elevate survival rate and improve qol. this should be done in the presence of completely resectable primary and metastatic lesions. for these patients, resection of a single lesion may pronouncedly affect the effectiveness of adjunctive therapy and survival of cancer - bearing patients. for patients with solitary metastatic bone cancer of the limbs, limb salvage surgery with wide or marginal resection is beneficial for the improvement of post - operative pain, qol and limb function. in addition, the control of local cancer is also favorable and the local recurrence rate not influenced. for patients with metastatic bone cancer with concomitant primary cancer, 1-stage resection of both lesions is recommended, which may delay disease progression and improve the survival rate. in our study, the time of follow - up was relatively short, and the effect of limb salvage surgery on the overall survival needs to be further investigated in studies with long - term follow - up. | summarybackgroundto evaluate the pain, quality of life (qol), and limb function of patients after en bloc resection of solitary metastatic bone cancer in the limbs.material/methodsa total of 27 patients with solitary metastatic bone cancer in the limbs were recruited. all these patients underwent limb - salvage surgery with en bloc resection of the metastatic tumor. pain and qol were evaluated before and after surgery. pain was assessed with a 10-point scale before and 1 month after surgery. the qol was evaluated with the sf-30 scale before and 3 months after surgery. limb function was evaluated with the musculoskeletal tumor society scale (msts) 3 months after surgery. follow - up was performed for 6~31 months (mean : 16.157.47 months).resultsall procedures were successfully performed. post - operative complications were found in 6 patients, including incision infection, prosthesis dislocation, deep vein thrombosis, and pulmonary infection. the pain score before and 1 month after surgery was 6.853.11 and 1.260.81, respectively, indicating obvious improvement (t=9.978, p<0.001). the qol score before and 3 months after surgery was 38.3013.05 and 65.7810.65, respectively, indicating pronounced improvement (t=18.550, p<0.001). the mean post - operative msts score was 233 (range : 1730) (t=1.450, p=0.016). no local recurrence was observed in any patient during the follow-up.conclusionslimb salvage surgery with wide or marginal resection for solitary metastatic bone cancer may significantly improve the pain, qol, and limb function, but there is no difference in local control between wide and marginal resection. |
they can be defined as aneurysmal dilatation of intra- and extra - hepatic ducts and are classified according to the involved segment of the biliary tree. the incidence of choledochal cysts is increasing due to the improvements in imaging techniques and choledochal cysts of adults get clinical attention due to various complications such as cholangitis, bile stasis, and malignancies of the biliary tree. at times, choledochal cysts may grow very large and differentiation from other cystic masses of the abdomen can be challenging. the correct diagnosis of choledochal cysts in pre - operative patients is essential since reconstruction of the biliary trees is mandatory in these patients. here, we discuss the imaging features and correlate them to gross specimen in a young female with type 1a giant choledochal cyst, which manifested as a huge cystic mass in the abdomen of unclear etiology. a previously healthy, 19-year - old female patient presented to the emergency room with intermittent abdominal pain and jaundice. ultrasound (us) images showed a large septated cystic and tubular structure in the abdomen [figure 1a ]. computed tomography (ct) scan was performed and it revealed a large, convoluted tubular and cystic structure in the right upper quadrant with associated dilated intrahepatic ducts [figures 1b d ]. the long diameter of the cystic structure was over 23 cm with associated dilatation of intrahepatic ducts. no solid, enhancing components were noted in the wall of this cystic mass. because of the compressive effect by this mass, the pancreatic head was not clearly seen and relationship between this mass and pancreatic duct was unclear. 19-year - old female with intermittent abdominal pain and jaundice diagnosed with type 1a giant choledochal cyst. (a) ultrasound scan axial view shows a large cystic mass () and dilated bile ducts (b). the mass was very large and the relationship of the biliary tree to the mass was not clear on ultrasound. (b) computed tomography (ct) scan, axial view of portal venous phase demonstrates dilated intrahepatic ducts (b). (c) computed tomography scan axial view at a lower level than 1b shows a large cystic mass () at the porta hepatis. (d) coronal reformatted computed tomography image delineates a huge cystic mass in right upper quadrant (). magnetic resonance cholangiopancreaticography (mrcp) demonstrated a huge septated cystic mass appearing to communicate with the intrahepatic ducts [figure 2a ]. differential diagnoses include gallbladder hydrops, duplication cyst, mesenteric or peritoneal cyst, and a huge choledochal cyst. in view of biliary dilatation 19-year - old female with intermittent abdominal pain and jaundice diagnosed with type 1a giant choledochal cyst. a) axial and b) coronal heavily t2-weighted images show a large cystic mass (), gallbladder (g) and intrahepatic ducts (b). roughly 2.5 l of bilious fluid was aspirated with subsequent decompression of the biliary tree. explorative laparotomy was scheduled and performed and severe dilatation of common bile duct and relatively small - sized gallbladder was found on surgery. intraoperative finding was a massive, thick walled cystic lesion measuring over 20 cm in maximum dimension arising from the common bile duct. the cystic lesion involved entire extra - hepatic ducts and there was no stricture or soft - tissue masses. the gross specimen and histopathological examination confirmed the diagnosis of a giant choledochal cyst, type ia according to todani classification [figure 3 ]. a schematic diagram shows the location of the cystic mass and its relationship to surrounding structures [figure 4 ]. 19-year - old female with intermittent abdominal pain and jaundice diagnosed with type 1a giant choledochal cyst. a) the gross specimen obtained after excision of the cystic mass shows a massive, thick walled cystic lesion (arrows) measuring over 20 cm in maximum dimension arising from the left hepatic duct and confirms the diagnosis of a giant choledochal cyst. (b) h and e stained tissue (400), shows the dilated bile duct with columnar epithelial lining, and dense fibrous wall ; scattered smooth muscle and elastic fibers are present (arrows) ; mild infiltration of chronic inflammatory cells are also seen (arrowheads). there is no evidence of dysplasia or malignancy schematic diagram shows the location of the choledochal cyst and resection margin (dotted lines). choledochal cysts are very rare lesions seen in people in western countries. in asian population the incidence is increasing due to the use of the state - of - the - art imaging techniques such as multidetector ct (mdct) or mrcp. patients with choledochal cysts can have several complications and are considered as a high risk group for cholangiocarcinomas and surgical excision of the entire cyst is recommended. us is the primary imaging tool for evaluation, however, this modality has its limitations. evaluating the entire biliary tree is difficult due to limited field of view and poor overview when compared with cross - sectional imaging modalities. therefore, for accurate diagnosis, cross - sectional imaging modalities with a larger field of view are superior to us. mdct can obtain volumetric data sets and multiplanar reformations (mpr) are readily available. furthermore, mrcp is the preferred imaging modality for the correct diagnosis of biliary anomalies. mrcp is non - invasive and can be obtained without the use of contrast agents. 2d or 3d mrcp images clearly delineate biliary anomalies. in adults patients, differential diagnosis for cystic masses at porta hepatis other than choledochal cysts include hepatic cysts, biliary cystadenomas, duplication cysts of the duodenum and colon, mesenchymal and peritoneal cysts and pseudocysts or cystic tumors from the pancreas.. however, huge masses can compress adjacent structures and therefore intrahepatic ducts can be dilated simply due to mass effect. in those cases, mrcp can also be helpful to make a correct diagnosis because mrcp can delineate the relationship between cystic masses and adjacent structures. the most common type of choledochal cyst is type i and accounts for more than 75% of all choledochal cysts. type i cysts involve the extrahepatic bile ducts and can be divided into three subtypes ; ia (cystic), ib (segmental) and ic (fusiform). type i cyst should be differentiated from type iva cysts which involve both intra- and extra - hepatic ducts., there was no stricture between intra- and extra - hepatic ducts and intrahepatic duct dilatation was continuous to the dilated extra - hepatic duct. furthermore, after percutaneous transhepatic biliary drainage (ptbd) insertion, intrahepatic ducts are seen collapsed and the diagnosis of type ia choledochal cyst can be made. one of the strongly suggested etiologies is reflux of pancreatic juice into the common bile duct and anomalous pancreaticobiliary ductal union (apbdu) can facilitate this phenomenon. actually, strong association of presence of apbdu and choledochal cysts was reported by many authors. however, a case report of choledochal cyst in a 15-week - old fetus may refute this theory ; i.e., 15-week - old fetus does not have pancreas tissue with exocrine function. however, high incidence of choledochal cysts in patients with apbdu can not be ignored. though endoscopic retrograde cholangiopancreaticography was considered as the gold standard for diagnosing abnormalities of the biliary tree and pancreatic ducts, mrcp is also excellent, non - invasive imaging modality for delineating and diagnosing congenital anomalies of pancreatic ducts and biliary trees including apbdu. however, in our case, apbdu was not clearly demonstrated on mrcp because the choledochal cyst was too large and pancreas head was markedly compressed. direct cholangiography via ptbd was not helpful also because the cystic mass was too large and filling of the pancreatic duct with contrast material was nearly impossible. therefore, mdct might be the primary imaging modality for huge cystic mass in the abdomen and mrcp can be a problem - solving tool. as mentioned above, adult patients with choledochal cysts have a higher risk of cholangiocarcinomas and other complications are commonly encountered in these patients. therefore, entire excision of choledochal cysts is recommended treatment option in most cases. in our case, mrcp or mdct also can be helpful for the planning of surgical excision and essential to pre - operative evaluation. we report a giant, type ia choledochal cyst presenting with a massive septated cystic mass in the abdomen in a young adult patient. state of the art cross - sectional imaging with mdct or mrcp can help to reach the correct diagnosis and formulate a robust surgical plan. | choledochal cysts are uncommon congenital anomalies of the biliary tree, commonly presenting in infancy, generally in the 1st year of life. presentation in adult life is less common, accounting for 20% of cases. a 19-year - old female patient presented to the emergency department with severe abdominal distension, a palpable abdominal mass, mild jaundice and low grade fever. ultrasound, computed tomography (ct) and magnetic resonance imaging of the abdomen showed a massive septated cystic lesion filling the entire abdomen with a significant mass effect on surrounding structures. origin of the lesion was unclear and diagnosis included a giant mesenteric or duplication cyst, massive gallbladder with hydrops, biliary cystadenoma and giant choledochal cyst, among others. final diagnosis was a type ia choledochal cyst with massive asymmetric cystic dilatation of the extra - hepatic segments of the left hepatic duct with asymmetric dilatation of the right hepatic duct. patient had an uneventful recovery after resection of the entire extrahepatic cyst and roux - en - y hepaticojejunostomy at the level of the hilum. in this article, we correlate ct and mri findings to gross and histopathological findings of this giant todani 's type ia choledochal cyst. |
lumbar spinal stenosis (lss) presents with intermittent claudication (ic) in the lower extremities as a typical symptom26,28). peripheral arterial disease (pad) also presents with similar symptoms as vascular ic17,21). neurogenic ic, on the other hand, caused by narrowing of the neural foramen and spinal canal, is aggravated by standing and relieved by sitting. however, it can be difficult to differentiate vascular and neurogenic ic due to atypical signs and variable symptoms. therefore, when patients report claudication, it is important to correctly identify the accompanying pathology. pad includes many pathologies, such as arteriosclerosis obliterans, buerger 's disease, and acute arterial obstruction. untreated pad is progressive and increases the risk of severe vascular events and even death8,13,24). therefore, early diagnosis and treatment of pad is important to reduce fatal or nonfatal vascular events17,21). in order to diagnose lss accompanied by pad, however, the argument arises as to whether pad can be ruled out only by arterial pulse examination. to establish a screening test for pad, ankle - brachial index (abi), abi has been found to be highly sensitive and specific for pad screening10,18,20,23,24). those reports presented the incidence of and risk factors for pad in patients with lss with atypical symptoms. if a patient has atypical symptoms, and diagnostic imaging shows no significant spinal canal stenosis, spine surgeons are always concerned about accompanying pad. however, spine surgeons tend to overlook the possibility of accompanying pad in patients with lss diagnosed by typical symptoms and diagnostic imaging, such as magnetic resonance imaging (mri). in order to prevent misdiagnosis of vascular disease, it is important to know the incidence of and risk factors for pad in patients with lss. therefore, the aim of our study was to evaluate the incidence of and risk factors for pad in patients with typical and severe lss who underwent spinal surgical treatment. the occurrence of pad was examined retrospectively in 171 consecutive patients with lss and severe ic who underwent surgical treatment at our hospital from june 2012 to june 2013. three diseases were included in this study : spinal stenosis (st), degenerative spondylolisthesis (ds) and isthmic spondylolisthesis (is). abi is the ratio of arm systolic blood pressure at the brachial artery to ankle systolic blood pressure at the posterior tibial or dorsalis pedis artery6,11,16,31). abi < 0.9 was considered abnormal according to transatlantic inter - society consensus (tasc) guidelines21) (fig. all patients with abnormal abi underwent computed tomography angiography (cta) and consultation with a vascular surgeon (fig. data were collected on background characteristics (sex, age) and known risk factors for pad, such as hypertension, diabetes mellitus, smoking, hyperlipidemia, stroke, and ischemic heart disease. quality of life (qol) was evaluated using a visual analog scale (vas) and the oswestry disability index (odi)2,3,7,22). patients with coexisting pad and lss were designated the lsspad group, while those with lss but no pad were denoted the lss group. using the lss group as a control, analyses were conducted to identify the characteristics of the lsspad group. to evaluate clinical characteristics at the time of enrollment, univariate analysis was performed using the student 's t test (v2 test). the mann - whitney u - test was used to compare group means of continuous data. any variable with a p value of < 0.05 on univariate analysis was included in multiple logistic regression models. the study population comprised 79 men and 92 women with a mean age of 64.68.6 years. cta was performed in these patients, and a final diagnosis of pad was established for all 7 patients. six of the 7 patients diagnosed with pad were treated with an endovascular stent, while the remaining 1 patient was recommended observation and regular follow - up. the results of univariate analysis of risk factors for pad, type of disease, and other clinical parameters are shown in table 1. the lsspad group was significantly older than the lss group (72.35.5 years vs 64.28.6 years, p<0.01). stroke and ischemic heart disease were significantly more common in the lsspad group compared with the lss group. the other known risk factors for pad(diabetes mellitus, hypertension, smoking history, hyperlipidemia) showed no significant differences between groups. of the 171 patients with lss, 37 (21.6%) smoked. among the 164 patients in the lss group, 100 (61%) had st, 53 (32.3%) had ds, and 11 (6.7%) had is. among the 7 patients in the lsspad group, 6 (85.7%) had st and 1 (14.3%) had ds. in multivariate analysis, the entered variables were age, diabetes mellitus, preoperative vas score, stroke and ischemic heart disease (p<0.2) (table 2). multiple logistic regression analyses with a forced - entry method revealed that the stroke (p<0.05) was independent risk factor for pad. pad is an atherosclerotic syndrome with a high prevalence of approximately 5.8% to 12% in older adults1,15). high incidence of fatal or nonfatal cardiovascular events associated with pad increases mortality. according to tasc guidelines for pad, the mortality rate of patients with ic associated with pad is 2 times that of patients with ic only2,7,22). besides the risk of death, the 10-year incidence of myocardial infarction and cerebral stroke in patients with pad are increased by about 4 and 3 times, respectively, compared with that in patients without pad5,29). however, pad is often misdiagnosed in older patients with lss because it is difficult to distinguish vascular ic from neurogenic ic. therefore, spine surgeons often overlook the possibility of pad in patients with severe lss18). in a previous study, the coincidence of pad with lss was similar to the prevalence of pad in older individuals (6.7%)27). therefore, it is very important to understand the risk factors and to evaluate pad in patients with lss. arteriography is considered the gold standard for diagnosis of pad. however, the use of arteriography has some limitations, including complications arising from ionizing radiation, its invasive nature, and nephrotoxic contrast media. among substitutable tests, abi is the simplest and most inexpensive25). abi also has high sensitivity (79 - 95%) and specificity (96 - 100%) for screening pad17). thus, we evaluated the coincidence of pad with lss and risk factors for pad using abi. there have been several studies to evaluate the incidence of pad in patients with lss using abi. their study showed that abi had high sensitivity and specificity for screening pad in cases with atypical claudication. twenty - two of 42 patients in their study were diagnosed with lss and pad, showing a high coincidence (54.8%) between diseases. however, their study did not analyze the risk factors for pad and was limited in that only mild cases of lss, determined by mri, were included. most spine surgeons easily can doubt a hidden disease, such as pad, in these cases. therefore, in our study, we enrolled patients with typical symptoms, severe lss, confirmed by mri, who underwent decompressive surgery. they also showed that comorbidity of diabetes mellitus, history of stroke, and history of ischemic heart disease were characteristics of patients with lss and pad. this was the first nationwide multicenter survey of the prevalence of pad in patients with lss in japan. however, the mean vas score of patients with lss and pad was 53.627.0, while that of patients with lss was 60.128.4 (vas 0 - 100 mm), indicating that patients with mild ic were included in this study as well as patients with severe ic who required surgical treatment. spine surgeons occasionally make a mistake in performing surgery in patients who have radiographically asymptomatic stenosis and vascular ic arising from pad, since it is difficult to distinguish between neurogenic and vascular ic. therefore, diagnosis of pad is not important for patients with mild ic, but is important for patients with typical neurogenic ic and definite lss who require surgical treatment. we analyzed the incidence of and risk factors for pad in patients with severe lss and intractable ic who underwent surgical treatment. the mean preoperative vas score of all patients was 7.21.9, and there was no response to conservative treatment, such as medication, physical therapy, and epidural nerve block. although the coincidence of pad with lss was not high (7 of 171 patients [4.1% ]), 6 of 7 patients had severe vascular insufficiency requiring endovascular stent surgery. thus, it can be very dangerous if pad is misdiagnosed in patients with lss. previous studies have presented several risk factors for pad, such as old age, sex, diabetes mellitus, dyslipidemia, hypertension, smoking history, and body mass index15,27,30). in this study, old age and history of stroke was the only significant predisposing factor for pad. according to this result, we propose abi examination in old patients with lss and history of stroke to rule out pad. if pad is diagnosed, spine surgeons may be able to decide whether to perform decompressive surgery for lss. in this decision making process, although imaging studies are often not specific for lss, electrodiagnostic testing is highly specific for diagnosis of lss4,12). the strength of this study is the severe ic of the patient population, which was analyzed by multivariate logistic regression to identify the incidence of and risk factors for pad. diagnosis of pad by abi enables spine surgeons to avoid fatal vascular events and unnecessary surgery in patients with radiographically asymptomatic lss. however, this study is limited by its retrospective nature, relatively small patient population, and short - term duration of follow - up. to further verify the incidence of and risk factors for pad in addition, this study did not reveal the sensitivity and specificity of abi because cta was not performed in all patients. misdiagnosis of pad may increases the risk of severe vascular events and even death. to prevent misdiagnosis of fatal pad and to avoid unnecessary surgery, we propose further evaluation | objectiveintermittent claudication (ic) is a typical symptom of peripheral arterial disease (pad) and lumbar spinal stenosis (lss). in order to prevent misdiagnosis of vascular disease, it is important to know the incidence of and risk factors for pad in patients with lss. therefore, the aim of our study was to evaluate the incidence of and risk factors for pad in patients with typical and severe lss who underwent spinal surgical treatment.methodsthe occurrence of pad was examined retrospectively in 171 consecutive patients with lss and severe ic who underwent surgical treatment at our hospital from june 2012 to june 2013. data were collected on background characteristics (sex, age) and known risk factors for pad, such as hypertension, diabetes mellitus, smoking, hyperlipidemia, stroke, and ischemic heart disease.resultsof the 171 patients enrolled, 7 had an abnormal ankle - brachial index (abi). computed tomography angiography (cta) was performed in these patients, and a final diagnosis of pad was established for all 7 patients. the incidence of pad in all patients with lss was 4.1%(7 of 171). stroke and ischemic heart disease were significantly more common in the lsspad group compared with the lss group. multiple logistic regression analyses with a forced - entry method revealed that age and stroke (p<0.05) were independent risk factors for pad.conclusionto prevent misdiagnosis of fatal pad, we recommend abi be assessed in patients with lss and history of stroke. |
hole argument is the english translation of the german phrase lochbetrachtung, used by albert einstein to describe his argument against the possibility of generally - covariant equations for the gravitational field, developed in late 1913 and accepted until late 1915. einstein realized the desirability of general covariance, and showed that it was easily implemented for the rest of physics ; but the hole argument purported to show why it could not be demanded of the gravitational field equations he was trying to formulate for the metric tensor.1 this article is a historical - critical study, in ernst mach s sense.2 it includes a review of the literature on the hole argument that concentrates on the interface between historical, philosophical and physical approaches. although recounting the history of the hole argument, the primary purpose is to discuss its contemporary significance in both physics and philosophy for the study of space - time structures. like mach, while presenting various other viewpoints, i have not hesitated to advocate my own. in physics, i believe the main lesson of the hole argument is that any future fundamental theory, such as some version of quantum gravity, should be background independent, with basic elements obeying the principle of maximal permutability. in the philosophy of space - time, this leads me to advocate a third way that i call dynamic structural realism, which differs from both the traditional absolutist and relationalist positions. one of the most crucial developments in theoretical physics was the move from theories dependent on fixed, non - dynamical background space - time structures to background - independent theories, in which the space - time structures themselves are dynamical entities. this move began in 1915 when einstein stated the case against his earlier hole argument. even today, many physicists and philosophers do not fully understand the significance of this development, accept it in practice. so it is of more than historical interest for physicists and philosophers of science to understand what initially motivated this move, as well as the later developments stemming from it. einstein s starting point was the search for a generalization of the special theory that would include gravitation. he quickly realized that the equivalence principle compelled the abandonment of the privileged role of inertial (i.e., non - accelerated) frames of reference, and started to investigate the widest class of accelerated frames that would be physically acceptable. his first impulse was to allow all possible frames of reference ; since he identified frames of reference and coordinate systems, this choice corresponds mathematically to a generally - covariant theory. but he soon developed an argument the hole argument purporting to show that generally - covariant equations for the metric tensor are incompatible with his concept of causality for the gravitational field. the argument hinged on his tacit assumption that the points of space - time are inherently individuated, quite apart from the nature of the metric tensor field at these points. only two years later, after other reasons compelled him to reconsider general covariance, did einstein finally recognize the way out of his dilemma : one must assume that, in an empty region of space - time, the points have no inherent individuating properties nor indeed are there any spatio - temporal relations between them that do not depend on the presence of some metric tensor field. thus, general relativity became the first fully dynamical, background - independent space - time theory. without some knowledge of this historical background, it is difficult to fully appreciate either the modern significance of the hole argument, or the compelling physical motives for the requirement of background independence. einstein s starting point in his search for a theory of gravitation was the theory we now call special relativity. from a contemporary viewpoint, its most important feature is that it has two fixed, kinematical space - time structures the chrono - geometry embodied in the minkowski metric tensor field and the inertial field embodied in the associated flat affine connection both of which are invariant under the ten - parameter lie group now called the poincare or inhomogenous lorentz group.3 in minkowski space - time, all dynamical theories must be based on geometric objects that form a representation (or more generally, a realization) of this group. there is a preferred class of spatial frames of reference in minkowski space - time, the inertial frames. einstein had shown how to define a class of physically preferred coordinate systems for each inertial frame of reference ; in particular, he defined a clock synchronization procedure that provides a preferred global time for each frame. this enabled him to show how the principle of relativity of all inertial frames could be reconciled with the universal properties of light propagation in vacuum. the lesson he drew was the need to find a physical interpretation of the coordinates associated with an inertial frame of reference a lesson that had to be painfully unlearned in his search for a generalized theory of relativity. in large part, the history of the hole argument is the story of that unlearning process. the end result was the formulation of the general theory of relativity, the first background - independent physical theory turning all space - time structures into dynamical fields. this was such a revolutionary break with all previous physical theories, in which space - time structures constitute a fixed, non - dynamical background, that its ultimate significance is still debated by physicists.4 understanding the hole argument in both its historical and contemporary aspects can help to clarify the issues at stake in this debate. the basic issue can be stated as follows : given a physical theory, when should an equivalence class of mathematically distinct models of the theory be identified as corresponding to single, unique physical model ? the hole argument shows that, for any theory defined by a set of generally - covariant field equations, the only way to make physical sense of the theory is to assume that the entire equivalence class of diffeomorphically - related solutions to the field equations represent a single physical solution. as will be seen later, mathematically this result can best be stated in the language of natural bundles. but a similar result holds for the even broader class of all gauge - invariant field theories, notably yang - mills theories : an equivalence class of gauge - related models of any such theory must be physically identified. mathematically, broadening the question in this way requires the language of gauge - natural bundles. general relativity itself may also be treated by the use of gauge - natural bundle techniques : its similarities to and differences from gauge theories of the yang - mills type will also be discussed. this move to natural and gauge - natural formulations of field theories also has important implications for the philosophy of space and time. the old conflict between absolute and relational interpretations of space and then space - time has been renewed on this new ground. but i shall argue that this reformulation of the question suggests a third position, around which a consensus is forming. this position has been given various names, but i prefer dynamic structural realism. sections 2.12.5 recount the developments leading up to einstein s adoption of the hole argument against general covariance in 1913, how it misled him for over two years, the reasons for his rejection of it in late 1915, and its replacement by the point - coincidence argument for general covariance. section 2.6 discusses kretschmann s 1917 critique of the concept of general covariance and einstein s 1918 reply ; decades later this debate led komar to propose the use of what are now called kretschmann - komar coordinates as a way of resolving the hole argument. finally, section 2.7 discusses hilbert s 1917 reformulation of the hole argument : he replaced the four - dimensional hole in space - time with a space - like hypersurface, on which he posed an initial value problem for the field equations ; this was the first step in a series of developments culminating a decade later in a fully satisfactory formulation by darmois of the general - relativistic cauchy problem. section 3 discusses the revival of interest in the hole argument in the 1970s, which grew out of an attempt to answer a historical question : why did three years (19121915) elapse between einstein s adoption of the metric tensor to represent the gravitational field and his adoption of what are now called the einstein equations for this field ? some highlights of this discussion are recalled, from the post - world war ii revival of interest in general relativity up to the present. section 4 presents a modern version of the hole argument in general relativity, and its generalization from metric theories of gravitation to gauge - natural field theories. by abstraction from continuity and differentiability, the concept of general covariance of a field theory is similarly extended to general permutability, a concept wide enough to include theories based on relations between the elements of any set. sections 5 and 6 focus on current discussions of philosophical and physical implications of the hole argument, respectively ; no attempt is made to rigidly separate issues that overlap both areas. section 5 discusses such issues as : the range of applicability of the hole argument, the correct mathematical definition of general covariance and its physical significance, the controversy between relationalists and substantivalists in discussions of space - time structures. the arguments of ear - man, pooley and stachel are reviewed, and their convergence on a third alternative, which i call dynamic structural realism, is stressed. section 6 discusses such issues as partially background - independent theories, including mini- and midi - solutions to the einstein field equations ; the reformulation of general relativity as a gauge natural theory ; and some implications of the hole argument for attempts to formulate a quantum theory of gravity. einstein attributed his success in formulating the special theory in 1905 in no small measure to his insistence on defining coordinate systems that allowed him to attach physical significance to spatial and temporal coordinate intervals : the theory to be developed like every other electrodynamics is based on the kinematics of rigid bodies, since the propositions of any such theory concern relations between rigid bodies (coordinate systems), clocks and electromagnetic processes. not taking this into account insufficiently is the root of the difficulties, with which the electrodynamics of moving bodies currently has to contend (einstein, 1905, my translation). the theory to be developed like every other electrodynamics is based on the kinematics of rigid bodies, since the propositions of any such theory concern relations between rigid bodies (coordinate systems), clocks and electromagnetic processes. not taking this into account insufficiently is the root of the difficulties, with which the electrodynamics of moving bodies currently has to contend (einstein, 1905, my translation). his subsequent attempt to include gravitation in his theory focused on the equality of gravitational and inertial mass, and led him to adopt the equivalence principle : inertia and gravitation are wesensgleich (the same in essence), and must be represented by a single inertio - gravitational field.5 the distinction between the two is not absolute (i.e., frame independent), but depends on the frame of reference adopted. in particular, he noted that a linearly accelerated (rigid) frame of reference in a space - time without a gravitational field is physically equivalent to an inertial frame of reference, in which there is a uniform, constant gravitational field : both result in equal acceleration of bodies moving relative to their respective frames. he concluded that, in order to include gravitation, one must go beyond the special theory, with its privileged role for inertial frames, and look for a generalized (verallgemeinerte) theory of relativity. in the simplest case, linearly accelerated frames in minkowski space, the usual time coordinate loses its direct physical significance ; and in uniformly rotating frames, a global time can not even be defined. in the latter case, the spatial coordinates also lose their direct significance : the measured spatial geometry is no longer flat. i soon saw that, according to the point of view about non - linear transformations required by the equivalence principle, the simple physical interpretation of the coordinates had to be abandoned. this recognition tormented me a great deal because for a long time i was not able to see just what are coordinates actually supposed to mean in physics ? (einstein 1933, translation from stachel, 2007, p. 86). i soon saw that, according to the point of view about non - linear transformations required by the equivalence principle, the simple physical interpretation of the coordinates had to be abandoned. this recognition tormented me a great deal because for a long time i was not able to see just what are coordinates actually supposed to mean in physics ? (einstein 1933, translation from stachel, 2007, p. 86). the equivalence principle made it not only probable that the laws of nature must be invariant with respect to a more general group of transformations than the lorentz group (extension of the principle of relativity), but also that this extension would lead to a more profound theory of the gravitational field. first of all, elementary arguments showed that the transition to a wider group of transformations is incompatible with a direct physical interpretation of the space - time coordinates, which had paved the way for the special theory of relativity. further, at the outset it was not clear how the enlarged group was to be chosen (einstein, 1956, my translation). made it not only probable that the laws of nature must be invariant with respect to a more general group of transformations than the lorentz group (extension of the principle of relativity), but also that this extension would lead to a more profound theory of the gravitational field. first of all, elementary arguments showed that the transition to a wider group of transformations is incompatible with a direct physical interpretation of the space - time coordinates, which had paved the way for the special theory of relativity. further, at the outset it was not clear how the enlarged group was to be chosen (einstein, 1956, my translation). einstein first attempted to develop a theory of the gravitational field produced by a static source, still based on the idea of a scalar gravitational potential. his earlier work had led him to consider non - flat spaces ; this work led him to consider non - flat space - times : he found that his equation of motion for a test particle in a static field can be derived from a variational principle : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \int { { { \{{{[c(x, y, z)]}^2}d{t^2 } - [d{x^2 } + d{y^2 } + d{z^2}]\}}^{1/2 } } = 0,}$$\end{document } where he interpreted c(x, y, z) as a spatially - variable speed of light.6 already familiar with minkowski s four - dimensional formulation of the special theory, he realized that this variational principle could be interpreted as the equation for a geodesic (i.e, an extremal) in a non - flat space - time with ds = { [c(x, y, z) ] dt [dx + dy + dz ] } as its line element. by explicitly introducing the flat minkowski pseudo - metric, einstein then made a big leap : he generalized the geodesic equation using a non - flat riemannian pseudo - metric g, and assumed that it would still describe the path of a test particle in an arbitrary non - static gravitational field. the gravitational theory he was seeking must be based on such a non - flat metric, which should both : determine the line element, ds = g dx dx representing the chrono - geometry of space - time;serve as the potentials for the inertio - gravitational field. determine the line element, ds = g dx dx representing the chrono - geometry of space - time ; serve as the potentials for the inertio - gravitational field. while a student at the swiss federal polytechnic, einstein had learned about gauss theory of non - flat surfaces, and realized he needed a four - dimensional generalization. his old classmate and new colleague, the mathematician marcel grossmann, told einstein about riemann s generalization of gauss theory and about the tensor calculus (absolute differential calculus), developed by ricci and levi - civita to facilitate calculations in an arbitrary coordinate system. still identifying a coordinate system with a physical frame of reference, his goal of extending the principle of the relativity led einstein to investigate the widest possible group of coordinate transformations. with grossman s help, he succeeded in formulating the influence of the inertio - gravitational field on the rest of physics by putting these equations into a generally covariant form. the one exception was the gravitational field equations, the problem to which they now turned. general covariance then meant covariance under arbitrary coordinate transformations,7 the concepts of covariant derivative and riemann tensor were based on the theory of differential invariants, and lacked a simple geometrical interpretation.8 nevertheless, einstein seriously considered the ricci tensor, the only second rank contraction of the riemann tensor, for use in the gravitational field equations. he tried, in linear approximation, setting it equal to the stress - energy - momentum tensor of the sources of the gravitational field ; and even realized that, in order to obtain consistency with the vanishing divergence of the source tensor, the ricci tensor would have to be modified by a trace term.9 however, after coming so close to the final form of field equations of gr, he retreated. his earlier work on static fields led him to conclude that, in adapted coordinates, the spatial part of the metric tensor must remain flat [see eq. (1) ], which is easily shown to be incompatible with field equations based on the ricci tensor. so, as he later put it, he abandoned these equations with a heavy heart, and began to search for non - generally - covariant field equations. einstein soon developed a meta - argument against a generally covariant set of field equations for the metric tensor. why did he formulate this argument in terms of a hole a finite region of space - time devoid of all non - gravitational sources?10 it was probably the influence of mach s ideas. one of einstein s main motivations in the search for a generalized theory of relativity was his interpretation of mach s critique of newtonian mechanics. according to mach, space is not absolute : it does not have any inherent properties of its own, and its apparent influence on the motion of a body as manifested in the law of inertia, for example must result from an interaction between the moving body and all the rest of the matter in the universe. mach suggested that the inertial behavior of matter in the region of empty space around us is the effect of all the matter in the universe that surrounds this region, or when einstein adopted the metric tensor as the representation of the potentials of the inertiogravitational field, he interpreted mach s idea as the requirement that metric field in such a hole be entirely determined by its sources that is by the stress - energy tensor of the surrounding matter.11 the hole argument purports to show that, if the field equations are generally covariant, this requirement can not be satisfied : even if the field and all its sources outside of and on the boundary of the hole are fully specified, such equations can not determine a unique field in the interior of the hole. we present the argument here in einstein s original coordinate - based formulation (see section 4 for a modern, coordinate - free form). let the metric tensor be symbolized by the single letter g, and a given four - dimensional coordinate system by a single letter x. suppose g(x) is a solution to a set of field equations. if both the coordinates and components of the metric tensor are subject to the transformation 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$x \rightarrow x^{\prime } = f(x)$$\end{document } from coordinates x to another coordinate system x, then g(x) represents the same solution in the new coordinate system. this presents no problem einstein is quite clear on this point.12 but if the field equations are generally covariant, einstein noted, it follows that g(x) must also be a solution. he emphasized clearly that g(x) and g(x) represent two mathematically distinct solutions in the same coordinate system.13 now consider a hole h a bounded, closed region of space - time, in which all non - gravitational sources of the field represented by the stress - energy tensor t vanish ; and suppose the field g(x) and all its sources t are specified everywhere outside the hole and on its boundary, together with any finite number of normal derivatives of field on the boundary. for there are coordinate transformations x x that reduce to the identity outside of and on the boundary of h, together with all their derivatives up to any finite order ; yet which differ from the identity inside h. such coordinate transformations will leave t unchanged, and the resulting g(x) will still equal g(x) outside of and on the boundary of h ; but g(x) will differ from g(x) inside h. in short, if the field equations are generally covariant, then specification of the gravitational field together with its sources outside of and on the boundary of such a hole do not suffice to determine the field inside. einstein concluded that generally - covariant field equations could not be used to describe the metric tensor field, and began a search for non - covariant field equations. now the question became : if lorentz invariance is too little (equivalence principle) and general covariance is too much (hole argument), what is the widest possible group (if it is a group !) of coordinate transformations, under which one can demand the invariance of such equations ? the hole argument is not valid for the inhomogeneous lorentz (poincar) group ; and einstein concluded that the invariance group of the field equations should be extended only up to but not including the invariance group, for which the hole argument becomes valid in coordinates adapted to this group. because of problems unrelated to the hole argument, in mid-1915 einstein abandoned the search for a non - covariant theory of gravitation and returned to the riemann tensor. after several intermediate steps, by november of that year he adopted the set of generally - covariant equations now known as the einstein equations. his successful explanation of the anomalous perihelion advance of mercury convinced him and many others of the profound significance of the resulting theory, known today as general relativity. what about the hole argument ? einstein realized that, to avoid it, he had only to drop one of the premises that he had tacitly adopted : the assumption that the points of space - time in the hole are individuated independently of the metric field. if that assumption is dropped, it follows that when the metric is dragged - along by a coordinate transformation, all the individuating properties and relations of the points of space - time are dragged along too.14 while g(x) does differ mathematically from g(x) inside h, they are merely different representations of the same physical solution. properly - specified conditions outside the hole will suffice to specify a unique physical solution inside the hole. in order to better illustrate the flaw in the hole argument, einstein developed a counter - argument, the point - coincidence argument.15 there are actually two versions of this argument, which have been called the private and the public one.16 first i shall cite the private version. in letters to friends, einstein explained why the argument no longer applies to general relativity : everything in the hole argument was correct up to the final conclusion. it has no physical content if, with respect to the same coordinate system k, two different solutions g(x) and g(x) exist [see section 2.3 ]. to imagine two solutions simultaneously on the same manifold has no meaning, and indeed the system k has no physical reality. if, for example, all physical events were to be built up from the motions of material points alone, then the meetings of these points, i.e., the points of intersection of their world lines, would be the only real things, i.e., observable in principle. these points of intersection naturally are preserved during all [coordinate ] transformations (and no new ones occur) if only certain uniqueness conditions are observed. it is therefore most natural to demand of the laws that they determine no more than the totality of space - time coincidences. from what has been said, this is already attained through the use of generally covariant equations (letter to michele besso, 3 january 1916, in schulmann., 1998, it has no physical content if, with respect to the same coordinate system k, two different solutions g(x) and g(x) exist [see section 2.3 ]. to imagine two solutions simultaneously on the same manifold has no meaning, and indeed the system k has no physical reality. if, for example, all physical events were to be built up from the motions of material points alone, then the meetings of these points, i.e., the points of intersection of their world lines, would be the only real things, i.e., observable in principle. these points of intersection naturally are preserved during all [coordinate ] transformations (and no new ones occur) if only certain uniqueness conditions are observed. it is therefore most natural to demand of the laws that they determine no more than the totality of space - time coincidences. from what has been said, this is already attained through the use of generally covariant equations (letter to michele besso, 3 january 1916, in schulmann., 1998, einstein s argument consists of three points ; in modern language, they are : if two metrics in their respective different coordinate systems differ only in that one is the carry - along of the other, then physically there is no distinction between them.generally covariant equations have the property that, given a solution, any carry - along of that solution in the same coordinate system is also a solution to these equations.in the absence of a metric tensor field, a coordinate system on a differentiable manifold has no intrinsic significance.17 if two metrics in their respective different coordinate systems differ only in that one is the carry - along of the other, then physically there is no distinction between them. generally covariant equations have the property that, given a solution, any carry - along of that solution in the same coordinate system is also a solution to these equations. in the absence of a metric tensor field, a coordinate system on a differentiable manifold has no intrinsic significance.17 note that points 1) and 2) were included in the original hole argument (see section 2.3). it follows from the three points that the entire equivalence class of carry - alongs of a given solution in the same coordinate system corresponds to one physical gravitational field. thus, the hole argument fails. as will be seen in section 4, point 1) constitutes a coordinate - dependent version, applied to the metric tensor, of what i call the basic or trivial identity ; point 2) constitutes the coordinate - dependent version, applied to the metric tensor, of my definition of covariant theories. i would apply the term generally covariant to the conclusion that an entire equivalence class of carry alongs corresponds to one physical solution. the coordinate - independent versions of all three concepts are obtained by substituting basis vectors for coordinates and diffeomorphisms for coordinate transformations. einstein s 1916 review paper presents the public version of the argument to justify the requirement that any physical theory be invariant under all coordinate transformations : our space - time verifications invariably amount to a determination of space - time coincidences. if, for example, events consisted merely in the motion of material points, then ultimately nothing would be observable but the meetings of two or more of these points. moreover, the results of our measurements are nothing but verifications of such meetings of the material points of our measuring instruments with other material points, coincidences between the hands of a clock and points on the clock - dial, and observed point - events happening at the same place at the same time.the introduction of a system of reference serves no other purpose than to facilitate the description of the totality of such coincidences. we allot to the universe four space - time variables x, x, x, x in such a way that for every point - event there is a corresponding system of values of the variables x x. to two coincident point - events there corresponds one system of values of the variables x x, i.e., coincidence is characterized by the identity of the co - ordinates. if, in place of the variables x x, we introduce functions of them, x, x, x, x, as a new system of co - ordinates, so that the systems of values are made to correspond to one another without ambiguity, the equality of all four co - ordinates in the new system will also serve as an expression for the space - time coincidence of the two point - events. as all our physical experience can be ultimately reduced to such coincidences, there is no immediate reason for preferring certain systems of coordinates to others, that is to say, we arrive at the requirement of general covariance (einstein, 1916, pp. 776777, reprinted in kox., 1996, pp. our space - time verifications invariably amount to a determination of space - time coincidences. if, for example, events consisted merely in the motion of material points, then ultimately nothing would be observable but the meetings of two or more of these points. moreover, the results of our measurements are nothing but verifications of such meetings of the material points of our measuring instruments with other material points, coincidences between the hands of a clock and points on the clock - dial, and observed point - events happening at the same place at the same time. the introduction of a system of reference serves no other purpose than to facilitate the description of the totality of such coincidences. we allot to the universe four space - time variables x, x, x, x in such a way that for every point - event there is a corresponding system of values of the variables x x. to two coincident point - events there corresponds one system of values of the variables x x, i.e., coincidence is characterized by the identity of the co - ordinates. if, in place of the variables x x, we introduce functions of them, x, x, x, x, as a new system of co - ordinates, so that the systems of values are made to correspond to one another without ambiguity, the equality of all four co - ordinates in the new system will also serve as an expression for the space - time coincidence of the two point - events. as all our physical experience can be ultimately reduced to such coincidences, there is no immediate reason for preferring certain systems of coordinates to others, that is to say, we arrive at the requirement of general covariance (einstein, 1916, pp. 776777, reprinted in kox., 1996, pp. indeed, he proceeds to illustrate it with a version of the trivial identity applied to a system of particles, rather than fields, the model being any set of particle world lines, without any requirement that they satisfy equations of motion. einstein also mentions the requirement of general covariance ; but here it amounts basically to point 3) together with a generalization of point 1) to any objects used in a physical theory, whether or not they obey any field equations. it is essentially a coordinate - dependent version of the basic identity, extended from metrics to all geometric object fields of a certain type (see section 4.2). we see here the origins of two differing usages of the term general covariance one involves the field equations, the other does not. he wrote to schlick : generally considered, your presentation of the [point coincidence ] argument does not correspond with my conception of it since i find your entire conception too positivistic, so to speak. physics is an attempt at the conceptual construction of a model of the real world, as well as of its lawful structure. indeed it must represent exactly the empirical relations between the sense experiences that are accessible to us ; but only in this way is it linked to the latter (einstein to moritz schlick, 28 november 1930 ; cited from engler and renn, 2013, p. 18 generally considered, your presentation of the [point coincidence ] argument does not correspond with my conception of it since i find your entire conception too positivistic, so to speak. physics is an attempt at the conceptual construction of a model of the real world, as well as of its lawful structure. indeed it must represent exactly the empirical relations between the sense experiences that are accessible to us ; but only in this way is it linked to the latter (einstein to moritz schlick, 28 november 1930 ; cited from engler and renn, 2013, p. 18 in 1915, even before einstein completed the general theory of relativity, erich kretschmann (1915a, b) had undertaken an investigation that led him to a version of the trivial identity. kretschmann (1917) uses einstein s public point coincidence argument to conclude that any theory could be put into a form satisfying einstein s principle of general covariance. einstein (1918) concedes the point, but argued, not very successfully,18 that an added criterion of simplicity gives the principle a heuristic significance. evidently, he was not himself clear on the difference between his two arguments : while the public point coincidence argument does not provide a criterion for singling out theories, the criterion of general covariance in the private argument does.19 of greater future significance was kretschmann s suggestion : use four invariants of the riemann tensor to fix a unique coordinate system (an individuating field in my terminology). section of kretschmann (1917) discusses the use of the principal directions of the riemann tensor to fix the coordinate directions;20 and section iii.2 proposes the use of four mutually - independent invariants of the riemann tensor and metric as the space - time coordinates.21 apparently unaware of kretschmann (1917), arthur komar (1958) also suggested the use of four invariants of the riemann tensor as coordinates. in subsequent discussions of the problem of true observables in general relativity, they are often referred to as kretschmann - komar coordinates. stachel (1989, 1993) noted their use as a way of individuating the points of space - time, and they have subsequently figured in many discussions of the hole argument. kretschmann (1917) notes that : this system of [principal ] directions naturally may be indeterminate or otherwise degenerate ; and that the four invariants may be used as coordinates only by postulating that in no finite four - dimensional region are [they ] mutually dependent. section 6.1 discusses the treatment of such cases, in which the symmetry or isometry group of an equivalence class of metrics is non - trivial. as we have seen, in 1913 einstein formulated his argument against generally covariant equations in terms of the non - uniqueness of the field in a hole in space - time. david hilbert, the renowned mathematician, became interested in the problem of a unified gravitational - electromagnetic theory and followed einstein in arguing against generally covariant field equations. instead of a hole, however, he formulated the argument in a mathematically more sophisticated way, using a spacelike hypersurface.22 he showed that there is no well - posed cauchy problem for generally covariant equations ; i.e., no finite set of initial values on such a hypersurface can determine a unique solution to these equations off the initial hypersurface.23 after einstein returned to generally covariant field equations, hilbert dropped this argument against them, and hilbert (1917) is the first discussion of the cauchy problem in general relativity ; but the analysis is far from complete.24 it was not until 1927 that georges darmois gave a reasonably complete treatment.25 his discussion included the role of null hypersurfaces as characteristics, the use of the first and second fundamental forms on a space - like hypersurface as initial data, and the division of the ten field equations into four constraints on the initial data and six evolution equations. most post world - war ii discussions of the cauchy problem in general relativity are based on the work of andre lichnerowicz,26 but he acknowledges his debt to darmois : in 1926 in belgium, darmois gave a course of four lectures on the equations of einsteinian gravitation in the presence of de donder. the monograph version (darmois, 1927) became my bedside reading. in this book is the first rigorous analysis of the hyperbolic nature of the einstein equations, i.e., the foundation of the relativistic theory of gravitation as a theory of wave propagation, with profound understanding, the splitting of the einstein equations relative to the cauchy problem into two sets is clearly discussed : one treats the initial conditions, and the other deals with time evolution (lichnerowicz, 1992, p. 104). in 1926 in belgium, darmois gave a course of four lectures on the equations of einsteinian gravitation in the presence of de donder. the monograph version (darmois, 1927) is the first rigorous analysis of the hyperbolic nature of the einstein equations, i.e., the foundation of the relativistic theory of gravitation as a theory of wave propagation, with profound understanding, the splitting of the einstein equations relative to the cauchy problem into two sets is clearly discussed : one treats the initial conditions, and the other deals with time evolution (lichnerowicz, 1992, p. 104). many current discussions of the non - uniqueness problem in general relativity are formulated in terms of the cauchy problem rather than the original hole argument (see, e.g., belot and earman, 2001 ; rickles, 2005 ; lusanna and pauri, 2006). its modern revival came about as the result of debates about the reason for the delay of over two years between einstein s adoption of the metric tensor in 1913 and his formulation of the generally - covariant field equations for the metric at the end of 1915 (see section 2). the answer to this question hinges on the answer given to the question of why einstein formulated the hole argument and held to it during this entire period. in 1982, pais summarized the generally - accepted view : in 1914 not only did he [einstein ] have some wrong physical ideas about causality but in addition he did not yet understand some elementary mathematical notions about tensors (pais, 1982, p. 224).27einstein still had to understand that this freedom [to make an arbitrary coordinate transformation ] expresses the fact that the choice of coordinates is a matter of convention without physical content (ibid., p. 222).28 in 1914 not only did he [einstein ] have some wrong physical ideas about causality but in addition he did not yet understand some elementary mathematical notions about tensors (pais, 1982, p. 224).27 einstein still had to understand that this freedom [to make an arbitrary coordinate transformation ] expresses the fact that the choice of coordinates is a matter of convention without physical content (ibid. stachel (1979) presented a version of the standard account, but by the following year it had become evident that this account was incorrect. at the 1980 jena meeting of the grg society, he presented a detailed analysis of the hole argument and its refutation by the point coincidence argument ; it circulated as a preprint, but was not published until 1989 (stachel, 1989). however, torretti (1983, chapter 5.6) gives a detailed account of the hole argument based on it;29 and norton (1984), the first detailed analysis of einstein s 1913 zurich notebook,30 also summarizes stachel s account. stachel (1987) contains a historical - critical account of the hole argument, and stachel (1986) uses the fiber bundle formalism to generalize the argument to any geometric object field obeying generally - covariant equations. these two talks helped to stimulate renewed interest in the meaning of diffeomorphism invariance among relativists, especially those working on quantum gravity (see, e.g., rovelli, 1991). earman and norton s presentations of the hole argument earman and norton (1987) ; earman (1989) provoked renewed discussion of absolute versus relational theories of space - time among philosophers of science, a discussion that continues to this day. 31 section 5 shows how several initially - different positions have converged on an approach that gives precise meaning to einstein s vision of general relativity, and section 6 reviews some physical topics related to the hole argument. as we have seen already, einstein often posed a problem, the solution to which required mathematical tools that went far beyond his current knowledge. another example is the vision of the nature of general relativity that replaced his earlier faith in mach s principle (see section 2.3). as we shall see, this new vision requires the theory of fiber bundles for its appropriate mathematical formulation. when asked by a reporter to sum up the theory of relativity in a sentence, einstein said, half jokingly : before my theory, people thought that if you removed all the matter from the universe, you would be left with empty space. my theory says that if you remove all the matter, space disappears, too ! 32 before my theory, people thought that if you removed all the matter from the universe, you would be left with empty space. my theory says that if you remove all the matter, space disappears, too ! (einstein, 1931). 32 in 1952, he developed the same idea at greater length : on the basis of the general theory of relativity space as opposed to if we imagine the gravitational field, i.e., the functions gik to be removed, there does not remain a space of the type (1) [minkowski space - time ], but absolutely nothing, and also no topological space. there is no such thing as an empty space, i.e., a space without field. space - time does not claim existence on its own, but only as a structural quality of the field (einstein, 1952, p. 155). on the basis of the general theory of relativity space as opposed to if we imagine the gravitational field, i.e., the functions gik to be removed, there does not remain a space of the type (1) [minkowski space - time ], but absolutely nothing, and also no topological space. there is no such thing as an empty space, i.e., a space without field. space - time does not claim existence on its own, but only as a structural quality of the field (einstein, 1952, p. 155). it is evident that this new approach to general relativity completely reverses his original machian vision. now the field is primary, and matter like everything else must be treated as an aspect of the field. einstein s comment occurs in the course of a discussion of the age - old conflict between absolute33 and relational interpretations of space, which relativity theory metamorphosed into a conflict between interpretations of space - time.34 the quotation above stresses the role of the metric tensor, but elsewhere einstein emphasizes the role of the affine connection, which he calls a displacement field : it is the essential achievement of the general theory of relativity that it freed physics from the necessity of introducing the inertial system (or inertial systems) the development of the mathematical theories essential for the setting up of general relativity had the result that at first the riemannian metric was considered the fundamental concept on which the general theory of relativity and thus the avoidance of the inertial system were based. later, however, levi - civita rightly pointed out that the element of the theory that makes it possible to avoid the inertial system is rather the infinitesimal displacement field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { jk}^i$\end{document}. the metric or the symmetric tensor field gik which defines it is only indirectly connected with the avoidance of the inertial system in so far as it determines a displacement field (einstein, 1955, pp. it is the essential achievement of the general theory of relativity that it freed physics from the necessity of introducing the inertial system (or inertial systems) the development of the mathematical theories essential for the setting up of general relativity had the result that at first the riemannian metric was considered the fundamental concept on which the general theory of relativity and thus the avoidance of the inertial system were based. later, however, levi - civita rightly pointed out that the element of the theory that makes it possible to avoid the inertial system is rather the infinitesimal displacement field \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { jk}^i$\end{document}. the metric or the symmetric tensor field gik which defines it is only indirectly connected with the avoidance of the inertial system in so far as it determines a displacement field (einstein, 1955, pp. einstein s vision can be summed in the sentence : space - time does not claim existence on its own, but only as a structural quality of the field. the two main elements of this vision are : if there is no field, there can be no space - time manifold.the spatio - temporal structural qualities of the field include the affine connection, which is actually of primary significance as compared to the metric tensor field. the spatio - temporal structural qualities of the field include the affine connection, which is actually of primary significance as compared to the metric tensor field. up until quite recently, the standard formulations of general relativity did not incorporate this vision. they start by postulating a four - dimensional differentiable manifold, which is described as a space - time before any metric tensor field is defined on it ; and all other space - time structures, such as the levi - civita connection, are defined in terms of this one field.35 but the concepts of fiber bundles and sheaves enable a mathematical formulation of general relativity consistent with einstein s vision36 (see section 4.3) : if there is no total space for the fields, then there is no base manifold.the conceptual distinction between the roles of the metric and connection becomes evident : the metric lives on the vertical fibers of the total space ; while the connection lives on the horizontal directions of the total space, connecting the fibers with each other. if there is no total space for the fields, then there is no base manifold. the conceptual distinction between the roles of the metric and connection becomes evident : the metric lives on the vertical fibers of the total space ; while the connection lives on the horizontal directions of the total space, connecting the fibers with each other. clearly, einstein s vision favors a non - absolutist view of space - time.37 while no formalism can resolve a philosophical issue, the traditional approach that starts from a manifold m and defines various geometric object fields over it gives manifold substantivalists an initial advantage : opponents must explain away somehow the apparent priority of m. the modern approach starts from a principal fiber bundle p with total space e and structure group g, and defines m as the quotient e / g ; this gives non - substantivalists an initial advantage : the whole bundle (pun intended), which includes some geometric object field, a connection and a manifold, is there from the start ; a manifold substantivalist must justify giving priority to m. after defining geometric and algebraic structures, a space is defined as a set of points with a geometric structure that is invariant under some group of transfomations of its points. then i discuss product and quotient spaces, fibered spaces, and theories based on these spaces, in particular permutable and generally permutable theories (section 4.1). up to this point but all definitions are still applicable appropriately modified, of course when additional structures are introduced. in particular, the case of most physical interest is that of geometric object fields defined on a differentiable manifold (section 4.2). they provide the framework for coordinate - independent definitions of covariant and generally covariant theories, followed by a precise formulation of the of the original hole argument against general covariance and of the way to avoid its conclusion, discussed informally in section 2. then i discuss fiber bundles, which consist of a total space, a base space, and a projection operator. under certain circumstances, the base space may be defined as the quotient of the total space divided by an equivalence relation defining its fibers (section 4.3) this approach allows a more precise formulation of einstein s vision of general relativity, discussed informally in section 3.2. finally, the distinction between natural and gauge natural bundles is discussed, and between the concepts of covariance and general covariance when applied to theories defined on each type of bundle (section 4.4). a number of philosophical concepts used but not defined in this section, such as : intrinsic and extrinsic properties, internal and external relations, and quiddity and haecceity, are discussed in the appendix b. consider a set s of elements x, y, z, etc., together with a set of relations r between its elements.39 there is a major distinction between a geometry and an algebra : in a geometry, the elements of s (hereafter called points and symbolized by p, q, etc.)40 all are of the same quiddity (i.e., of the same nature) but lack haecceity (i.e., are not inherently individuated) : the only distinctions between the points arise from a set of internal relations r between them. if we abstract from these relations, the set s is invariant under perm(s), the group of all permutations of the points of s.41 the set of relations defining a geometry structure or geometry the maximal subgroup aut rg(s) of perm(s) that preserves all these relations between the points of s is called the symmetry or automorphism group of this geometry, and could just as well be used to define it.42 obviously, perm(s) is the maximal possible automorphism group ; so a study of its subgroups and their relation to each other is equivalent to a study of all possible geometries on s and their relation to each other. in contrast to a geometry, in an algebra each element (symbolized by a, b, etc.) in addition to having the same quiddity also has an intrinsic haecceity (individuality). an algebraic structure or algebra a on a set is also defined by a set of relations ra between its elements ; but these are external relations, which do not affect the intrinsic individuality of each element.43 since descartes introduced analytic geometry, it has proved convenient and often necessary to apply algebraic methods in the solution of geometrical problems. this is done by a coordinatization of the geometry (see weyl, 1946, for this term) : a one - one correspondence is set up between the points of the geometry and certain elements of an appropriately chosen algebra. this coordinatization assigns to each point p of the geometry an element a of the algebra, called its coordinate and symbolized by a(p). but, by individuating the points of a geometry, a coordinatization negates their homogeneity, turning the geometry into an algebra. the only way to restore their homogeneity is to negate the coordinatization as follows : introduce the class of all admissible coordinatizations of the geometry44 based on the given algebra, so that each point of the geometry will have every admissible element of the algebra as its coordinate in at least one admissible coordinate system. transformations between two admissible coordinate systems are called admissible coordinate transformations ; they usually form a group that includes a subgroup isomorphic to the automorphism group of the geometry. there are two distinct ways in which the assignment of all admissible coordinates to each point of a geometry may be accomplished. active point transformations : keep the coordinate system fixed, a a, and permute the points of the geometry : p q, a(p) a(q).passive coordinate transformations : keep the points of the geometry fixed, p p, and carry out an admissible coordinate transformation of the elements of the algebra : a b, a(p) b(p). active point transformations : keep the coordinate system fixed, a a, and permute the points of the geometry : p q, a(p) a(q). passive coordinate transformations : keep the points of the geometry fixed, p p, and carry out an admissible coordinate transformation of the elements of the algebra : a b, a(p) b(p). the terms active and passive refer to the effects of a transformation on the points of the geometry. a passive coordinate transformation is an active transformation of the elements of the algebra.45 two active permutation groups of any geometry have already been introduced : perm(s), the group of all permutations of the elements of s;aut rg(s), the subgroup of perm(s) consisting of the permutations that belong to the automorphism group rg of a particular geometry g. perm(s), the group of all permutations of the elements of s ; aut rg(s), the subgroup of perm(s) consisting of the permutations that belong to the automorphism group rg of a particular geometry g. relations may also be permuted. let r(p) symbolize a relation between the set x of all points of s.46 consider a permutation px of the elements of s. define the permuted relation pr as follows : pr(x) holds iff r(px) does. when a permutation px is carried out, the relation r will be said to be carried along if it is also permuted into the relation pr.47 it follows that, if r(x) is valid, then so is pr(px). by virtue of the intrinsic homogeneity of its points, a geometry g remains unchanged if, for each permutation p of perm(s), the corresponding permutation prg of the set of relations rg defining g is also carried out. for any relation rg rg, it is clear that prg(pp) holds if and only if rg(p) holds, so prg rg ; thus rg and prg describe the same geometry. i shall refer to this result as the basic or trivial identity for the group perm(s) : it holds for any geometry based on a subgroup of perm(s). an equivalence relation req on any set s is a two - place relation having the following three properties : for all x, y, z in s, it is reflexive : req(x, x) holds ; symmetric : if req(x, y) holds, then so does req(x, y) ; and transitive : if req(x, y) and req(y, z) both hold, then so does req(x, z). if the context is clear, one often abbreviates req(x, y) by x y. an equivalence relation divides s into equivalence classes sr, often also called its orbits (see neumann., 1994, chapter v). the quotient set of s by req, often called the orbit space and abbreviated sq = s / req, is defined by the condition that each element of the quotient set corresponds to one and only one such equivalence class. given two sets a and b, we can form the product set a b, consisting of all pairs of elements (x, y), with x a and y b. a mapping from the domain a to the range or codomain b (see lawvere and schanuel, 1997, pp. 1314), often symbolized : a b, is defined as a subset of a b, such that for each x in the domain there is one and only one y in its range. in various contexts, mappings might also be called functions, transformations, or operators. homomorphisms, isomorphisms, homeomorphisms, diffeomorphisms, continuous or differentiable maps will be more attached to certain classes of mappings, which certain structures on the sets which are their domains and ranges (hermann, 1973, p. 3). in various contexts, mappings might also be called functions, transformations, or operators. homomorphisms, isomorphisms, homeomorphisms, diffeomorphisms, continuous or differentiable maps will be more attached to certain classes of mappings, which preserve certain structures on the sets which are their domains and ranges (hermann, 1973, p. 3). the mapping is surjective if, for every element y b, there is at least - one element x a that maps onto y. if both mappings a b and b a are surjective, the mapping is called bijective. if the bijective map preserves all structures on a and b, a and b are said to be isomorphic. if the mapping : a b is surjective, the set b is isomorphic to the quotient set a/ ; so b can actually be defined as the quotient set. this passing to the quotient is a way of defining new spaces and mappings that is very important in all of mathematics, particularly in algebra and differential geometry. this passing to the quotient is a way of defining new spaces and mappings that is very important in all of mathematics, particularly in algebra and differential geometry. this possibility allows us to realize einstein s vision of general relativity (see section 3.2), which in this context is simply : if no a, then no b. we can define the mapping or morphism from s to sq, : s sq, which projects each element x of s into the element (x) of sq corresponding to the equivalence class that includes x. conversely a section of s is an inverse mapping from the point (x) of sq to a unique point y in the equivalence class of s that maps into that point of sq : (x) = y, with x y. so far, these concepts can be applied to any set. if s is a geometry with automorphism group g, it is referred to as a g - space (see neumann., 1994, pp. the equivalence relation is said to be g - invariant if, whenever p q holds for two points of s, then it follows that g(p) g(q) for all g g. consequently, the action of an element g g on s preserves the orbits of s but permutes them ; so all orbits, henceforth called fibers, must be isomorphic to what is called a typical fiber. the quotient set sq = s / req is itself a g - space called the quotient space. a fibered space consists of a total space e ; a base space b ; and a projection operator : e b that is a surjective mapping, as defined above. the fiber fb over each point b b is the set of all inverse elements (b) e ; that is, all elements p e such that (p) = b. a section of a fiber space is a choice of one element on each fiber fb for every b b. to convert a homogeneous set s with an equivalence relation into a fibered space, let s constitute the total space e ; then sq forms the base space b, and the mapping becomes the projection operator. if s is a geometry with automorphism group g, then g preserves the equivalence classes ; so all the fibers are isomorphic, resulting in a fiber bundle : a fiber bundle (e, b,) consists of : 1) a total space e, divided into fibers by an equivalence relation, all of these fibers being isomorphic to a typical fiber ; 2) a base space b that is isomorphic to the quotient e/ ; and 3) a projection operator : e b that takes each point of its domain e into the point p of its range b that corresponds to the fiber including p. a section of the bundle is a mapping that takes each point p of its domain b into a unique point of its range, consisting of the set of fibers of e. the point of on the fiber p over p is symbolized by p(). if e has the automorphism group g, the action of an element g g on the points of any section will result in a new section ; symbolically : g() =. so, given one section, the action of the elements of g produces a whole equivalence class of sections g(). a theory of a certain type is a procedure for producing models of that type. a particular theory of that type is a rule for selecting a subset of these models. one type of theory is defined by the choice of a fiber bundle with automorphism group g ; its models are the sections of this bundle. a particular theory is a rule for choosing a subset of sections of the bundle as models. if the rule is such that, when is a model, then so is the entire equivalence class of sections g(), the theory is permutable. it is generally permutable if this entire equivalence class is interpreted as a single model of the theory. in terms of the distinction between syntax and semantics, one may say : while each section of a theory is always syntactically distinguishable from the others, in a permutable theory they may also be semantically distinguishable. however, in a generally permutable theory they are not ; only an entire equivalence class of sections has a unique semantic interpretation. all assertions about geometric figures, such as right triangles, rectangles, circles, etc., are invariant under its automorphism group, which consists of translations and rotations ; so it is certainly a permutable theory. but these assertions actually apply to the whole equivalence class of geometric figures satisfying any of these definitions ; so it is a generally permutable theory. on the other hand, plane analytic geometry includes a choice of origin, unit of length, and a pair of rectangular axes. so all of its assertions are still permutable ; but some of them include references to the origin, axes, etc. we can distinguish, for example, between a circle of radius r centered at the origin, and one of the same radius centered at some other point. the reason, of course, is that the choice of a unique preferred coordinate system converts the euclidean plane from a geometry into an algebra. for the space - time theories forming the main topic of this review, s is often a four - dimensional differentiable manifold m ; and the analogue of perm(s) is diff(m), the diffeomorphism group consisting of all differentiable point transformations of the points x of m. any given, fixed geometric structures defined on m, such as a metric tensor field, will be symbolized by (x) ; they represent the analogue of the relations rg. the -geometry of m is also defined by the invariance of these (x) under the action of some lie subgroup g of diff(m). in other words, g plays the role, analogous to that of aut(rg), of the automophism group aut(m,) of the -geometry of m. and just as in that case, here every g - space can also be defined as a quotient or orbit space : every g - space can be expressed as in just one way as a disjoint union of a family of orbits. p. 51) every g - space can be expressed as in just one way as a disjoint union of a family of orbits. p. 51) just as in analytic geometry, one may set up ordinary and partial differential equations for various particles and fields on m. denote a set of such geometric object fields on m collectively by the symbol (x), and consider the effect of an element g(x) g on (x).50 from the definition of a geometric object (see schouten, 1954, pp. 6768) it follows that if x g(x) = x, then (x) (x). their transformation law under g(x) is linear and homogeneous in the components of (x) and homogeneous in the derivatives of g(x). in both galilean space - time (see yaglom, 1979) and in special relativistic space - time (minkowski space), g is a ten parameter lie group. four of these parameters generate spatial and temporal translations of the points, making these space - time geometries homogeneous. and in both, the six remaining parameters act at each point of space - time : three generate spatial rotations and three generate but they do so in different ways because their boosts differ : for galilean space time, they are galilei transformations that preserve the invariance of the absolute time. for minkowski space - time, they are lorentz transformations that combine spatial and temporal intervals into an invariant, truly four - dimensional space - time interval. both of these groups are subgroups of sl(4, r), the group of four - volume - preserving transformations.51 and both theories have a homogeneous, flat affine connection in common that is the mathematical expression of the newton s first law of inertia. its invariance group is al(4, r), which is a subgroup of sl(4, r). newtonian gravitational theory, in the form which incorporates the equivalence principle, preserves the global space - time structure of galilean space - time, but abandons the homogeneous flatness of the affine connection in favor of a non - flat affine connection that is the mathematical expression of the dynamical inertia - gravitational field. this field is non - homogeneous, but its compatibility with the space - time structure requires that locally it remain invariant under al(4, r), which means that its globally automorphism group must be sdiff(m), the group of unimodular diffeomorphisms. general relativity similarly abandons the homogeneous flatness of the affine connection in favor of a non - flat affine connection that is the mathematical expression of the dynamical inertia - gravitational field. but, in order to preserve its compatibility with the special - relativistic chrono - geometry expressed by the metric tensor, the latter must also become a dynamical field. it preserves the local space - time structure of the special theory at each point. but globally both dynamical fields must have automorphism groups consisting of diffeomorphisms of m, the space - tme manifold, now itself no longer globally fixed. traditionally, diff(m), the full diffeomorphism group, has been assumed to be the correct automorphism group for general - relativistic theories. however, there are good arguments for restricting this group to sdiff(m), the group of unimodular diffeomorphisms with determinant one. but first some definitions are needed (see, e.g., wikipedia : group action). the action of g is said to be effective if its identity element is the only one that takes each point into itself : that is, if g g, x m and : x g(x) is such that g(x) = x for all x, then g = e, the identity element of g. the action is transitive if is a map onto m that connects any two of its points : that is for any two points x, y m, there is always a g g for which g(x) = y. the stabilizer group hx at a point x of m is the subgroup of transformations of g that leave the point x invariant : that is, g hx if and only if g(x) = x.52 since g is a lie group, hx is a closed subgroup at each point of g and these stabilizer groups are conjugate subgroups of g. indeed, m is isomorphic to g / hx ; so one may actually define a geometry by the pair (g, h), where h is a closed subgroup of g. the action of g is free or semiregular if its stabilizer group is the identity : that is, if gx = x for some point x, then g = e, the identity element of g ; equivalently, if gx = hx for some x, then g = h. for example, the translation groups discussed above act freely on galilean and special relativistic space - times. now we are ready to return to the question of automorphism groups for general relativistic theories. the action of the stabilizer of diff(m) on the tangent space at each point x of m is lx = gl(n), the group of all linear transformations at x. but consider the objects defining the geometry of a general - relativistic space - time with n = 4 : again, if one wants to preserve the four - volumes of space - time, which are needed to formulate meaningful physical averages, one must restrict these transformations to sl(4), the group of 23 special linear transformations with unit determinant. the linear affine connection at x, which represents the inertio - gravitational field, is only invariant under the subgroup asl(4), the group of affine transformations with unit determinant. and the invariance group of the metric tensor, which represents the chrono - geometry, is even further restricted to the pseudo - orthogonal subgroup so(3,1) of sl(4). in short, globally physical considerations suggest the need to start from sdiff(m) as the automorphism group of general - relativistic theories. so physically, diff(m) overshoots the mark by allowing non - unimodular transformations, i.e, transformations with any value of their determinant at a point. geometrically, they correspond to similarity transformations, which preserve the shape but not the size of four - volumes in space - time. usually, one compensates for this unwanted change of size by introducing tensor densities : when appropriate weights are introduced for various tensors, these densities can undo the effects of the size changes produced by non - unimodular transformations. the action of its stabilizer on the tangent space at each point of m is slx, the maximal symmetry group that preserves the size of four - volumes, thus avoiding the need to introduce densities, among its many other advantages (see stachel, 2011 ; bradonji and stachel, 2012). for much of the following discussion, however, the distinction between diff(m) and sdiff(m) is inessential, so i shall continue to discuss diff(m), and only point out the distinction at some places where it is really important. by definition, aut rg(s), the group of permutations of the points of s defining the geometry g, leaves the relations rg (which equally well define the geometry of s) unchanged ; so the rg do not need to be permuted when the points of s are. whichever lie subgroup g of diff(m) is chosen as the automorphism group defining the geometry of a differentiable manifold m, similar comments hold for it. as we shall see, the important difference for the hole argument is that between geometries based on finite - parameter lie groups and those based on lie groups depending on one or more functions (functional lie groups). since it is no more than a relabeling of its points, any admissible passive coordinate transformation has no effect on a geometry (see section 4.1) however, if one restricts the group of coordinate transformations to a subgroup of those corresponding elements of the automorphism group of the geometry, then there is an isomorphism between this subgroup of passive coordinate transformations and the group of active point transformations defining the geometry. hence, it is possible to reformulate any statement about the geometry in terms of relations between the coordinate components of the geometric object fields that are invariant under this subgroup of restricted coordinate transformations. in the past, this is how coordinate - dependent techniques were used to arrive at geometric results ; and many contemporary treatments still utilize this technique. if one permutes the points of m by a diffeomorphism, carries along the fields defining its geometry and the fields defining the theory, and also carries out the corresponding coordinate transformations, then clearly the new fields at the new points will have the same coordinate components in the new coordinate system as the old fields at the old points in the old coordinate system. this observation is another, coordinate - dependent variant of the basic or trivial identity. it holds for any fields, quite independently of any theory, or any field equations that these fields may obey. geometrically, a coordinate system corresponds to the choice of a holonomic basis ei at each point of m : that is, there is a local coordinate system such that ei = /x. but the essential element geometrically is the choice of a basis, not its holonomicity. so, introduce an ordered set of basis vectors ei(x)(i = 1,2,,n), holonomic or not, at each point x of m together with the associated dual basis of covectors or one - forms e(x), such that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\langle { e_i}{e^j}\rangle = \delta _ i^j$\end{document}.53 associated with the geometric object fields and on m are their components with respect to such a pair of basis vectors, which will be symbolized by [e(x) ] and [e(x) ] : is is a set of coordinate - independent scalars that result from saturating all the free covariant and contravariant indices of and with the ei and e respectively. of course, under a change of basis : e(x) e(x) these scalars transform appropriately. a diffeomorphism d : x x induces such a change of basis : e(x) ed(x), and corresponding changes in the geometric object fields (e) d(x) and (x) ed(x). however, the values of these scalars remain unchanged if one carries out the associated push forwards and pull backs of and, as well of the basis vectors and covectors. that is, if we take the new basis vectors at the new point : ed(x) ; then the new components with respect to the new basis vectors at the new points will equal the old components with respect to the old basis vectors at the old points : [e(x) ] = d[ed(x) ] and [e(x) ] = d[ed(x) ]. this observation is a coordinate - independent formulation of the basic identity. since any model of a physical theory can only fix the values of such coordinate - independent scalars with respect to some basis for all geometric objects in that model, this identity can not fail to hold for any theory based on the -geometry of m. suppose we perform the push forwards and pull packs on the geometric object fields, but not on the -geometry or the basis vectors and convectors. that is, let e(x) e(x) and [e(x) ] [e(x) ], but [e(x) ] d[e(x) ]. in general, [e(x) ] d[e(x) ], so this results in a set of scalars that is distinct from [e(x) ] at each point x of m. a theory is covariant under the the -geometry s automorphism group if, whenever [e(x) ] is a model of the theory, then so is d [e(x) ]. covariance clearly defines an equivalence relation between models of the theory ; so covariance divides all models of a theory into equivalence classes.54 a covariant theory is generally covariant under the -geometry s automorphism group if an entire equivalence class of its mathematically distinct models corresponds to a single physical model of the theory. an ordered set of basis vectors ei(x) at a point of m is called a linear frame, and the set of all such linear frames at a point of m constitutes one fiber of the bundle of linear frames over m. as kobayashi explains, the bundle concept can be used to formulate any geometry on m as a g - structure:56 let m be a differentiable manifold of dimension n and l(m) the bundle of linear frames over m. then l(m) is the principal fibre bundle over m with group gl(n ; r). let g be a lie subgroup of gl(n ; r). by a g - structure on m we shall mean a differentiable subbundle p of l(m) with structure group g. (kobayashi, 1972, p. 1) let m be a differentiable manifold of dimension n and l(m) the bundle of linear frames over m. then l(m) is the principal fibre bundle over m with group gl(n ; r). let g be a lie subgroup of gl(n ; r). by a g - structure on m we shall mean a differentiable subbundle p of l(m) with structure group g. (kobayashi, 1972, p. 1). such a fiber bundle formulation of geometries has several crucial advantages : it makes evident the fundamental distinction between vertical geometrical quantities, such as metric tensors, that live on the fibers of the bundle ; and horizontal geometrical objects, such as linear affine connections, that serve to connect these fibers. this is the case whether the metric and/or connection are fixed and given components of ; or are components of, themselves subject to dynamical field equations. it enables us to go from global to local formulations of background - independent theories, such as general relativity, in which the global topology of the base manifold m can not be specified a priori, because it differs for different solutions to the field equations.57 the concept of fibered spaces for a set, discussed in section 4.1, can now be applied to differentiable manifolds (see section 4.2). after a fibered manifold is defined, the important cases of principal bundles, vector bundles, natural bundles and gauge - natural bundles and their physical applications are discussed, stressing the importance for general relativity of quotient bundles and local considerations. a fibered manifold (e, m,) consists of a total manifold e, a base manifold m, and a projection operator : e m. e is a differentiable manifold, the points of which, u,, etc., are grouped into fibers by an equivalence relation between its points. m is also a differentiable manifold, the points of which are symbolized by x, y, etc. note that, if the relation is given initially, sometimes the base manifold m may be defined as the quotient of the total manifold e by : m = e/ ; in other words as the orbit space of g (see sections 4.1 and 4.2). but the situation is generally somewhat more complicated : usually, when symmetries and invariance groups are considered, a problem reduces to the corresponding orbit space, and therefore the structure of these spaces has to be investigated. this structure theory is quite complicated in general, since these spaces usually are singular spaces and not again manifolds. in fact, only if the action of the lie group is free (i.e., all isotropy subgroups of single points are trivial), the resulting orbit space bears a manifold structure and forms together with the manifold and the quotient map a principal fiber bundle, whose structure is well known. more often, the orbit space admits a stratification into smooth manifolds with an open and dense largest stratum, the set of principal orbits. this stratified space can then be treated almost like a manifold when taking special care. the existence of such a stratification is usually shown by proving the existence of slices at every point for the group action (schichl, 1997, p. 1). usually, when symmetries and invariance groups are considered, a problem reduces to the corresponding orbit space, and therefore the structure of these spaces has to be investigated. this structure theory is quite complicated in general, since these spaces usually are singular spaces and not again manifolds. in fact, only if the action of the lie group is free (i.e., all isotropy subgroups of single points are trivial), the resulting orbit space bears a manifold structure and forms together with the manifold and the quotient map a principal fiber bundle, whose structure is well known. more often, the orbit space admits a stratification into smooth manifolds with an open and dense largest stratum, the set of principal orbits. this stratified space can then be treated almost like a manifold when taking special care. the existence of such a stratification is usually shown by proving the existence of slices at every point for the group action (schichl, 1997, p. 1). i shall assume that as in general relativity in any theory considered, the quotient space is either a manifold or a stratified manifold ; and that any local solution to its field equations can be extended to a global solution.58 a fiber bundle is a fibered manifold in which all its fibers are isomorphic to a typical fiber f, itself a manifold ; that is, for all x, fx f. suppose f is q - dimensional and m is p - dimensional one can always introduce a local trivialization of the bundle : let x be an open subspace of m. locally, the total space e is a product space (f x), and one can introduce p variables (x, x, x)as local coordinates of a point x of x, and q variables (u, u,, u) as coordinates of a point u of f. so (f x) is coordinatized by the (p+q) coordinates (x, u) of a point ux of e lying on the fiber fx over the point x. let g be a lie group of diffeomorphisms that acts on e.59 the action of an element g g on the point (x, u) of e is symbolized by (x, u) g(x, u) = (x,u) = [(x, u), (x, u) ]. two subgroups of g are especially important : the base transformations (diffeomorphisms of x) that do not affect the fibers : x x = (x), u = u.the pure fiber or pure gauge transformations on the fiber at each point : x = x, u = (x, u). both of these are included in a third subgroup : the fiber - preserving transformations : (x, u) (x,u) = [(x),(x, u) ]. the base transformations (diffeomorphisms of x) that do not affect the fibers : x x = (x), u = u. the pure fiber or pure gauge transformations on the fiber at each point : x = x, u = (x, u). the fiber - preserving transformations : (x, u) (x,u) = [(x),(x, u) ]. if g is a connected lie group, all of its actions can be constructed from iterations of the action of its lie algebra, composed of its infinitesimal generators : the vector fields v on e, each of which generates a one - parameter group of point transformations, or flow, on e. locally v may be written in terms of the coordinates (x, u) : 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v = \sum\limits_i { { \xi ^i}(x, u)\partial/\partial { x^i } + \sum\limits_\alpha { { \varphi ^\alpha}(x, u)\partial/\partial { u^\alpha}\quad i = 1, \ldots, p\;\;\alpha = 1, \ldots, q.}}$$\end{document } the generator v is called : horizontal if = 0, i.e., it generates only base transformations. vertical if = 0, i.e., if it generates only pure fiber or pure gauge transformations. the flow generated by v will be fiber preserving if and only if = (x). a fiber - preserving diffeomorphism projects naturally into a unique diffeomorphism of the base manifold m ; but generally the converse does not hold. if it does, i.e., if a base diffeomorphism of m lifts uniquely to a fiber - preserving diffeomorphism of e, then the bundle is a natural bundle. a geometric object defined on such a bundle is called a natural object.60 this is the fiber bundle version of the definition of geometric objects in section 4.2. if the the typical fiber f is isomorphic to the structure group g : f g, then the bundle is a principal fiber bundle p with structure group g : p = (e, m, ; g). corresponding to any p with structure group g, there is a class of associated vector bundles. in such an associated bundle, each fiber forms a vector representation of g. this vector representation need not be irreducible, so the class of associated vector bundles includes all tensor fields. the use of fibered manifolds allows a precise formulation of the concepts of covariance and general covariance for any physical theory ; and of the hole argument for background - independent theories, and even with appropriate modifications for some partially - background - dependent theories. every natural physical theory can be formulated in terms of some natural geometric object(s)61 that lives on an appropriate fibered differentiable manifold,62 the nature of which depends on these geometric object(s). if the theory is defined on a differentiable manifold m that is the quotient of the fibered manifold e divided by the equivalence relation defining the fibration, m = e/ ; then there is an operator, projecting each fiber onto the corresponding point of m : : e m. since the fibered manifold represents a natural object, there is a one - one correspondence between fiber - preserving diffeomorphisms of e and diffeomorphisms of m. a number of most important gauge natural theories can not be so formulated, but require the broader concept of gauge natural bundles for their precise formulation. indeed, every classical physical theory can be reformulated as the jet prolongation of some gauge natural bundle by adjoining the derivatives of the geometric object fields to the original bundle.63 a theory based on such fixed -fields on m is called background - dependent, with aut(m,) as its symmetry group.64 any geometric object fields (x) can then be introduced on this fixed - background space - time together with a set of field equations governing their dynamics, which generally involve some or all of the (x). in many theories, the fixed geometric object fields on m consist of a vertical chrono - geometric metric tensor on each fiber and the corresponding horizontal inertio - gravitational linear connection. any non - gravitational theory can be formulated on a fiber bundle associated with the principal bundle determined by the metric and connection : the (x) break up into two subclasses : the fields of massive objects (such as charged bodies) are represented by geometric quantities living on the vertical fibers ; and the gauge fields transmitting the forces between these objects (such as the electromagnetic field) are represented by verical connections along the fibers ; these connections are only fixed up to some group of gauge transformations. in the case of general relativity and other background - independent theories (such as the coupled einstein - maxwell equations), reduces to the identity and there are no fixed background space - time structures on m. diff(m) is chosen as aut(m) in the usual formulations ; but, as suggested in section 4.2, sdiff(m), the unimodular subgroup, may be chosen. in that case, the space - time structures subdivide further : the pseudo - metric splits into a conformal metric with determinant 1 and a scalar field, both of which live on the vertical fibers ; while the linear affine connection splits into a trace - free projective connection and a one form, both of which serve to connect the fibers. to define the gauge symmetries of a certain type of theory, one must consider the sections of the corresponding fiber bundle. a local section : m x e or global cross section : m e is a map taking each point p of x or e, respectively, into a unique point of the fiber fp over p.65 for each type of physical theory, a section represents a particular configuration of the corresponding physical field. however, in theories of the gauge - field type, this representation is not unique. there is a group of gauge transformations, each element of which maps one mathematical representative of some field configuration into another representative of the same configuration. a gauge symmetry is an equivalence relation on the set of sections : two sections and are gauge equivalent if there is a gauge transformation taking one into the other. this equivalence relation divides the set of all sections into equivalence classes, the gauge orbits ; each section belongs to one and only one such orbit. if the gauge group of some type of theory consists entirely of fiber - preserving transformations, then the theory can be formulated on a natural bundle. but if its gauge group includes non - fiber - preserving transformations, then a gauge - natural bundle is needed to formulate this type of theory correctly. the field equations of a particular gauge field theory serve to pick out a class of preferred sections consisting of the solutions to these equations. for a gauge theory, these equations must be of such a form that, if one section is a solution, then so are all members of the entire gauge orbit of that section. in other words, the gauge transformations must form a symmetry group of the field equations. this group is the automorphism group aut(p) of the principal gauge - natural bundle p corresponding to the theory (see, e.g., fatibene and francaviglia, 2003, p. 223). fatibene and his collaborators explain the distinction between the two types of theory well : the main technical difference between natural and gauge natural theories is that [base ] diffeomorphisms are completely replaced by gauge transformations. in gauge natural theories spacetime diffeomorphisms do not act at all on fields, since the only action one can define in general is that of gauge transformations. this is due to the fact that although pure gauge transformations are canonically embedded into the group of generalized gauge transformations, there is no canonical horizontal complement to be identified with diff(m). horizontal symmetries, in fact, are generally associated to physically relevant conservation laws, such as energy, momentum and angular momentum. the definition of such quantities is almost trivial in natural theories ; on the contrary, in gauge natural theories pure gauge transformations are easily associated to gauge charges (e.g., the electric charge in electromagnetism), while the absence of horizontal gauge transformations is a problem to be solved to appropriately define energy, momentum and angular momentum. for this reason, in gauge natural theories the dynamical connection plays an extra role in determining horizontal infinitesimal symmetries as the gauge generators which are horizontal with respect to the principal connection (fatibene., 2001, pp. the main technical difference between natural and gauge natural theories is that [base ] diffeomorphisms are completely replaced by gauge transformations. in gauge natural theories spacetime diffeomorphisms do not act at all on fields, since the only action one can define in general is that of gauge transformations. this is due to the fact that although pure gauge transformations are canonically embedded into the group of generalized gauge transformations, there is no canonical horizontal complement to be identified with diff(m). horizontal symmetries, in fact, are generally associated to physically relevant conservation laws, such as energy, momentum and angular momentum. the definition of such quantities is almost trivial in natural theories ; on the contrary, in gauge natural theories pure gauge transformations are easily associated to gauge charges (e.g., the electric charge in electromagnetism), while the absence of horizontal gauge transformations is a problem to be solved to appropriately define energy, momentum and angular momentum. for this reason, in gauge natural theories the dynamical connection plays an extra role in determining horizontal infinitesimal symmetries as the gauge generators which are horizontal with respect to the principal connection (fatibene., 2001, pp. 34). while bundle formulations of the hole argument originally dealt only with natural bundles, lyre (1999) develops a generalized version that can be applied to gauge - natural bundles : the generalized hole argument is motivated and extended from the spacetime hole argument. [it ] rules out fiber bundle substantivalism and, thus, a relationalistic interpretation of the geometry of fiber bundles is favored (lyre, 1999, p. 1). [it ] rules out fiber bundle substantivalism and, thus, a relationalistic interpretation of the geometry of fiber bundles is favored (lyre, 1999, p. 1). healey (2001) also argues that fiber bundle substantivalism is subject to an analogue of the to recapitulate : the choice of a bundle (e, m,) selects a certain type of physical theory but does not picked out a particular theory of that type, nor introduce any space - time structures on e or m. the points of m form a geometry (see section 4.1). as points of the space - time manifold, they have quiddity but they lack haecceity : a priori there is nothing to distinguish one such point from the others. their automorphism group is the diffeomorphism group of m or some appropriate subgroup, such as the unimodular group (see stachel, 2011 ; bradonji and stachel, 2012). for example, a metric - free formulation of electromagnetic theory can be based on a bundle of one - forms. a particular theory is a rule for choosing a preferred class of cross sections of the fiber bundle. this rule generally includes specification of some space - time structures on m. for example, in addition to a bundle of one - forms, source - free maxwell electromagnetic theory, requires the specification of a conformal structure on m. in general relativity, an equivalence class of diffeomorphically - equivalent pseudo - metrics on a four - dimensional manifold,66 often referred to as a four - geometry, is regarded as corresponding to a single inertia - gravitational field. while the fiber space consisting of all four - metrics over a given manifold forms a manifold,67 the space of all four - geometries does not form a simple manifold, but a stratified manifold. that is, it is partitioned into slices, each of which is itself a manifold, consisting of all four - geometries having the same symmetry or isometry group. the largest slice is the manifold of generic geometries having no nontrivial symmetries ; it contains the vast majority of geometries. thence one descends slice by slice down to the slice consisting of all four - geometries having the maximal symmetry group (see stachel, 2009 and section 6.1). the rule specifying the choice of a preferred class of space time structures may or may not include some restriction on diff(m), the maximal possible automorphism group of m. obviously, diffeomorphisms always remain unrestricted in the sense of the trivial identity. a true restriction on the theory arises with the imposition of a finite - parameter lie group as the symmetry group of the class of space - time structures picked out by the rule.68 if there are no such restrictions, the theory is background independent. if the rule includes a lie group involving some functions as well as parameters, the theory is partially background dependent. if the lie group is maximal (ten - parameters in four dimensions), then the theory is totally background dependent. if the rule restricts the preferred class maximally, i.e., to the identity, then the theory specifies an individuating field on the space - time, turning it into an algebra. in this formulation, the symmetry group is included in the rule defining a physical theory, rather than being imposed a priori on the space - time structures defined on m. this change enlarges the class of physical solutions : for example, not fixing the global topology of m allows several possibilities for the global topology associated with a given local metric. but this does not alter the fact that the symmetry group of the space - time structures must be preserved by all such solutions. to what extent the hole argument applies to a non background - independent theory depends on the degree of background dependence that has been imposed (see stachel, 2009) ; but if a theory is background independent, the hole argument certainly applies. on the basis of the analysis developed in the previous sections,69 i shall re - examine some of the issues currently being discussed in the philosophy of science. rather than attempting to cover the vast literature on this subject, the discussion is limited to a few representative samples of what i consider to be the most important trends, and will show that they are converging towards variations around a common denominator. since earman and norton (1987) (see section 3), philosophical discussion of the hole argument has centered largely around the issue of space - time absolutism now often called substantivalism70 versus the opposing viewpoint, usually denominated relationalism or relationism. einstein summarized an earlier version, the age - old controversy over the nature of space : two concepts of space may be contrasted as follows : space as positional quality of the world of material objects;space as container of all material objects. space as positional quality of the world of material objects ; space as container of all material objects. in case (a), (b), a material object can only be conceived as existing in space ; space then appears as a reality which in a certain sense is superior to the material world (foreword to jammer, 1954). relativity theory metamorphosed the object of controversy from space to space - time, and einstein made is his own viewpoint quite clear : on the basis of the general theory of relativity space as opposed to what fills space has no separate existence. if we imagine the gravitational field to be removed, there does not remain a space of the type [of the minkowski space of sr ], but absolutely nothing, not even a topological space [i.e., a manifold ] there is no such thing as an empty space, i.e., a space without field. space - time does not claim existence on its own, but only as a structural quality of the field (einstein, relativity and the problem of space, in einstein, 1952). on the basis of the general theory of relativity space as opposed to what fills space has no separate existence. if we imagine the gravitational field to be removed, there does not remain a space of the type [of the minkowski space of sr ], but absolutely nothing, not even a topological space [i.e., a manifold ] there is no such thing as an empty space, i.e., a space without field. space - time does not claim existence on its own, but only as a structural quality of the field (einstein, relativity and the problem of space, in einstein, 1952). here are a couple of recent statements on the nature of the controversy : substantivalists understand the existence of spacetime in terms of the existence of its pointlike parts, and gloss spatiotemporal relations between material events in terms of the spatiotemporal relations between points at which they occur. relationists will deny that spacetime points enjoy this robust sort of existence, and will accept spatiotemporal relations between events as primitive (belot and earman, 2001, p. 227).a modern - day substantivalist thinks that spacetime is a kind of thing which can, in consistency with the laws of nature, exist independently of material things (ordinary matter, light, and so on) and which is properly described as having its own properties, over and above the properties of any material things that may occupy parts of it (hoefer, 1996).what is space ? what is time ? do they exist independently of the things and processes in them ? or is their existence parasitic on these things and processes ? are they like a canvas onto which an artist paints ; they exist whether or not the artist paints on them ? or are they akin to parenthood ; there is no parenthood until there are parents and children ? that is, is there no space and time until there are things with spatial properties and processes with temporal durations ? the hole argument arose when these questions were asked in the context of modern spacetime physics. in that context, space and time are fused into a single entity, spacetime, and we inquire into its status. one view is that spacetime is a substance, a thing that exists independently of the processes occurring within spacetime. substantivalists understand the existence of spacetime in terms of the existence of its pointlike parts, and gloss spatiotemporal relations between material events in terms of the spatiotemporal relations between points at which they occur. relationists will deny that spacetime points enjoy this robust sort of existence, and will accept spatiotemporal relations between events as primitive (belot and earman, 2001, p. 227). a modern - day substantivalist thinks that spacetime is a kind of thing which can, in consistency with the laws of nature, exist independently of material things (ordinary matter, light, and so on) and which is properly described as having its own properties, over and above the properties of any material things that may occupy parts of it (hoefer, 1996). do they exist independently of the things and processes in them ? or is their existence parasitic on these things and processes ? are they like a canvas onto which an artist paints ; they exist whether or not the artist paints on them ? or are they akin to parenthood ; there is no parenthood until there are parents and children ? that is, is there no space and time until there are things with spatial properties and processes with temporal durations ? the hole argument arose when these questions were asked in the context of modern spacetime physics. in that context, space and time are fused into a single entity, spacetime, and we inquire into its status. one view is that spacetime is a substance, a thing that exists independently of the processes occurring within spacetime. this is spacetime substantivalism (norton, 2011). in the light of the hole argument, i find it more fruitful to frame discussion in terms of two other distinctions, leading to a point of view about space - time distinct from either substantivalism or relationalism as traditionally defined. these are the distinctions between : internal and external relations, and betweenquiddity and haecceity. these concepts are discussed in appendix b and briefly reviewed in section 5.5. when applied to mathematical structures, they lead to succinct discussions of algebraic and geometric structures and the nature of coordinatization in section 5.6, which establishes a correspondence between the two (for a fuller discussion, see section 4.1). these concepts lead to a viewpoint on the nature of space - time that has been given various names, such as structural spacetime realism71 and sophisticated substantivalism (see pooley, 2000, summarized in section 5.3). i have called it dynamic structural realism (see stachel, 2006a), which has several advantages. it avoids use of the words substantivalism and relationalism, fraught with so many unwanted implications ; it places emphasis on diachronic aspects of structure ; and its application is not confined to theories of space - time structure (see stachel, 2005). the fiber bundle approach, motivated in section 3 and treated in more detail in section 4.3, allows a rigorous formulation of this viewpoint in section 5.4. but first, i shall give a brief account of the controversy between relationalists and substantivalists provoked by the hole argument and how it has led a number of participants from each camp to adopt this new viewpoint. rather than attempting a (necessarily superficial) review of the vast philosophical literature on the controversy, i shall focus on an account of the views of one important relationalist and one important substantivalist. earman (1989) is a standard reference, so i shall employ its terminology and notation in discussing his views. discussing a modified form of absolutism, he states that the only plausible candidate for the role of supporting the nonrelational structures [of a physical theory ] is the space - time manifold m the only plausible candidate for the role of supporting the nonrelational structures [of a physical theory ] is the space - time manifold m (ibid. manifold substantivalism the view that m is a basic object of predication, he sets out to show that this view lays itself open to leibniz s argument (p. 126). in his formulation of the problem, earman uses the standard pre - bundle approach to theories (see sections 3.2 and 4.3) : a model of a theory consists of the manifold m, together with [geometric ] object fields on m, which he denotes by ai and pj, characterizing respectively the space - time structure and the physical contents of space - time. symbolically, = m, ai, pj, with i and j each running through a finite sequence of integers. a manifold diffeomorphism d : m m then results in a different model d = m, d ai, d pj, where d ai, d pj denote the pull backs or the push forwards of ai, pj. it is important to note that earman (1989) defines general covariance in a way that is equivalent to my definition of covariance (see section 4.4) : let us say that the laws of [a theory ] t are generally covariant just in case whenever [is a model of the theory ] then also [is a model ] for any manifold diffeomorphism let us say that the laws of [a theory ] t are generally covariant just in case whenever [is a model of the theory ] then also [is a model ] for any manifold diffeomorphism 175208), earman applies this concept of model to the formulation of general relativity in terms of the metric tensor and its first and second derivatives. thus his ai is restricted to the metric field gik ; while the pj correspond to components of the stress energy tensor tik. he presents a version of einstein s hole argument, involving a diffeomorphism d, such that d = i d outside the hole h but i d inside h and such that the two pieces join smoothly on the boundary the upshot is that we have produced two solutions, m, g, t and m, d g, t, which have identical t fields but different g fields an apparent violation of the kausalgesetz that the t field determines the g field (ibid. d = i d outside the hole h but i d inside h and such that the two pieces join smoothly on the boundary the upshot is that we have produced two solutions, m, g, t and m, d g, t, which have identical t fields but different g fields an apparent violation of the kausalgesetz that the t field determines the g field (ibid. he then presents a version of hilbert s cauchy problem argument (see section 2.7). assuming the existence of a cauchy surface, parameterized by t = 0, he considers : a diffeomorphism d such that d = i d for all t 0 and i d for t > 0 and such that there is a smooth join at t = 0 (ibid. a diffeomorphism d such that d = i d for all t 0 and i d for t > 0 and such that there is a smooth join at t = 0 (ibid. one can then construct two solutions, m, g, t and m, d g, t, that do not differ for t 0 and sharing the same initial data to any finite order of differentiability on t = 0. [this provides ] a seeming violation of the weakest form of laplacian determinism indeed, any nontrivial form of determinism suffers equally (ibid. [this provides ] a seeming violation of the weakest form of laplacian determinism indeed, any nontrivial form of determinism suffers equally (ibid., the discussion of the range of applicability of the hole argument in earman (1989) differs significantly from that in earman and norton (1987), which maintained that the hole argument applied to every classical spacetime field theory [that ] can be formulated as a local space - time theory. they included all special - relativistic field theories, which they maintained were made local by adjoining the riemann tensor rabcd(g) to the set of geometric objects included in any model, and adding the equation rabcd = 0 to the set of field equations defining acceptable special - relativistic models. i must linger a bit longer on earman and norton (1987), because their discussion of general covariance has led to much confusion. they divide geometric object fields o1, on into two classes : o1,,ok1 and ok,,on. the second, dynamical class is assumed to obey field equations 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${o_k } = 0,\quad { o_{k + 1 } } = 0 \ldots, \quad { o_n } = 0,$$\end{document } while the first class may include non - dynamical, fixed background fields.72 when applied to special relativistic field theories, the minkowski metric and the associated flat affine connection are included among these non - dynamical fields. in order to demonstrate that the hole argument applies to all such theories (and in contrast to the definition of general covariance in earman (1989), cited above), earman and norton (1987) prove a gauge theorem (general covariance) : if m, o1,, on is a model of a local spacetime theory and h is a diffeomorphism from m to m, then the carried along n - tuple m, h o1,,h on is also a model of the theory (ibid., p. 520). if m, o1,, on is a model of a local spacetime theory and h is a diffeomorphism from m to m, then the carried along n - tuple m, h o1,,h on is also a model of the theory (ibid.,,on fields of both classes are subject to arbitrary diffeomorphisms, which need not be symmetries of the non - dynamical fields. is essentially the trivial identity (see sections 2.5, 4.2 and 5.7). indeed, we can reformulate the trivial identity in their notation : if m, o1,, on is a model of a local spacetime theory and h is a diffeomorphism from m to m, taking a point p m into a point p m : p p, then the carried along n - tuple is m, h o1,,h on. if we now carry out a coordinate transformation x x, such that x(p) = x(p), i.e., the new coordinates of the new point equal the old coordinates of the old point ; then [h oi] = oi, i.e, the new components of the new geometric objects are numerically equal to the old components of the old objects, then clearly nothing has changed. so it is not clear why the authors feel any : need to establish that the vanishing of the field equations ok = 0,ok+1 = 0,on = 0 is preserved under a diffeomorphism (ibid., p. 520). need to establish that the vanishing of the field equations ok = 0,ok+1 = 0,on = 0 is preserved under a diffeomorphism (ibid., p. 520). while earman (1989) avoids this confusion by silently renouncing this position, much of the later literature on the hole argument still falls into this error. earman now argues that one can not simply take a special relativistic theory of motion and rewrite the equations using covariant derivatives with respect to an undetermined lorentz metric g. then write the field equation for g, namely, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{jkl}^i(g) = 0$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{jkl}^i$\end{document } is the riemann curvature tensor (earman, 1989, p. 183), and then apply the hole argument to show that the theory is non - deterministic. [t]his distinction corresponds to the distinction between the [geometric ] object fields (ai) that characterize the structure of space - time and those (pj) that characterize the physical contents of space - time (ibid. he then requires that, for any two dynamically possible models of the theory = m, ai, pj and = m,ai, pi there is a diffeomorphism d : m m such that d ai = ai for all i (ibid., p. 184) take a special relativistic theory of motion and rewrite the equations using covariant derivatives with respect to an undetermined lorentz metric g. then write the field equation for g, namely, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{jkl}^i(g) = 0$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{jkl}^i$\end{document } is the riemann curvature tensor (earman, 1989, p. 183), a distinction between absolute and dynamical objects [t]his distinction corresponds to the distinction between the [geometric ] object fields (ai) that characterize the structure of space - time and those (pj) that characterize the physical contents of space - time (ibid., for any two dynamically possible models of the theory = m, ai, pj and = m,ai, pi there is a diffeomorphism d : m m such that d ai = ai for all i (ibid., p. 184) letting m = m, ones sees that the condition d ai = ai singles out those diffeomorphisms d of the manifold that are symmetries of the ai fields.73 without going into further detail (see ibid., p. 184), earman essentially argues that the hole argument does not apply if the symmetry group makes the absolute - space time structures sufficiently rigid. it is also clear (although earman does not make the point) that the trivial identity (earman and norton s gauge theorem) is of no help in an attempt to apply the hole argument to such cases. earman (2004) continues the line of reasoning in earman (1989), but with some further evolution : it emphases from the start the difference between finite - parameter lie symmetry groups, covered by noether s first theorem ; and symmetry groups that are function groups, covered by noether s second theorem. earman (2006) starts off in a way reminiscent of earman and norton (1987) : it will be assumed that the spacetime theories to be discusses have been formulated in such a way that (a) their models have the form (, o1, o2,, on), where is a differentiable manifold and the on are geometric object fields that live on m, and (b) their laws of motion / field equations have the form f(1,2,, k) = 0, where f is some functional and the k are geometric object fields constructed from the on (ibid. it will be assumed that the spacetime theories to be discusses have been formulated in such a way that (a) their models have the form (, o1, o2,, on), where is a differentiable manifold and the on are geometric object fields that live on m, and (b) their laws of motion / field equations have the form f(1,2,, k) = 0, where f is some functional and the k are geometric object fields constructed from the on (ibid., p. 446). the difference is that now he separates the field equations f = 0 from the quantities defining the model, and relaxes the demand that they be tensorial equations. he introduces the concept of gauge symmetry as a transformation, in which : the physical situation is not being changed ; rather different but equivalent descriptions of one and the same physical situation are being generated. the physical situation is not being changed ; rather different but equivalent descriptions of one and the same physical situation are being generated. he then defines substantive general covariance (sgc) : the equations of motion / field equations of the theory display diffeomorphism invariance ; that is, if (,ol, o2,,on) is a solution, then so is (, dol,o2,, on) for any d diff(). and the equations of motion / field equations of the theory display diffeomorphism invariance ; that is, if (,ol, o2,,on) is a solution, then so is (, dol,o2,, on) for any d diff(). and in other words, the two solutions are mathematically distinct descriptions of the same physical solution. formal general covariance is what bergmann (1957) calls weak covariance, trivial general covariance ; and stachel and iftime (2005) call covariance tout court. now corresponds to the stachel and iftime (2005) definition of general covariance but what s in a name?75 the two positions are now substantively the same. the remaining difference is mathematical : rather than using fiber bundles, earman still works with fields on a manifold, so his formalism is still vulnerable to the substantivalists attack. as he noted in another context, formalism generated the problem and formalism is needed to resolve it (earman, 1989, p. 184). or perhaps it would be better to say : if you adopt a certain philosophical stance, you should adopt the formalism best suited to it. pooley describes sophisticated substantivalism succinctly as a combination of anti - haecceitism and realism about spacetime points (pooley, 2006, p. 103). a frequent response [to the argument from leibniz equivalence ] is that one can regard all isomorphic models of general relativity as representing the same physical possibility (leibniz equvalence) and regard spacetime as a basic, substantival and concrete entity.sophisticated substantivalism : isomorphic models and 0 represent the same physical possibility (= l[eibniz ] e[quivalence ]) and spacetime points exist as fundamental entities. le accords with the practice of physicsthe metric (plus manifold) gets its natural interpretation as spacetime and 0 can only be regarded as representing distinct possible worlds if spacetime points have primitive identity. denying that they do is good metaphysics independently of the hole argument (pooley, 2000). sophisticated substantivalism may be compatible with taking seriously physicists concerns, but does it have a coherent motivation ? the obvious thing to be said for the position is that one thereby avoids the indeterminism of the hole argument. a less ad hoc motivation would involve a metaphysics of individual substances that does not sanction haecceitistic differences, perhaps because the individuals are individuated by their numerical distinctness is grounded by their positions in a structure. stachel has recently sought to embed his response to the hole argument in exactly this type of more general framework. i hope enough has been said to indicate the coherence of such a point of view ; it is perhaps a modest structuralism about spacetime points, but it is a far cry from the objectless ontology of the ontic structural realist (pooley, 2006, p. 102). a frequent response [to the argument from leibniz equivalence ] is that one can regard all isomorphic models of general relativity as representing the same physical possibility (leibniz equvalence) and regard spacetime as a basic, substantival and concrete entity. sophisticated substantivalism : isomorphic models and 0 represent the same physical possibility (= l[eibniz ] e[quivalence ]) and spacetime points exist as fundamental entities. le accords with the practice of physicsthe metric (plus manifold) gets its natural interpretation as spacetime and 0 can only be regarded as representing distinct possible worlds if spacetime points have primitive identity. denying that they do is good metaphysics independently of the hole argument (pooley, 2000). sophisticated substantivalism may be compatible with taking seriously physicists concerns, but does it have a coherent motivation ? the obvious thing to be said for the position is that one thereby avoids the indeterminism of the hole argument. a less ad hoc motivation would involve a metaphysics of individual substances that does not sanction haecceitistic differences, perhaps because the individuals are individuated by their numerical distinctness is grounded by their positions in a structure. stachel has recently sought to embed his response to the hole argument in exactly this type of more general framework. i hope enough has been said to indicate the coherence of such a point of view ; it is perhaps a modest structuralism about spacetime points, but it is a far cry from the objectless ontology of the ontic structural realist (pooley, 2006, p. 102). le accords with the practice of physics the metric (plus manifold) gets its natural interpretation as spacetime and 0 can only be regarded as representing distinct possible worlds if spacetime points have primitive identity. denying that they do is good metaphysics independently of the hole argument (pooley, 2000). again, there is sophisticated substantial agreement between pooley s viewpoint and those of earman and stachel (see pooley, 2013, for a more recent account of his position). my earliest discussions of the hole argument were based on a purely relationalist approach to space time, which denied any physical significance to points of the four - dimensional manifold m ; they only became elements of space - time after a metric tensor field was specified. this was largely in response to mathematical formulations of physical field theories in terms of geometric object fields on a given m. if one conceded that the points of this manifold represented elements of space - time, this seemed to hand victory to the absolutists (subsequently metamorphosed into substantivalists). when i realized the full implications of the fiber bundle approach, which allows the definition of m as the quotient of the total manifold of the bundle by the equivalence relation defining the fibration (see section 4.2) ; and of schouten s (1951) observation that, in contrast to mathematical tensor fields, physical tensor fields have physical dimensions ; i came to recognize that the points of m, so defined, do have the physical character of elements of space - time even before the choice of a particular field (cross section of the bundle).76 what they lack is individuality, or haecceity as i put it after adopting teller s (1998) terminology (see section 5.5). this led me to a structuralist account of physical theories, but not the kind of structuralism espoused by ladyman and french (see, e.g., ladyman, 1998 ; french and ladyman, 2003) which they call ontic structural realism ; but which is really a kind of hyper - relationalism.77 stachel (2006a) espouses a form of traditional realism as a philosophical position, and also stresses the priority of processes over states, hence it names this position dynamic structural realism. to summarize the last three sections, starting from various relationalist or substantivalist positions, earman, pooley and stachel have been led to a third position, which earman calls substantive general covariance, pooley calls sophisticated substantivalism, and stachel calls dynamic structural realism ; but all three positions are essentially the same. the major difference is stachel s emphasis on the utility of the fiber bundle approach for the mathematical expression of this position. after this lengthy historical - critical excursus, i shall turn to some philosophical arguments for this approach, starting with the definition of some terms already given in section 4 and appendix b, but repeated here for the benefit of those who did not read that section. a relation is said to be internal if one or more essential properties of the relata78 depend on the relation. it is said to be external if no essential property so depends.79 this distinction is in turn based on the distinction between intrinsic and extrinsic properties of an entity. some of its intrinsic properties serve to characterize what has been variously called the essence, nature or natural kind of the entity ; if any of these essential intrinsic properties depend on its relation(s) to other entities, then these relations are internal. no extrinsic property can depend on an internal relation. whether a relation is internal or external is theory - dependent, and hence may depend on the theoretical level at which the objects are treated. in any physical theory, for example, a set of units must be adopted before a mathematical form can be given to any physical quantity. its numerical expression is actually a relation the ratio of the quantity to its unit. at this level, it is an external relation based on the properties of the quantity and its unit. whether these properties themselves are intrinsic or extrinsic may depend on the theoretical level considered. in the system of units adopted, haecceity refers to those properties of the relata that enable us to individuate entities of the same quiddity. up until the last century, it was assumed that entities of the same quiddity could always be individuated by some of their intrinsic properties, independently of any relations, into which they entered. this is leibniz s principle : the identity of indiscernibles.80 any further individuation due to such relations was supposed to supervene on this basic individuation.81 with the advent of quantum statistics, it was argued that there are entities the elementary particles that have quiddity (any particle with charge e, mass me and spin 1/2 is an electron) but no inherent haecceity (one can not distinguish one electron from another by any intrinsic property). and the refutation of hole argument can be similarly formulated : the points of space - time have quiddity but no inherent haecceity. so theoretical physics led to the introduction of a new category : entities having quiddity but no inherent haecceity. an important example of the utility of this category in mathematics is the fundamental distinction between geometric and algebraic structures. geometry deals with elements that have (the same) quiddity but lack inherent haecceity ; a set of internal relations between these elements then defines a particular geometric structure. the group of permutations of these elements preserving the defining internal relations is the symmetry or automorphism group of the geometry. each geometry has such a group of transformations of its elements, under which all geometrical relations of that geometry remain invariant.82 algebra deals with elements that possess both quiddity and haecceity ; a set of external relations between these elements defines a particular algebraic structure. 83 coordinatization of a geometry by an (appropriate) algebra is the assignment of a unique element of this algebra to each point of the geometry ; one can carry out certain algebraic operations and then give the result a geometric interpretation. coordinate transformations : any coordinatization of a geometry gives each of its elements a haecceity, thus negating their homogeneity. this is restored by negating in turn any individual coordinatization : a group of coordinate transformations between all admissible coordinate systems is introduced. an admissible coordinate transformation is one that corresponds to an element of the automorphism group of the geometry. it follow that each point of the geometry will have every element of the algebra as its coordinate in (at least) one admissible coordinate system. to talk about a principle of relativity only makes sense if one has first defined a frame of reference. one then asserts that the laws of physics take the same form in all members of some class of frames of reference. in special relativity, this class of frames (actually a group in the case) consists of the inertial frames of reference. given the minkowski metric and its associated flat inertial connection, such a frame may be defined by taking any time - like autoparallel (straight) line, and constructing the family of such lines, one through each point of the manifold (i.e., a fibration of the space - time), each of which is parallel to the initial line.84 one may then pick a fiduciary point on each such line, and use the proper time along this world line, counted forwards and backwards starting from that point = 0, to individuate the points along the line. assuming that each line is itself physically individuated (given haecceity) in some way, all the points of the space - time are now individuated. it is customary to choose all the fiduciary points to lie on the same space - like hyperplane orthogonal to the time - like fibration (einstein convention for defining distant simultaneity). then the entire group of inertial frames may be generated from the initial one by the action of the poincar group on the points of that inertial frame. of course, the trivial identity holds : if we move everything together with some diffeomorphism of the manifold, nothing has changed. but given that we move only the world lines with respect to the metric and connection, the poincar group is the automorphism group of the inertial frames. the inertial frames thus form a rigid structure, individuating the points of minkowski space - time, and the hole argument fails, as it will for any finite - parameter lie group. in general relativity, a spatial frame of reference also corresponds to a fibration of the four - dimensional manifold m with the stipulation that, when a metric tensor field g is introduced, the fibration consist of curves with a unit time - like vector field tangent to the fibration : = dx / d.85 we may then define projection operators p along the foliation and p orthogonal to it. the vector field represents the four - velocities of observers in the chosen reference frame and the orthogonal projection of the metric field g represents the instantaneous spatial rest - frame of each observer. again, one may pick a fiduciary point on each time - like world line and use the proper time, forwards and backwards starting from that point, to individuate the points along the line. evolution of any geometric object field along the congruence will be represented by its lie derivatives with respect to,. one will usually pick the fiduciary points so that they fit together smoothly to form a space - like hypersurface that transvects the fibration. now there are two possibilities : holonomic case : if one has chosen a congruence, the tangent field of which has vanishing rotation, there will be a foliation of space - time consisting of a one - parameter family hypersurfaces orthogonal to the fibration. the fiduciary points can be chosen to lie on one hypersurface of the foliation, and the local spatial rest - frames of each observer will fit together to form a global spatial rest - frame ; so that the local spatial rest - frames of each observer fit together to form a one - parameter family of global spatial rest - frames. this is the geometric basis of the traditional approach to the cauchy problem in general relativity (see section 2.7).86 non - holonomic case : but there is no need to impose this requirement. it is customary to introduce a triad of orthonormal space - like vectors ei(i = 1, 2, 3) that, together with, span the tangent space at each point of the manifold. then, the components with respect to this tetrad of any geometric object field, called the physical components by pirani, are assumed to be the physically measurable quantities by an observer in that frame at that point. on the assumption that each curve in the three - parameter fibration is physically individuated (given haecceity) in some way, and that some foliation is introduced to provides the fourth individuating quantity, the hole argument still fails, because a fibration and foliation provide an individuating field (see section 4.4), whether or not the rotation of the foliation vanishes. indeed, one does not even need a foliation : just as in the case of sr, if one hypersurface intersecting all the fibers is chosen as the origin for the proper time on each fiber (i.e., = 0 on this hypersurface) ; then the proper time on each fiber provides the fourth individuating quantity. the transformation from one fibration with associated proper times to another is merely a change of labeling of the individuation.87 this individuation evades the hole argument and allows the formulation of the cauchy problem for the einstein field equations in terms of lie derivatives of the tetrad components of the appropriate quantities with respect to any time - like congruence, holonomic (see stachel, 1969) or non - holonomic (see stachel, 1980).88 thus, the principle of relativity has been extended beyond inertial frames in minkowski spacetime : the laws of any physical theory based on a geometric object field, or indeed the laws governing any particle world - lines introduced into the theory, can be formulated with respect to any reference frame based on any such fibration and the associated proper times. the automorphisms of these reference frames now form a function group, which can be defined by its action on the orthonormal tetrad field (, ei) (i = 1, 2, 3) characterizing some initial frame. at any point x, an element of the group so(1, 3) will take one such tetrad into another (,ei) ; such an element depends on six position - dependent parameters (three rotations and three pseudorotations). since any smooth vector field is holonomic, the resulting field will generate a new fibration. (i.e., diffeomorphisms of the manifold m that are transitive and effective) will take the origin of the first reference frame into the origin of the second.89 in this sense, the general theory does extend the principle of relativity from inertial frames in minkowski space - time to arbitrary orthonormal tetrad frames in pseudo - riemannian space - times, either given a priori (background - dependent theories) or constructed from a solution to the einstein equations (background - independent theories such as general relativity). as noted in section 2.6, in the case of a generic metric (i.e., one having no symmetries), the kretschmann - komar coordinates may be used to individuate the points of space - time. while non - generic metrics constitute a subset of measure zero, all known solutions to the einstein field equations belong to this subset. only the a priori imposition of some fixed, background symmetry group on the pseudo - metric tensor enables construction of such solutions (see, e.g., stephani., 2003). the symmetry group, also called the isometry group of the metric, determines a portion of the metric field non - dynamically ; the remaining portion obeys a reduced set of dynamical einstein equations. one must examine each symmetry group to see how much freedom remains in the class of solutions to these reduced equations ; in particular, whether enough freedom remains for a restricted version of the hole argument to apply to these solutions. the possible isometries of a four - dimensional pseudo - riemannian manifold have been classified : they are characterized by two integers (m, o) (see, e.g., stephani., 2003 ; hall, 2004):91 the dimension m 10 of the isometry group.the dimension o min(4, m) of the highest - dimensional orbits of this group. the dimension o min(4, m) of the highest - dimensional orbits of this group. the two extreme cases are : the maximal symmetry group (m = 10, o = 4). minkowski s - t is the unique ricci - flat space - time in this class. its isometry group is the poincar or inhomogeneous lorentz group, which acts transitively on the entire space - time manifold. the field equations of special - relativistic field theories must be invariant under this group ; they provide the most important physical example of background - dependent theories. at the other extreme (m = 0, o = 0) is the class of generic metrics already mentioned. field theories that are covariant under the group of all diffeomorphisms of the underlying four - dimensional differentiable manifold (see section 4), will include a subclass of the generic metrics among their models. if such a theory is generally covariant, so that all diffeomorphically - related models represent one physical model, it is called a background - independent theory. in between these two extremes lie all models of a background - independent theory that are restricted by the further requirement that they have some fixed non - maximal, non trivial symmetry group. if this symmetry group is a function group, we shall say the theory has a partially - fixed background. as noted above, all known exact models of general relativity fall into this category.92 considerable work has been done on two classes of such models : the mini - superspace cosmological solutions, in which so much symmetry is imposed that only functions of one parameter (the time) are subject to dynamical equations (see, e.g., ashtekar., quantization here resembles more the quantization of a system of particles than of waves, and does not seem likely to shed too much light on the generic case.the midi - superspace (see torre, 1999) solutions, notably the cylindrical wave metrics (see ashtekar and pierri, 1996 ; ashtekar., 1997a, b ; bik, 2000). here, sufficient freedom remains to include both degrees of freedom of the gravitational field. diffeomorphisms of a two - dimensional manifold with pseudo - metric are still possible, so a two - dimensional version of the hole argument can be formulated. the mini - superspace cosmological solutions, in which so much symmetry is imposed that only functions of one parameter (the time) are subject to dynamical equations (see, e.g., ashtekar., quantization here resembles more the quantization of a system of particles than of waves, and does not seem likely to shed too much light on the generic case. the midi - superspace (see torre, 1999) solutions, notably the cylindrical wave metrics (see ashtekar and pierri, 1996 ; ashtekar., 1997a, b ; bik, 2000). here, sufficient freedom remains to include both degrees of freedom of the gravitational field. diffeomorphisms of a two - dimensional manifold with pseudo - metric are still possible, so a two - dimensional version of the hole argument can be formulated. taking advantage of fact that any two - dimensional pseudo - metric is conformally related to two - dimensional minkowski space, one can adopt a coordinate system in which the two degrees of freedom are represented by a pair of scalar fields obeying non - linear, coupled wave equations in this flat space - time (stachel, 1966). in addition to static and stationary fields, the solutions include gravitational radiation fields having both states of polarization. their quantization can be carried out formally as if they were two interacting two - dimensional fields (see kouletsis., 2003). but, of course, the invariance of any results under the remaining diffeomorphism freedom must be carefully examined, as well as possible implications for the generic case. as noted in section 4.3, the space of all four - geometries forms a stratified manifold, partitioned into slices. if a metric in one such slice is then perturbed, unless the perturbation is restricted to lie within the same (or some other) symmetry group, it will move the geometry into the generic slice of the stratified manifold. this observation is often neglected when perturbation - theoretic quantization techniques, developed for special relativistic field theories, are applied to perturbations of the minkowski metric. diffeomorphisms of such perturbations can not be treated as pure gauge transformations on a fixed background minkowki s - t ; they modify the entire causal and inertio - gravitational structure of space - time (see, e.g., doughty, 1990, chapter 21). this seems to be the fundamental reason behind the problems that plague the application of special - relativistic quantization techniques to such perturbations. an important class of solutions to the field equations lacks global symmetries, but does have asymptotic symmetries as infinity is approached in null directions. this results in the possibility of separating kinematics from dynamics, and the asymptotic quantization of such solutions (see komar, 1973, section vi, and ashtekar, 1987). the imposition of certain conditions on the behavior of the weyl (conformal curvature) tensor in the future (past) null limit allows conformal compactification of a large class of space - times (penrose, 1963) by adjoining boundary null hypersurfaces scri plus + (scri minus) to the space - time manifold. both boundaries have a symmetry group that is independent of particular dynamical solutions to the field equations in this class. thus, there is a separation of kinematics and dynamics on, and a quantization based on this asymptotic symmetry group can be carried out in more or less conventional fashion. more or less because the asymptotic symmetry group, the bondi - metzner - sachs (bms) group, is not a finite - parameter lie group, as is the poincar group ; it includes four so - called supertranslation functions, which depend on two angular variables. nevertheless, asymptotic gravitons may be defined as representations of the bms group, independently of how strong the gravitational field may be in the interior region (ashtekar, 1987). consider a natural or gauge - natural fiber bundle involving a geometric object field with base manifold m, and a covariant theory t that picks out a valid class of sections of the -bundle. since the theory is assumed covariant, if is a model of the theory, then so is d, where d is the unique fiber - preserving bundle diffeomorphism corresponding to a base diffeomorphism dm if the bundle is natural ; or to any member of the class of such diffeomorphisms if the bundle is gauge - natural. consider the equation = 0, where is a vector field generating a one - parameter family of base diffeomorphisms. we say that such a family of diffeomorphisms is a symmetry of generated by. the class of all models of the theory is divided into equivalence classes by the following equivalence relation : two models and are equivalent if any that generates a symmetry of also generates a symmetry of and vice versa. if there is more than one generator of the symmetries in such an equivalence class, they form a group under addition with constant coefficients. the poisson bracket of two symmetry generators : [, ] is always a generator of a symmetry ; so the generators form a lie algebra.93 if we assume the theory based on to be background - independent, i.e., generally covariant, we can impose a further condition on models of the theory : that they all have the symmetries generated by some particular lie sub - algebra. this results in a class of partially background - dependent theories, each based on the background independent theory. there are a number of ways to treat general relativity as a gauge theory (see sections 4.3 and 4.4 ; for a survey that includes some generalizations of general relativity, see hehl., 1995). we shall follow the discussion in trautman (1980) : for me, a gauge theory is any physical theory of a dynamic variable which, at the classical level, may be identified with a connection on a principal bundle. the structure group g of the bundle p is the group of gauge transformations of the first kind ; the group of gauge transformations of the second kind may be identified with a subgroup of the group aut p of all automorphisms of p. in this sense, gravitation is a gauge theory. the basic gauge field is a linear connection (or a connection closely related to the linear connection). in addition to, there is a metric tensor g which plays the role of a higgs field. the most important difference between gravitation and other gauge theories is due to the soldering of the bundle of frames lm to the base manifold m. the bundle lm is constructed in a natural and unique way from m, whereas a noncontractible m may be the base of inequivalent bundles with the same structure group. the soldering form leads to a torsion which has no analogue in nongravitational theories. moreover, it affects the group, which now consists of the automorphisms of lm preserving. this group contains no vertical automorphism other than the identity ; it is isomorphic to the group diff m of all diffeomorphisms of m (ibid., for me, a gauge theory is any physical theory of a dynamic variable which, at the classical level, may be identified with a connection on a principal bundle. the structure group g of the bundle p is the group of gauge transformations of the first kind ; the group of gauge transformations of the second kind may be identified with a subgroup of the group aut p of all automorphisms of p. in this sense, gravitation is a gauge theory. the basic gauge field is a linear connection (or a connection closely related to the linear connection). in addition to, there is a metric tensor g which plays the role of a higgs field. the most important difference between gravitation and other gauge theories is due to the soldering of the bundle of frames lm to the base manifold m. the bundle lm is constructed in a natural and unique way from m, whereas a noncontractible m may be the base of inequivalent bundles with the same structure group. the soldering form leads to a torsion which has no analogue in nongravitational theories. moreover, it affects the group, which now consists of the automorphisms of lm preserving. this group contains no vertical automorphism other than the identity ; it is isomorphic to the group diff m of all diffeomorphisms of m (ibid. theory : in a gauge theory of the yang - mills type over minkowski space - time, the group is isomorphic to the semi - direct product of the poincar group by the group 0 of vertical automorphisms of p. in other words, in the theory of gravitation, the group of pure gauge transformations reduces to the identity ; all elements of correspond to diffeomorphisms of m (ibid. in a gauge theory of the yang - mills type over minkowski space - time, the group is isomorphic to the semi - direct product of the poincar group by the group 0 of vertical automorphisms of p. in other words, in the theory of gravitation, the group of pure gauge transformations reduces to the identity ; all elements of correspond to diffeomorphisms of m (ibid. trautman points out that, even for a given theory, the choice of structure group g is not unique. since space - time m is four - dimensional, if p = lm then g = gl(4, r). but one can equally well take for p the bundle am of affine frames ; in this case g is the affine group. there is a simple correspondence between affine and linear connections which makes it really immaterial whether one works with lm or am. if one assumes as usually one does that and g are compatible, then the structure group of lm or am can be restricted to the lorentz or poincar group, respectively. it is also possible to take, as the underlying bundle for a theory of gravitation, another bundle attached in a natural way to space - time, such as the bundle of projective frames or the first jet extension of lm. the corresponding structure groups are natural extensions of gl(4, r), o(1, 3) or the poincar group (ibid. since space - time m is four - dimensional, if p = lm then g = gl(4, r). but one can equally well take for p the bundle am of affine frames ; in this case g is the affine group. there is a simple correspondence between affine and linear connections which makes it really immaterial whether one works with lm or am. if one assumes as usually one does that and g are compatible, then the structure group of lm or am can be restricted to the lorentz or poincar group, respectively. it is also possible to take, as the underlying bundle for a theory of gravitation, another bundle attached in a natural way to space - time, such as the bundle of projective frames or the first jet extension of lm. the corresponding structure groups are natural extensions of gl(4, r), o(1, 3) or the poincar group (ibid. as indicated earlier (see section 4.2), for all relevant physical theories there are good arguments for considering sl(4, r) as the frame automorphism group, with the unimodular diffeomorphism group as the corresponding structural group (see stachel, 2011 ; bradonji and stachel, 2012). to recapitulate the main results of section 4.1 : the essence of the hole argument is independent of the differentiability or even the continuity properties of a manifold. when one abstracts from these properties,94 a manifold diffeomorphism becomes a permutation of the members of the set ; so one can use fibered sets to formulate the hole argument for permutable theories. the covariance of a theory defined on a fibered manifold any valid model of it is turned into another valid model by a diffeomorphism acting on its base manifold becomes the permutability of a theory defined on a fibered set : a theory is permutable if any valid model of it is turned into another valid model by a permutation of the elements of its base set. the theory is generally permutable if an equivalence class of such mathematically distinct models corresponds to a single model of the theory. consider a system consisting of n so - called identical, or better, indistinguishable elementary particles, sharing a common quiddity. by nature, these particles lack an inherent haecceity ; but in order to formulate a dynamical theory for the system (e.g., in order to write down a lagrangian or hamiltonian for it in non - relativistic quantum mechanics), one needs to enumerate them (i.e., assign a number from 1 to n to each of the particles). this is a discrete example of coordinatization (see section 4.1), and one must undo this individuation by requiring invariance under all possible permutations of the initial enumeration. as discussed in section 4.1 for sets in general, such permutations can be done in either of two ways : active : fix the enumeration and permute the particles ; orpassive : fix the particles and permute the enumeration. active : fix the enumeration and permute the particles ; or passive : fix the particles and permute the enumeration. either way, it is obvious that each of the n particles will have each of the integers from 1 to n assigned to it in some of these permutations. like space - time points, particles of the same natural kind (quiddity) can only be individuated (to the extent that they are) by their position in some relational structure in a theory that is permutable in both the active and passive senses : if some state of affairs is possible for a system that includes n indistinguishable particles, then the state of affairs resulting from the action of any element of the permutation group perm(n) on these n particles must be an identical state of affairs. possible state of affairs in quantum mechanics ? as discussed in the appendix b, one may adopt a synchronic or diachronic approach to such questions. the term state of affairs is often interpreted synchronically, as referring to the instantaneous state of the system (e.g., its state vector or wave function). but quantum theory can only treat open systems (again see the appendix b) ; and its task is diachronic : to compute the probability amplitude for a complete process (or phenomenon in bohr s terminology). in short, only a complete process constitutes a possible state of affairs for a quantum - mechanical system (see stachel, 1997). if the system is generally permutable and a certain value of the probability for such a process is calculated, then the same value must be calculated for any process that results from this one by a permutation of the indistinguishable particles in either the initial act of preparation or (inclusive or) the final act of registration. in order to verify these probabilities, it seems that, for such individuation of an object, a level of structural complexity must be reached, at which it can be uniquely and irreversibly marked in a way that distinguishes it from other objects of the same nature (quiddity). my argument is based on an approach, according to which quantum mechanics does not deal with quantum systems in isolation, but only with processes that such a system can undergo a process (feynman uses process, but bohr uses phenomenon to describe the same thing) starts with the preparation of the system, which then undergoes some interaction(s), and ends with the registration of some result (a measurement). in this approach, a quantum system is defined by certain essential properties (its quiddity) ; but manifests other, non - essential properties (its haecceity) only at the beginning (preparation) and end (registration) of some process. (note that the initially - prepared properties need not be the same as the finally - registered ones.) the basic task of quantum mechanics is to calculate a probability amplitude for the process leading from the initially prepared values to the finally - registered ones. (i assume a maximal preparation and registration the complications of the non - maximal cases are easily handled) (stachel, 2006a). it seems that, for such individuation of an object, a level of structural complexity must be reached, at which it can be uniquely and irreversibly marked in a way that distinguishes it from other objects of the same nature (quiddity). my argument is based on an approach, according to which quantum mechanics does not deal with quantum systems in isolation, but only with processes that such a system can undergo a process (feynman uses process, but bohr uses phenomenon to describe the same thing) starts with the preparation of the system, which then undergoes some interaction(s), and ends with the registration of some result (a measurement). in this approach, a quantum system is defined by certain essential properties (its quiddity) ; but manifests other, non - essential properties (its haecceity) only at the beginning (preparation) and end (registration) of some process. (note that the initially - prepared properties need not be the same as the finally - registered ones.) the basic task of quantum mechanics is to calculate a probability amplitude for the process leading from the initially prepared values to the finally - registered ones. (i assume a maximal preparation and registration the complications of the non - maximal cases are easily handled) (stachel, 2006a). consider a scattering process for a system of indistinguishable particles, for example. the process consists of the transition from some initial free in - state of the particles to some final free out - state of the particles after their interaction with the target producing the scattering. the cross section for this process depends on the choice of initial in- and out - states.95 for a permutable theory, if some value for such a cross section is a possible result, then the same value must result for the processes that arise from separate permutations of the particles in both the in - state and the out - state. then specification of a unique initial preparation for some process (an experiment) could never result in a unique prediction of the outcome of the registration : every possible permutation of the particles in the registration result would be equally probable. conversely, registration of a unique result (an observation) could never produce a unique retrodiction of the preparation that led to this outcome : every possible permutation of the particles in the act of preparation would be equally probable. the way out of this non - uniqueness paradox, of course, is to require that any permutable theory involving indistinguishable particles be generally permutable. the basic entities are elementary particles (fermions) and field quanta (bosons). i reserve the term elementary particles for fermions and field quanta for bosons, although both are treated as field quanta in quantum field theory. i aim thereby to recall the important difference between the two in the classical limit : classical particles for fermions and classical fields for bosons. [i]n the special - relativistic theories, a preparation or registration may involve either gauge - invariant field quantities or particle numbers.at the level of non - relativistic quantum mechanics for a system consisting of a fixed number of particles of the same type, this [difference ] is seen in the need to take into account the bosonic or fermionic nature of the particle in question by the appropriate symmetrization or anti - symmetrization procedure on the product of the one - particle hilbert spaces. at the level of special - relativistic quantum field theory, in which interactions may change particle numbers, it is seen in the notion of field quanta, represented by occupation numbers (arbitrary for bosons, either zero or one for fermions) in the appropriately constructed fock space ; these quanta clearly lack individuality (stachel, 2006a). i reserve the term elementary particles for fermions and field quanta for bosons, although both are treated as field quanta in quantum field theory. i aim thereby to recall the important difference between the two in the classical limit : classical particles for fermions and classical fields for bosons. [i]n the special - relativistic theories, a preparation or registration may involve either gauge - invariant field quantities or particle numbers. at the level of non - relativistic quantum mechanics for a system consisting of a fixed number of particles of the same type, this [difference ] is seen in the need to take into account the bosonic or fermionic nature of the particle in question by the appropriate symmetrization or anti - symmetrization procedure on the product of the one - particle hilbert spaces. at the level of special - relativistic quantum field theory, in which interactions may change particle numbers, it is seen in the notion of field quanta, represented by occupation numbers (arbitrary for bosons, either zero or one for fermions) in the appropriately constructed fock space ; these quanta clearly lack individuality (stachel, 2006a). as discussed in section 4.3, in background - independent theories such as general relativity, an analogous principle of general covariance holds for the points of space - time. this common lack of haecceity suggests that, whatever their nature, the fundamental entities of any theory purporting to explain a deeper physical level should satisfy the principle of maximum permutability. thiemann has pointed out that the concept of a smooth space - time should not have any meaning in a quantum theory of the gravitational field where probing distances beyond the planck length must result in black hole creation which then evaporate in planck time, that is, spacetime should be fundamentally discrete. the fundamental symmetry is probably something else, maybe a combinatorial one, that looks like a diffeomorphism group at large scales (thiemann, 2001). the concept of a smooth space - time should not have any meaning in a quantum theory of the gravitational field where probing distances beyond the planck length must result in black hole creation which then evaporate in planck time, that is, spacetime should be fundamentally discrete. the fundamental symmetry is probably something else, maybe a combinatorial one, that looks like a diffeomorphism group at large scales (thiemann, 2001). it is hard to believe that, having had to renounce intrinsic individuality at the level of field quanta in qft and at the level of events in gr, that it will reemerge as we go to a deeper level, from which both qft and gr will emerge in the appropriate limits. [t]he way to assure the inherent indistinguishability of the fundamental entities of the theory is to require the theory to be formulated in such a way that physical results are invariant under all possible permutations of the basic entities of the same kind the exact content of the principle depends on the nature of the fundamental entities. for theories, such as non - relativistic quantum mechanics, that are based on a finite number of discrete fundamental entities, the permutations will also be finite in number, and maximal permutability becomes invariance under the full symmetric group. for theories such as general relativity, that are based on fundamental entities that are continuously, and even differentiably related to each other, so that they form a differentiable manifold, permutations become diffeomorphisms. for a diffeomorphism of a manifold further extensions to an infinite number of discrete entities or mixed cases of discrete - continuous entities, if needed, are obviously possible (stachel, 2006a). [t]he way to assure the inherent indistinguishability of the fundamental entities of the theory is to require the theory to be formulated in such a way that physical results are invariant under all possible permutations of the basic entities of the same kind the exact content of the principle depends on the nature of the fundamental entities. for theories, such as non - relativistic quantum mechanics, that are based on a finite number of discrete fundamental entities, the permutations will also be finite in number, and maximal permutability becomes invariance under the full symmetric group. for theories such as general relativity, that are based on fundamental entities that are continuously, and even differentiably related to each other, so that they form a differentiable manifold, permutations become diffeomorphisms. for a diffeomorphism of a manifold is nothing but a continuous and differentiable permutation of the points of that manifold. further extensions to an infinite number of discrete entities or mixed cases of discrete - continuous entities, if needed, are obviously possible (stachel, 2006a). current versions of string theory, for example, do not meet this criterion, and it has been suggested that an ultimately satisfactory version of that theory will have to be background independent (see the discussions in stachel, 2006a, b ; greene, 2004). on the other hand, various discretized space - time theories, such as causal set theory, do seem to meet this criterion ; but have other problems (see the discussion in stachel, 2006a). the next section 6.4 deals with a much more modest problem : attempts to quantize the field equations of general relativity based on the application of some variant of the standard techniques for the quantization of field theories. there is a well known tension between the methods of quantum field theory and the nature of general relativity. the methods of quantization of pre - general - relativistic theories are based on the existence of some fixed, background space - time structure(s) with a given symmetry group.96 the space - time structure is needed both for the development of the formalism and equally importantly for its physical interpretation (see dosch., 2005). it provides a fixed kinematical background for the dynamical theory to be quantized : the dynamical equations for particle or fields must be invariant under all automorphisms of the symmetry group. it is a background - independent theory, with no fixed, non - dynamical structures. to recapitulate, its field equations are generally covariant under all differentiable automorphisms (diffeomorphisms) of the underlying manifold, the points of which have no haecceity (and hence are indistinguishable) until the dynamical fields are specified. in a background - independent theory, there is no kinematics independent of the dynamics. this applies to both the homogeneous einstein equations, and to the inhomogeneous einstein equations coupled to the dynamical equations for any non - gravitational fields. and this is still the case if the automorphism group is restricted to the unimodular diffeomorphisms (see section 4.2). however general relativity and (special - relativistic) quantum field theory do share one fundamental feature that often is not sufficiently stressed : the primacy of processes over states. the four - dimensional approach, emphasizing processes in regions of space - time, is basic to both (see, e.g., stachel, 2006a ; reisenberger and rovelli, 2002 ; dewitt, 2003). the ideal approach to quantum gravity would be a diffeomorphism - invariant method of quantization that takes process as primary. however, the most successful approach so far, loop quantum gravity, only goes a certain way in this direction : it singles out a preferred fibration and foliation (see section 5.7) ; and by adopting a (3 + 1) hamiltonian approach and a set of redundant variables subject to constraints on the initial hypersurface, it effects a certain separation between kinematics and dynamics. but given these limitations, it has produced a mathematically rigorous and surprisingly a unique (but see nicolai and peeters, 2007) kinematic hilbert space (see ashtekar, 2010, for a brief review). even in non - relativistic quantum mechanics, the basic goal is to calculate a probability amplitude for a process connecting some initial preparation to some final registration. however, the existence of an absolute time allows one to choose a temporal slice of space - time so thin that it is meaningful to speak of instantaneous measurements of the initial and final states of the system (see micanek and hartle, 1996). but this is not so for measurements in (special - relativistic) quantum field theory, nor in general relativity (see, e.g., bohr and rosenfeld, 1933, 1979 ; bergmann and smith, 1982 ; reisenberger and rovelli, 2002 ; stachel and bradonji, 2013). the breakup of a four - dimensional space - time region into lower - dimensional sub - regions in particular, into a one parameter family of three - dimensional hypersurfaces raises a problem for measurements in both quantum field theory (see bohr and rosenfeld, 1933, 1979 ; dewitt, 2003) and general relativity (see bergmann and smith, 1982). a breakup of a process into the evolution of instantaneous states on a family of space - like hypersurfaces is a useful, perhaps sometimes indispensable, calculational tool. but no fundamental significance should be attached to such breakups, and mathematical results obtained from them should be carefully examined for their physical significance from the four - dimensional, process standpoint (see, e.g., nicolai and peeters, 2007). (see bergmann and smith, 1982), based on the relation between formalism and observation (see reisenberger and rovelli, 2002), sheds light on the physical implications of any formalism. the possibility of the definition of some physically significant quantity within a theoretical framework should coincide with the possibility of its measurement in principle ; i.e., by means of idealized measurement procedures consistent with that theoretical framework. this is true at the classical level, at which any complete classical set of physical observables should be measurable in principle by a single compound procedure.97 this criterion is easily met by unconstrained systems, such as a set of non - relativistic particles interacting via two - body potentials, or a scalar field obeying the classical klein - gordon equation. but delicate problems arise in applying it to constrained dynamical systems, and in particular to gauge field theories, including general relativity (see section 6.2). the problem becomes even more delicate for quantum systems, in which the existence of the quantum of action h is taken into account. the finite value of h precludes the measurement of a complete set of classical observables by a single compound procedure. it becomes important to show that a complete set of quantum observables, as defined by the theory, can indeed be so measured in principle. non - relativistic quantum mechanics and quantum electrodynamics, have been show to meet this criterion ; and it has been employed as a test of proposals for what should be the fundamental physical quantities defined in quantum gravity (see bergmann and smith, 1982 ; amelino - camelia and stachel, 2009). rovelli (2004) and oeckl (2008, 2013) have shown how to define such measurements on the hypersurface bounding a four - dimensional region of space - time, even in a background - independent theory. in field theory, the analog of the data set (x, t, x,t) is the couple [, ], where is a 3d surface bounding a finite spacetime region, and is a field configuration on. the data from a local experiment (measurements, preparation, or just assumptions) must in fact refer to the state of the system on the entire boundary of a finite spacetime region. the field theoretical space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal g}$\end{document } is therefore the space of surfaces and field configurations on. quantum dynamics can be expressed in terms of an [probability ] amplitude w[, ]. following feynman s intuition, we can formally define w[, ] in terms of a sum over bulk field configurations that take the value on the boundary. notice that the dependence of w[, ] on the geometry of codes the spacetime position of the measuring apparatus. in fact, the relative position of the components of the apparatus is determined by their physical distance and the physical time elapsed between measurements, and these data are contained in the metric of. what is happening is that in background - dependent qft we have two kinds of measurements : those that determine the distances of the parts of the apparatus and the time elapsed between measurements, and the actual measurements of the fields dynamical variables. in quantum gravity, instead, distances and time separations are on an equal footing with the dynamical fields. this is the core of the general relativistic revolution, and the key for background - independent qft (rovelli, 2004, p. 23). in field theory, the analog of the data set (x, t, x,t) is the couple [, ], where is a 3d surface bounding a finite spacetime region, and is a field configuration on. the data from a local experiment (measurements, preparation, or just assumptions) must in fact refer to the state of the system on the entire boundary of a finite spacetime region. the field theoretical space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal g}$\end{document } is therefore the space of surfaces and field configurations on. quantum dynamics can be expressed in terms of an [probability ] amplitude w[, ]. following feynman s intuition, we can formally define w[, ] in terms of a sum over bulk field configurations that take the value on the boundary. notice that the dependence of w[, ] on the geometry of codes the spacetime position of the measuring apparatus. in fact, the relative position of the components of the apparatus is determined by their physical distance and the physical time elapsed between measurements, and these data are contained in the metric of. what is happening is that in background - dependent qft we have two kinds of measurements : those that determine the distances of the parts of the apparatus and the time elapsed between measurements, and the actual measurements of the fields dynamical variables. in quantum gravity, instead this is the core of the general relativistic revolution, and the key for background - independent qft (rovelli, 2004, p. 23). in this sense, einstein s hole, as a symbol of process, has reasserted its physical primacy over hilbert s cauchy surface, as a symbol of instantaneous state (see section 2.7). | this is a historical - critical study of the hole argument, concentrating on the interface between historical, philosophical and physical issues. although it includes a review of its history, its primary aim is a discussion of the contemporary implications of the hole argument for physical theories based on dynamical, background - independent space - time structures.the historical review includes einstein s formulations of the hole argument, kretschmann s critique, as well as hilbert s reformulation and darmois formulation of the general - relativistic cauchy problem. the 1970s saw a revival of interest in the hole argument, growing out of attempts to answer the question : why did three years elapse between einstein s adoption of the metric tensor to represent the gravitational field and his adoption of the einstein field equations?the main part presents some modern mathematical versions of the hole argument, including both coordinate - dependent and coordinate - independent definitions of covariance and general covariance ; and the fiber bundle formulation of both natural and gauge natural theories. by abstraction from continuity and differentiability, these formulations can be extended from differentiable manifolds to any set ; and the concepts of permutability and general permutability applied to theories based on relations between the elements of a set, such as elementary particle theories.we are closing with an overview of current discussions of philosophical and physical implications of the hole argument. |
in this multicenter, prospective, open - label, randomized study, we enrolled 375 adult patients admitted to general medicine and surgery services. we recruited patients with a known history of t2d and with a bg before randomization between 140 and 400 mg / dl, a known history of t2d for > 3 months, age between 18 and 80 years, and treatment at home with, any combination of oral antidiabetic agents, or low - dose insulin therapy at a daily dose 0.4 units / kg before admission. on admission, we stopped oral antidiabetic agents, and bg was measured before meals and at bedtime. patients treated with low - dose insulin before admission had the same insulin regimen continued and were invited to participate if bg exceeded 140 mg / dl. we excluded patients with an admission, patients with any bg > 400 mg / dl before randomization or with a history of hyperglycemic crises, patients with hyperglycemia without a known history of diabetes, patients admitted to or expected to require icu admission, patients undergoing cardiac surgery, patients receiving corticosteroid therapy, patients with clinically relevant hepatic disease or impaired renal function (serum creatinine 3.0 mg / dl), patients with a history of diabetic ketoacidosis (19), pregnant patients, and patients with any mental condition rendering them unable to give informed consent. patients were randomized according a 2:2:1 ratio to one of three regimens : a basal - bolus regimen with insulin glargine given once daily and glulisine before meals plus corrective doses of glulisine by sliding scale for bg > 140 mg / dl (lantus and apidra ; sanofi), a basal plus regimen with a daily dose of glargine and corrective doses of glulisine by sliding scale before meals for bg > 140 mg / dl, or ssi for bg > 140 mg / dl (novolin r ; novo nordisk). patients in the basal - bolus group were started at a total daily dose (tdd) of 0.5 units / kg divided with half as insulin glargine once daily and half as insulin glulisine before meals. patients in the basal plus group received 0.25 units / kg of glargine plus corrective doses of glulisine before meals. in patients 70 years of age and those with a serum creatinine 2.0 mg / dl, the starting tdd in the basal - bolus group was reduced to 0.3 units / kg in the basal - bolus, and tdd of glargine was reduced to 0.15 units / kg in the basal plus regimen (10,20). the goal of insulin therapy was to maintain fasting and premeal glucose concentrations between 100 and 140 mg / dl. the doses of insulin were adjusted daily according to protocol (supplementary table 1). during treatment, treatment failure was arbitrarily defined as a mean daily bg level > 240 mg / dl or two consecutive values > 240 mg / dl (9,10). in case of treatment failure, patients in the basal plus and ssi groups were switched to a basal - bolus regimen starting at a tdd of 0.5 units / kg (9,10). this study was conducted at grady memorial hospital, emory university hospital, and the veterans administration medical center in atlanta, georgia ; medical university of south carolina medical center in charleston, south carolina ; and tulane medical center in new orleans, louisiana. the study protocol and consent form were approved by the institutional review board at each participating institution. a research pharmacist at each institution followed a computer - generated randomization table to coordinate the randomization and treatment assignment. all patients were managed for medical and surgical problems by their primary care team, who received a copy of the assigned treatment protocol. the primary outcome of the study was difference in glycemic control, as measured by mean daily bg concentration, among patients treated with basal - bolus, basal plus, and ssi regimens (fig. secondary outcomes included differences between treatment groups in any of the following measures : number of hypoglycemic events (bg 200 mg / dl) after the first day of treatment, tdd of insulin, hospital stay, and hospital complications. differences in glycemic control in medical and surgical patients with t2d treated with basal - bolus () and basal plus () regimens. this noninferiority study design was based on the hypothesis that the difference in mean daily bg between basal plus and basal - bolus would be no greater than 18 mg / dl (1 mmol / l) (9,10). a bg difference of such a magnitude has been reported as clinically insignificant and is typically smaller than the significant treatment effects detected in other superiority trials (9,10). in light of the data from the rabbit 2 (randomized study of basal - bolus insulin therapy in the inpatient management of patients with type 2 diabetes) medical and surgical trials (9,10), it is reasonable to assume that the sd of mean daily bg is bounded by 50 mg / dl. on the basis of two - sample t tests or wilcoxon tests (one - sided, = 0.05), 147 subjects were required for both the basal plus and the basal - bolus groups to ensure 80% power to reject the noninferiority hypothesis, with bonferroni correction applied to adjust for multiple comparisons across four time points. we compared baseline and clinical characteristics and outcomes, such as mean daily bg after day 1, occurrence of hypoglycemia, and occurrence of complications, both among treatment groups and between medical and surgical patients. the comparisons were made with the use of wilcoxon tests (or kruskal - wallis tests) for continuous variables and tests (or fisher exact tests) for discrete variables. multivariate analysis was conducted with a repeated measures linear model, which accounted for within - subject bg correlation through an autoregressive model of order 1 (ar 1) correlation structure. the data are generally presented as mean sd for continuous variables and n (%) for discrete variables. the primary outcome of the study was difference in glycemic control, as measured by mean daily bg concentration, among patients treated with basal - bolus, basal plus, and ssi regimens (fig. secondary outcomes included differences between treatment groups in any of the following measures : number of hypoglycemic events (bg 200 mg / dl) after the first day of treatment, tdd of insulin, hospital stay, and hospital complications. differences in glycemic control in medical and surgical patients with t2d treated with basal - bolus () and basal plus () regimens. this noninferiority study design was based on the hypothesis that the difference in mean daily bg between basal plus and basal - bolus would be no greater than 18 mg / dl (1 mmol / l) (9,10). a bg difference of such a magnitude has been reported as clinically insignificant and is typically smaller than the significant treatment effects detected in other superiority trials (9,10). in light of the data from the rabbit 2 (randomized study of basal - bolus insulin therapy in the inpatient management of patients with type 2 diabetes) medical and surgical trials (9,10), it is reasonable to assume that the sd of mean daily bg is bounded by 50 mg / dl. on the basis of two - sample t tests or wilcoxon tests (one - sided, = 0.05), 147 subjects were required for both the basal plus and the basal - bolus groups to ensure 80% power to reject the noninferiority hypothesis, with bonferroni correction applied to adjust for multiple comparisons across four time points. we compared baseline and clinical characteristics and outcomes, such as mean daily bg after day 1, occurrence of hypoglycemia, and occurrence of complications, both among treatment groups and between medical and surgical patients. the comparisons were made with the use of wilcoxon tests (or kruskal - wallis tests) for continuous variables and tests (or fisher exact tests) for discrete variables. multivariate analysis was conducted with a repeated measures linear model, which accounted for within - subject bg correlation through an autoregressive model of order 1 (ar 1) correlation structure. the data are generally presented as mean sd for continuous variables and n (%) for discrete variables. a total of 375 patients with t2d gave consent (210 medical and 165 surgical patients) ; of them, 22 patients were excluded from further analysis because they received 180 mg / dl were lower in the basal - bolus and basal plus groups than in the ssi group (27, 32, and 38%, respectively) ; however, the difference was not statistically significant (p = 0.11). in addition, ssi resulted in higher number of treatment failures than did the basal plus and basal - bolus regimens (19, 2, and 0%, respectively ; p 5 vs. 5 years), bg concentration at randomization (180 vs. > 180 mg / dl), and admission hba1c (7.5% vs. > 7.5%). in this model, we observed that treatment with ssi versus basal bolus, a longer history of diabetes, a higher bg at randomization, and a higher hba1c were significantly associated with worse glycemic control during the hospital stay. compared with patients with glucose 180 mg / dl, those with a bg > 180 mg / dl had significant higher mean daily glucose after the first day of therapy in the three treatment groups (all p 7.5% had significant higher mean daily glucose : basal bolus, 142 32 vs. 166 35 mg / dl ; basal plus, 147 30 vs. 181 37 mg / dl ; and ssi, 152 26 vs. 197 43 mg / dl (all p 7.5% (33% vs. 7% ; p = 0.007). the tdd of insulin was higher in the basal - bolus group (32.2 16 units / day) than in the basal plus group (27.3 11 units / day ; p 180 mg / dl and hba1c > 7.5% at admission had significant higher mean daily bg and experienced more treatment failures during therapy. this observation has important implications in clinical practice and indicates that t2d patients admitted with poor glycemic control (bg > 180 mg / dl or hba1c > 7.5%) should be treated with basal - bolus or basal plus regimens, and the use of ssi alone should be avoided because a third of all patients will continue to have significant hyperglycemia, which may increase the risk of hospital complications (10). we excluded patients admitted to the icu, those with clinically relevant hepatic disease or with serum creatinine 3.0 mg / dl, patients with severe hyperglycemia, and those receiving a total dose of insulin > 0.4 units / kg / day before admission. for such patients, higher insulin doses or a standard basal - bolus approach may be the preferred approach in achieving glycemic control. in addition, our study was not powered to determine differences in hospital complications across groups. the recent rabbit 2 surgical trial, which included a larger number of general surgical patients, reported a significant reduction in the frequency of hospital complications in patients treated with a basal - bolus regimen compared with ssi treatment (10). finally, in this study we observed a lower rate of bg within target range compared with previous randomized inpatient trials, in which we observed 55% of bg within goal in patients treated with a basal - bolus regimen (9,10). in this trial, we followed a similar starting insulin dose and escalation protocol, increasing insulin dose by 10% if bg was between 140 and 180 mg / dl and by 20% if bg was > 200 mg / dl. it is possible that increasing the starting insulin dose and increasing insulin titration will result in better glycemic control ; however, it may also increase the risk of hypoglycemic events (28). in summary, most general medical and surgical patients with t2d treated with diet, oral antidiabetic agents, or low - dose insulin (0.4 units / day) can be managed with a single daily dose of basal insulin with supplemental (corrective) doses of rapid - acting insulin analogs before meals. this basal plus regimen resulted in improvements in glycemic control and frequency of hypoglycemic events similar to those seen with a standard basal - bolus insulin regimen, and both regimens resulted in better glycemic control and in fewer treatment failures than did the use of ssi alone. these results indicate that basal insulin is necessary for stable glucose control in the hospital and that the basal plus approach is an effective alternative to the basal - bolus regimen for the initial management of hyperglycemia in general medical and surgical patients with t2d. | objectiveeffective and easily implemented insulin regimens are needed to facilitate hospital glycemic control in general medical and surgical patients with type 2 diabetes (t2d).research design and methodsthis multicenter trial randomized 375 patients with t2d treated with diet, oral antidiabetic agents, or low - dose insulin (0.4 units / kg / day) to receive a basal - bolus regimen with glargine once daily and glulisine before meals, a basal plus regimen with glargine once daily and supplemental doses of glulisine, and sliding scale regular insulin (ssi).resultsimprovement in mean daily blood glucose (bg) after the first day of therapy was similar between basal - bolus and basal plus groups (p = 0.16), and both regimens resulted in a lower mean daily bg than did ssi (p = 0.04). in addition, treatment with basal - bolus and basal plus regimens resulted in less treatment failure (defined as > 2 consecutive bg > 240 mg / dl or a mean daily bg > 240 mg / dl) than did treatment with ssi (0 vs. 2 vs. 19%, respectively ; p < 0.001). a bg < 70 mg / dl occurred in 16% of patients in the basal - bolus group, 13% in the basal plus group, and 3% in the ssi group (p = 0.02). there was no difference among the groups in the frequency of severe hypoglycemia (< 40 mg / dl ; p = 0.76).conclusionsthe use of a basal plus regimen with glargine once daily plus corrective doses with glulisine insulin before meals resulted in glycemic control similar to a standard basal - bolus regimen. the basal plus approach is an effective alternative to the use of a basal - bolus regimen in general medical and surgical patients with t2d. |
darier disease (dd) is a rare, autosomal dominant, inherited acantholytic dermatosis caused by a mutation in the atp2a2 gene on chromosome 12. the diagnosis of dd is suggested by the clinical findings of a persistent eruption of greasy hyperkeratotic papules and plaques usually occurring over seborrheic areas as well as distinctive nail abnormalities. numerous treatments such as topical corticosteroids, topical retinoids, topical 5-fluorouracil, diclofenac sodium 3% gel, dermabrasion and laser therapy have reportedly been used for the treatment of dd, with limited success. systemic retinoids, including acitretin, isotretinoin and alitretinoin, are the most effective treatment for dd. however, their use is limited by potential deleterious side effects. considering the recently reported efficacy of doxycycline for hailey - hailey disease, an inherited acantholytic skin disorder pathogenetically similar to dd, we report the case of a patient with extensive dd who showed a dramatic response to oral doxycycline monotherapy. a 77-year - old patient with a 57-year history of dd was referred to our department for a severe exacerbation of dd that was recalcitrant to conventional topical treatments, including emollients, topical corticosteroids and topical mupirocin ointment. physical examination revealed malodorous eroded hyperkeratotic papules and plaques on the axillae, groin, perineum and lower back (fig. classical nail changes, which included white and red longitudinal bands, were present. the mucosa was not affected. a skin biopsy specimen from a lower back lesion showed characteristic histopathological findings consistent with a diagnosis of dd (fig. 2). a therapeutic regimen of oral doxycycline (100 mg / day) was started. the skin lesions showed marked improvement and were in complete remission after 1 month (fig. 3). the patient was maintained on a half - dose therapy (50 mg / day) and did not experience any relapse within 3 months of follow - up. dd is caused by mutations in the atp2a2 gene that encodes the sarcoplasmic / endoplasmic reticulum calcium atpase 2 (serca2), which is a calcium pump. serca2 plays a central role in cellular calcium homeostasis and actively transports calcium ions from the cytosol into the lumen of the endoplasmic reticulum in order to maintain a low cytoplasmic calcium level. the molecular mechanisms by which specific atp2a2 mutations alter the function of the serca2 protein and cause acantholysis (loss of cell - to - cell adhesion) and dyskeratosis (premature and abnormal keratinization), the histological hallmarks of dd, remain unclear. as a consequence of the loss of serca2 calcium transport, darier keratinocytes display depleted endoplasmic reticulum calcium stores. in addition to their antibiotic potential, tetracyclines and their analogues exhibit anti - inflammatory properties by inhibiting granulocyte chemotaxis and secretion. interestingly, matrix metalloproteinase 9 and its inhibitor, tissue inhibitor of metalloproteinase 1, have been shown to be involved in dd and hailey - hailey disease. tetracyclines bind divalent metal cations and circulate in blood plasma primarily as calcium and magnesium chelators. furthermore, once chelated, tetracyclines can act as ionophores that are capable of transporting bound calcium and magnesium through lipophilic phases, such as cellular membranes, and delivering ions into intracellular compartments. after intracellular incorporation, calcium can act as a secondary messenger and affect pathways, such as those involved in secretory processes, receptor activation or inhibition, cell division and metabolic reactions. it can be hypothesized that these chelation properties can affect the release of calcium from storage organelles such as sarcoplasmic / endoplasmic reticulum and act on the molecular mechanisms involved in the disruption of calcium homeostasis in darier keratinocytes, leading to the clinical symptoms of the disease. due to its non - antibiotic properties and favorable safety profile compared to oral retinoids further studies are warranted to confirm these findings and to assess the efficacy of oral doxycycline in the treatment of dd. | darier disease (dd) is a rare dominantly inherited genodermatosis characterized by loss of intercellular adhesion (acantholysis) and abnormal keratinization. dd is often difficult to manage. numerous treatments have reportedly been used for the treatment of dd, with limited success. systemic retinoids are considered the drug of choice for treating dd. however, their use is limited by potential deleterious side effects. considering the recently reported efficacy of doxycycline for hailey - hailey disease, an inherited acantholytic skin disorder pathogenetically similar to dd, we report the case of a patient with extensive dd who showed a dramatic response to oral doxycycline monotherapy. |
eye diseases such as diabetic retinopathy (dr) and diabetic macular edema (dme) are the most common causes of irreversible vision loss in individuals with diabetes. just in united states alone, health care and associated costs related to eye diseases are estimated at almost $ 500 m. moreover, the prevalent cases of dr are expected to grow exponentially affecting over 300 m people worldwide by 2025. given this scenario, early detection and treatment of dr and dme play a major role in preventing adverse effects such as blindness. dme is characterized as an increase in retinal thickness within 1-disk diameter of the fovea center with or without hard exudates and sometimes associated with cysts. fundus images which have proven to be very useful in revealing most of the eye pathologies [4, 5 ] are not as good as oct images which provide information about cross - sectional retinal morphology. many of the previous works on oct image analysis have focused on the problem of retinal layers segmentation, which is a necessary step for retinal thickness measurements [7, 8 ]. however, few have addressed the specific problem of dme and its associated features detection from oct images. proposed a classification method to distinguish dme, age - related macular degeneration (amd), and normal sd - oct volumes. the oct images are preprocessed by reducing the speckle noise by enhancing the sparsity in a transform - domain and flattening the retinal curvature to reduce the interpatient variations. then, histograms of oriented gradients (hog) are extracted for each slice of a volume and linear support vector machine (svm) is used for classification. on a dataset of 45 patients equally subdivided into the three aforementioned classes, this method leads to a correct classification rate of 100%, 100%, and 86.67% for normal, dme, and amd patients, respectively. the images that have been used in their paper are publicly available but are already preprocessed (i.e., denoised), have different sizes for the oct volumes, and do not offer a huge variability in terms of dme lesions, and some of them, without specifying which, have been excluded for the training phase ; all these reasons prevent us from using this dataset to benchmark our work. proposed a method for oct images classification using the bag - of - words (bow) model. the method starts with the detection and selection of key points in each individual b - scan, by keeping the most salient points corresponding to the top 3% of the vertical gradient values. then, a texton of size 9 9 pixels is extracted around each key point, and principal component analysis (pca) is applied to reduce the dimension of every texton to get a feature vector of size 9. all extracted feature vectors are used to create a codebook using k - means clustering. then, each oct volume is represented in terms of this codebook and is characterized as a histogram that captures the codebook occurrences. these histograms are used as feature vector to train a random forest (rf) with a maximum of 100 trees. the method was used to classify oct volumes between amd and normal cases and achieved an area under the curve (auc) of 0.984 with a dataset of 384 oct volumes. liu. proposed a methodology for detecting macular pathology in oct images using lbp and gradient information as attributes. the method starts by aligning and flattening the images and creating a 3-level multiscale spatial pyramid. the edge and lbp histograms are then extracted from each block of every level of the pyramid. all the obtained histograms are concatenated into a global descriptor whose dimensions are reduced using pca. finally a svm with a radial basis function (rbf) kernel is used as classifier. the method achieved good results in detection oct scan containing different pathologies such as dme or amd, with an auc of 0.93 using a dataset of 326 oct scans. proposed using 2d and 3d lbp features extracted from denoised volumes and dictionary learning using the bow models. in the proposed method the work described in this paper is an extension of our previous work. in this research, beside the comparison of 2d and 3d features, we explore different possible representations of the features and different preprocessing steps for oct data (i.e., aligning, flattening, and denoising). this paper is organized as follows : the proposed framework is explained in section 2, while the experiments and results are discussed through sections 3 and 4. the proposed method, as well as its experimental setup, for oct volume classification is outlined in figure 2. first, the oct volumes are preprocessed as presented in detail in section 2.1. then, lbp and lbp - top features are detected, mapped, and represented as discussed in depth in sections 2.2, 2.3, and 2.4, respectively. this section describes the set of preprocessing techniques which aim at enhancing the oct volume. the influences of these preprocessing methods and their possible combinations are extensively studied in section 3. oct images suffer from speckle noise, like other image modalities such as ultrasound (us). the oct volumes are enhanced by denoising each b - scan (i.e., each (x - z) slice) using the nlm, as shown in figure 3. nlm has been successfully applied to us images to reduce speckle noise and outperforms other common denoising methods. nlm filtering preserves fine structures as well as flat zones, by using all the possible self - predictions that the image can provide rather than local or frequency filters such as gaussian, anisotropic, or wiener filters. however, the oct scans suffer from large type of variations : inclination angles, positioning, and natural curvature of the retina. therefore, these variations have to be taken into account to ensure a consistent characterization of the tissue disposition, regardless of the location in the retina. this invariance can be achieved in different manners : (i) using a rotation invariant descriptor (cf. this latter correction is known as image flattening which theoretically consists of two distinct steps : (i) estimate and fit the curvature of the retinal pigment epithelium (rpe) and (ii) warp the oct volume such that the rpe becomes flat. each b - scan is thresholded using otsu 's method followed by a median filtering to detect the different retina layers (see figures 4(c) and 4(d)). then, a morphological closing and opening is applied to fill the holes and the resulting area is fitted using a second - order polynomial (see figure 4(d)). finally, the scan is warped such that the curve becomes a line as presented in figures 4(e) and 4(f). thus, in addition to the flattening correction, the warped curves of each b - scan are positioned at the same altitude in the z - axis. in this research, we choose to detect simple and efficient lbp texture features with regard to each oct slice and volume. lbp is a texture descriptor based on the signs of the differences of a central pixel with respect to its neighboring pixels. these differences are encoded in terms of binary patterns as follows:(1)lbpp, r=p=0p1sgpgc2p, sx=1if x00otherwise, where gc, gp are the intensities of the central pixel and a given neighbor pixel, respectively, and p is the number of sampling points in the circle of radius r. ojala. further extended the original lbp formulation to achieve rotation invariance at the expense of limiting the texture description to the notion of circular uniformity. referring to the coordinate system defined in figure 3(a), the lbp codes are computed on each (x - z) slice, leading to a set of lbp maps, a map for each (x - z) slice. volume encoding is later proposed by zhao. by computing lbp descriptors in three orthogonal planes, so - called lbp - top. more precisely, the lbp codes are computed considering the (x - z) plane, (x - y) plane, and (y - z) plane, independently. thus, three sets of lbp maps are obtained, one for each orthogonal plane. in this work, we consider rotation invariant and uniform lbp and lbp - top features with various sampling points (i.e., { 8,16,24 }) with respect to different radius (i.e., { 1,2, 3 }). the number of patterns (lbp#pat) in regard to each configuration is reported in table 2. the mapping stage is used to partition the previously computed lbp maps ; for this work, two mapping strategies are defined : (i) global and (ii) local mapping. global mapping extracts the final descriptors from the 2d feature image for lbp and 3d volume for lbp - top. therefore, for a volume with d slices, the global - lbp mapping will lead to the extraction of d elements, while the global - lbp - top represents the whole volume as a single element. the global mapping for 2d images and 3d volume is shown in figures 5(a) and 5(b). local mapping extracts the final descriptors from a set of (m m) 2d patches for lbp and a set of (m m m) subvolumes for lbp - top. given n and n as the total number of 2d patches and 3d subvolumes, respectively, the local - lbp approach provides n d elements, while local - lbp - top provides n elements. the texture descriptor of an oct volume is defined as the concatenation of the lbp histograms with the global mapping. the lbp histograms are extracted from the previously computed lbp maps (see section 2.2). therefore, the lbp - top final descriptor is computed through the concatenation of the lbp histograms of the three orthogonal planes with the final size of 3 lbp#pat. more precisely, an lbp histogram is computed for each set of lbp maps (x - z) plane, (x - y) plane, and (y - z) plane, respectively. similarly, the lbp descriptor is defined through concatenation of the lbp histograms per each (x - z) slice with the final size of d lbp#pat. the concatenation of histograms employed in the low - level representation in conjunction with either global or local mapping can lead to a high - dimensional feature space. for instance, local mapping results in a size of n d lbp#pat for the final lbp descriptor and n lbp#pat for the final lbp - top descriptor, where n and n are the total number of 2d patches and 3d subvolumes, respectively. high - level representation simplifies this high - dimensional feature space into a more discriminant lower space. this model represents the features by creating a codebook or visual dictionary, from the set of low - level features. the set of low - level features are clustered using k - means to create the codebook with k clusters or visual words. after creating the codebook from the training set, the final descriptor is a histogram of size k which represents the codebook occurrences for a given mapping. the last step of our framework consists in the classification of sd - oct volumes as normal or dme. for that matter, five different classifiers are used : (i) k - nearest neighbor (nn), (ii) logistic regression (lr), (iii) random forest (rf), (iv) gradient boosting (gb) [21, 22 ], and (v) support vector machines (svm) [23, 24 ]. a set of three experiments is designed to test the influence of the different blocks of the proposed framework in comparison to our previous work. these experiments are designed as follows : experiment 1 evaluates the effects of number of words used in bow (high - level representation).experiment 2 evaluates the effects of different preprocessing steps and classifiers on high - level representation.experiment 3 evaluates the effects of different preprocessing steps and classifiers on low - level representation.table 4 reports the experiments which have been carried out in as a baseline and outlines the complementary experimentation here proposed. the reminder of this section details the common configuration parameters across the experiments, while the detailed explanations are presented in the following subsections. experiment 1 evaluates the effects of number of words used in bow (high - level representation). experiment 2 evaluates the effects of different preprocessing steps and classifiers on high - level representation. experiment 3 evaluates the effects of different preprocessing steps and classifiers on low - level representation. all the experiments are performed using a private dataset (see section 3.1) and are reported as presented in section 3.2. in all the experiments, lbp and lbp - top features are extracted using both local and global mapping for different sampling points of 8, 16, and 24 for radius of 1, 2, and 3 pixels, respectively. the partitioning for local - mapping is set to (7 7)-pixel patch for 2d lbp and (7 7 7)-pixel subvolume for lbp - top. this dataset was acquired by the singapore eye research institute (seri), using cirrus (carl zeiss meditec, inc., the dataset consists of 32 oct volumes (16 dme and 16 normal cases). all sd - oct images are read and assessed by trained graders and identified as normal or dme cases based on evaluation of retinal thickening, hard exudates, intraretinal cystoid space formation, and subretinal fluid. all the experiments are evaluated in terms of sensitivity (se) and specificity (sp) using the lopo - cv strategy, in line with. se and sp are statistics driven from the confusion matrix as depicted in figure 6. the se evaluates the performance of the classifier with respect to the positive class, while the sp evaluates its performance with respect to negative class. the use of lopo - cv implies that, at each round, a pair of dme - normal volumes is selected for testing while the remaining volumes are used for training. however, lopo - cv strategy has been adopted despite this limitation due to the reduced size of the dataset. this experiment intends to find the optimal number of words and its effect on the different configurations (i.e., preprocessing and feature representation), on the contrary to, where the codebook size was arbitrarily set to k = 32. several preprocessing strategies are used : (i) nlm, (ii) a combination of nlm and flattening (nlm+f), and (iii) a combination of nlm, flattening, and aligning (nlm+f+a). volumes are represented using bow, where the codebook size ranges within k { 10,20,30,, 100,200,, 500, 1000}. finally, the volumes are classified using lr. the choice of this linear classifier avoids the case that the results get boosted by the classifier. in this manner, any improvement would be linked to the preprocessing and the size of the codebook. the usual build of the codebook consists of clustering the samples in the feature space using k - means (see section 2.4). however, this operation is rather computationally expensive and the convergence of the k - means algorithm for all codebook sizes is not granted. nonetheless, nowak. pointed out that randomly generated codebooks can be used at the expense of accuracy. thus, the codebook is randomly generated since the final aim is to assess the influence of the codebook size and not the performance of the framework. for this experiment, the codebook building is carried out using random initialization using k - means++ algorithm, which is usually used as a k - means initialization algorithm. for this experiment, se and sp are complemented with acc and f1 score (see (2)). acc offers an overall sense of the classifier performance, and f1 illustrates the trade - off between se and precision. precision or positive predictive value is a measure of algorithm exactness and is defined as a ratio of true positive over the total predicted positive samples:(2)acc = tp+tntp+tn+fp+fn, f1=2tp2tp+fp+fn.table 6 in appendix shows the results obtained for the optimal dictionary size while the complete set of all acc and f1 graphics can be found at. according to the obtained results, it is observed that the optimum number of words is smaller for local - lbp features in comparison to local - lbp - top and global - lbp, respectively. using lr classifier, the best performances were achieved using local - lbp with 70 words (se and sp of 75.0%) and local - lbp - top with 500 words (se and sp of 75.0% as well). this experiment explores the improvement associated with (i) different preprocessing methods and (ii) using larger range of classifiers (i.e., linear and nonlinear) on the high - level representation. all the preprocessing stages are evaluated (nlm, nlm+f, and nlm+f+a). in this experiment, the codebooks for the bow representation of lbp and lbp - top features are computed using regular k - means algorithm which is initialized using k - means++, where k is chosen according to the findings of experiment 1. finally, the volumes are classified using k - nn, rf, gb, and svm. the k - nn classifier is used in conjunction with the 3 nearest neighbors rule to classify the test set. the rf and gb classifiers are trained using 100 unpruned trees, while svm classifier is trained using an rbf kernel and its parameters c and are optimized through grid - search. complete list of the obtained results from this experiment is shown in table 7 in appendix. despite the fact that highest performances are achieved when nlm+f or nlm+f+a is used, most configurations decline when applied with extra preprocessing stages. this experiment replicates experiment 2 for the case of low - level representation of lbp and lbp - top features extracted using global mapping. the obtained results from this experiment are listed in table 8 in appendix. in this experiment, flattening the b - scan boosts the results of the best performing configuration. however, its effects is not consistent across all the configurations. rf has a better performance by achieving better se (81.2%, 75.0%, and 68.7%), while svm achieves the highest sp (93.7%), see table 8 in the appendix. in terms of classifier, rf has a better performance than the others despite the fact that the highest sp is achieved using svm. table 5 combines the obtained results from section 3 with those reported by lemaitre., while detailing the frameworks configurations. the obtained results indicate that expansion and tuning of our previous framework improve the results. tuning the codebook size, based on the finding of experiment 1, leads to an improvement of 6% in terms of se (see table 5 at lines 7 and 13). furthermore, the fine - tuning of our framework (see section 2) also leads to an improvement of 6% in both se and sp (see table 5 at lines 1 and 13). our framework also outperforms the proposed method of with an improvement of 20% and 36% in terms of se and sp, respectively. note that although the effects of preprocessing are not consistent through all the performances, the best results are achieved with nlm+f and nlm+f+a configurations as preprocessing stages. in general, the configurations presented in experiment 2 outperform the others, in particular the high - level representation of locally mapped features with an svm classifier. focusing on the most desirable radius and sampling point configuration, smaller radius and sampling points are more effective in conjunction with local mapping, while global mapping benefits from larger radius and sampling points. the work presented here addresses automatic classification of sd - oct volumes as normal or dme. in this regard, an extensive study is carried out covering the (i) effects of different preprocessing steps, (ii) influence of different mapping and feature extraction strategies, (iii) impact of the codebook size in bow, and (iv) comparison of different classification strategies. while outperforming the previous studies [10, 12 ], the obtained results in this research showed the impact and importance of optimal codebook size, the potential of 3d features, and high - level representation of 2d features while extracting from local patches. the strengths of svm while being used along with bow approach and rf classifier while being used with global mapping were shown. in terms of preprocessing steps, although the highest performances are achieved while alignment and flattening were used in the preprocessing, it was shown that the effects of these extra steps are not consistent for all the cases and do not guarantee a better performance. the flattening method proposed by liu. flattens roughly the rpe due to the fact that the rpe is not segmented. thus, in order to have a more accurate flattening preprocessing, the rpe layer should be presegmented as proposed by garvin.. in this work, the lbp invariant to rotation was used and the number of patterns encoded is reduced. once the data are flattened, the nonrotation invariant lbp could be studied since this descriptor encodes more patterns. | this paper addresses the problem of automatic classification of spectral domain oct (sd - oct) data for automatic identification of patients with dme versus normal subjects. optical coherence tomography (oct) has been a valuable diagnostic tool for dme, which is among the most common causes of irreversible vision loss in individuals with diabetes. here, a classification framework with five distinctive steps is proposed and we present an extensive study of each step. our method considers combination of various preprocessing steps in conjunction with local binary patterns (lbp) features and different mapping strategies. using linear and nonlinear classifiers, we tested the developed framework on a balanced cohort of 32 patients. experimental results show that the proposed method outperforms the previous studies by achieving a sensitivity (se) and a specificity (sp) of 81.2% and 93.7%, respectively. our study concludes that the 3d features and high - level representation of 2d features using patches achieve the best results. however, the effects of preprocessing are inconsistent with different classifiers and feature configurations. |
global digital pathology networks between pathology laboratories, academic centers and commercial entities have capitalized on whole slide imaging (wsi) technology, the internet and cloud services. at the university of pittsburgh medical center (upmc) we launched a telepathology consultation service with kingmed diagnostics laboratory in guangzhou, china. wsi submitted for consultation resided on the client 's server (hammamatsu ndp.serve) in china. pathology consultants from pittsburgh in the usa used the web portal to securely access these images on the client 's server. workflow (e.g., case triage and transcription) and reporting was incorporated into this web - based application. with the aforementioned information technology client - specific infrastructure, we were able to initially avoid lengthy transfers of large wsi files over the internet. however, after 2 years of practice network latency issues increased that negatively impacted viewing of wsis and hence interpretation of digital consultation cases. reasons for the delay in processing network data were often hard to determine and, therefore, resolve. ping tools identified decreased download and upload speeds for connections to servers in china compared with those located locally. although the current telepathology workload is manageable, the delays experienced by pathologists (lengthy time waiting for images to open, pixelated images when panning and zooming) were frustrating. therefore, the aim of our study was to explore various image file transfer solutions to improve the viewing experience of digital consult cases. to solve ongoing latency issues, two attempts were made to transfer wsi files from china to an image server at upmc in our data center. the first solution employed an open source product (fast data transfer [fdt ], by cern. a command line utility was established to facilitate a batch transfer process of image files [figure 1 ]. aspera uses user datagram protocol (udp), does not involve data compression, and permitted immediate file transfers that did not need to be initiated by a user [figure 2 ]. the aspera software was configured using a hot folder for automated file transfer. with this set up, every time a file was dropped into the folder on the server in china it was immediately transferred over to the upmc server. since aspera can be configured to take up as much of the sender 's bandwidth network pipe as needed, our team set it up to use 80 mbps of the client 's 100 mbps internet connection to transfer files off business hours. once images were successfully transferred to the usa, when consultants logged into the web - based portal to access their digital pathology cases they were offered the option to view digital slides launched either from the server in china or locally in pittsburgh. process steps involved using an open source tool to transfer images process steps involved using aspera to transfer images table 1 compares file sizes, transfer times, and image quality parameters for the two transfer methods used. transferring files with the open source product fdt provided fast transfer speeds of 23 mbps, but suffered from intermittent dropped connections. although these interruptions varied throughout the day, they were most marked at 6 am in pittsburgh (eastern time zone). the image transfers with fdt required 515 min of extra processing time to zip files and involved a batch process. it was also noted that the open source zip utility did not compress files much more than they were already being compressed by the nanozoomer scanner (nanozoomer 2.0-ht, hamamatsu). employing the commercial file transfer software, aspera permitted much faster (75100 mbps) transfer speeds. use of hot folders did not add any extra processing time to the transfers and avoided files having to sit in a queue. in addition, the file sizes being transferred with fdt were larger because the open source software zipped up multiple images (anywhere from 10 to 30 files) into one file, whereas aspera sent over one image file at a time. fdt was used to transfer 8 cases (101 slides) including hematopathology and breast / gynecologic consults. all of these cases comprised large specimens that contained anywhere from 8 - 15 slides, except for 1 biopsy that included 2 slides. the open source software file sizes represent multiple whole slide images batched together, whereas only one file at a time was transmitted with the commercial software successful global telepathology requires dedicated image management, which includes fast and reliable movement of digital data. traditional transmission of data over the internet can be slow due to congestion, degradation, erratic transfer speed, and bottlenecks (e.g., packet loss). attempts in radiology to improve data transmission time between medical imaging systems have been based mainly on image compression. according to the american telemedicine association (ata) clinical guidelines for telepathology, compression technology may be applied with telepathology so long as it does not compromise the image for clinical use. the guidelines state that reversible (lossless) compression may always be used as there is no impact on the image. however, irreversible (lossy) compression may be used to reduce transmission time or storage space only if the resulting quality is sufficient to reliably perform the clinical task. fortunately, prior studies have shown that image compression does not appear to noticeably affect image quality and diagnostic accuracy, at least for store - and - forward telepathology. use of compression methods to improve data transmission time may not always be effective in high - speed networks. newer transmission methods and bulk data transport tools, such as aspera, which use alternative technology (e.g. udp - based application - level protocols vs. traditional tcp - based transport) offer innovative solutions. for example, some investigators were able to improve radiology image transmission time by using parallelism (increased number of parallel network streams) without compression. it is important to be aware, however, that overwhelming a network with too many streams may cause congestion and therefore actually have a negative effect on throughput achieved. despite the fact that transferring files from china to our local server delayed the availability of images for consultants to view by up to 24 h, this measure improved the overall turn - around time of digital consultations because pathologists found it easier to work with locally stored wsis. although we resolved the latency issue impacting our consulting pathologists, the exact reason causing this latency has not been clarified. network latency is not limited to file transfers between china and the usa, but can be an issue anywhere in the world, even within the usa. although biological materials (slides and blocks with human tissue) can not leave china to be sent to our institution, we are not aware of any regulations restricting the movement of images out of china. it is unclear how our telepathology experience was impacted by the golden shield project (i.e. great firewall of china), which is china 's censorship of the internet. using a combination of firewalls and proxy servers at internet gateways china are able to analyze and manipulate internet traffic. nonetheless, transfer of digital files helped us overcome network latency issues, which in turn enhanced the viewing experience for end - user digital consultants. | background : telepathology practice across international borders has become increasingly popular. our telepathology consultation service with a laboratory in china was hampered by latency issues when viewing whole slide images.objective:the aim was to explore data transfer solutions to improve the viewing experience of digital consult cases.methods:whole slide image files residing on a server in china were transferred to our data center in the usa using an open source product (fast data transfer). a faster more automated commercial high speed file transfer software solution (aspera) was also tested.results:transferring files with the open source product provided transfer speeds of 23 mbps, but suffered from intermittent dropped connections. employing commercial software permitted more reliable transmission of digital files with 75100 mbps transfer speeds.conclusions:successful global telepathology requires dedicated image management. transfer of files to local servers by employing high speed data transfer tools helped overcome network latency issues, improved the overall turn - around time of digital consultations, and enhanced the viewing experience for end - user digital consultants. |
blue nevus is an acquired benign melanocytic nevus described as a blue or blue - black, firm papule, nodule or plaque - like lesion occurring on the skin and mucous membranes. malignant blue nevus is rare, and a common blue nevus rarely needs a differential diagnosis from malignant melanoma. although a melanocytic nevus with a satellite lesion is usually suggestive of a peripherally disseminating malignant melanoma, very few cases of blue nevus with satellite lesions have been reported thus far [1, 2, 3, 4, 5, 6 ]. to our knowledge, this is the seventh case of a blue nevus with satellitosis. a healthy 24-year - old japanese man was referred to our hospital with a blue - black skin lesion on his left forearm. as confirmed by the patient, this lesion had developed after he accidentally pricked his forearm with a pencil 13 years earlier. physical examination revealed a 10 7-mm, blue - black nodule with an irregular border, which was accompanied by 12-mm guttate macular satellite lesions (fig. dermoscopic examination showed a homogeneous, blue - white structure in the absence of any other dermoscopic structures (fig. an excisional biopsy was performed with the suspicion of a common blue nevus versus a malignant melanoma. the lesion was excised with a 3-mm margin of surrounding normal skin down to the level of the superficial fascia. we recognized a black - colored restiform structure in the dermis during the operation and added another 3-mm margin. in the histopathological examination of the excised lesion, nevus cells were dispersed in the dermis, and hyperpigmented, spindle - shaped melanocytes infiltrated among the collagen bundles (fig. there were no features suggestive of malignancy, such as cytological atypia, atypical mitoses or necrosis. common blue nevi are usually solitary, blue - black, dome - shaped papules, and are not difficult to diagnose. however, in a very small number of cases [1, 2, 3, 4, 5, 6 ], including our case, a blue nevus can be accompanied by satellite lesions. this is when the physician needs to differentially diagnose it from malignant melanoma, especially the nodular type. under a dermatoscope, a blue nevus usually shows a typical steel - blue, homogeneous coloration generated by the presence of heavily pigmented melanocytes in the dermis, in the absence of any other dermoscopic structure. however, a recent study showed that a wide spectrum of local dermatoscopic features (whitish, scar - like depigmentation, dots / globules, peripheral streaks or vessels) may also be observed in blue nevi. in such cases, clinical and dermatoscopic distinction from a malignant melanoma may be difficult, or impossible, and surgical excision is necessary. kang and chung reported that nevus cells aggregated densely around the blood vessels in the main papule, and also in the satellite lesions, suggesting that nevus cells may spread along the perivascular space to manifest clinically as guttate or linear satellite lesions. in our case, periappendageal and perivascular concentrations of nevus cells were observed in the main papule as well as in the satellite lesions. clinically, we recognized a black - colored restiform structure in the dermis during the operation. these findings suggest that blue nevus cells could infiltrate along the perivascular area in the dermis and form multiple satellite lesions. in conclusion, we suggest that a blue nevus should be considered as a differential diagnosis when a locally disseminating malignant melanoma is suspected. | malignant blue nevus is rare, and a common blue nevus rarely needs a differential diagnosis from malignant melanoma. although a melanocytic nevus with a satellite lesion is usually suggestive of a peripherally disseminating malignant melanoma, very few cases of blue nevus with satellite lesions have been reported thus far. to our knowledge, this is the seventh case of a blue nevus with satellitosis. periappendageal and perivascular concentrations of the nevus cells were observed in the main papule as well as in the satellite lesions. these findings suggest that blue nevus cells could infiltrate along the perivascular area in the dermis and form multiple satellite lesions. blue nevus should be considered as a differential diagnosis when a locally disseminating malignant melanoma is suspected. |
it develops most of the time on the skin ; however, other parts of the body could be involved, including the oral cavities. oral malignant melanoma (omm), unlike cutaneous melanoma, is considered a rare lesion with an incidence of 0.4 - 1.8% of all melanomas and 0.5% of all oral malignancies. the etiology of omm is still not defined, contrarily to cutaneous melanoma for which sunlight and genetic background are considered etiologic factors. the most frequently affected oral sites are the palate and the maxillary gingiva ; other less frequently affected sites include the labial and buccal mucosa as well as the tongue. black africans, japanese, and native americans are more commonly affected than the european population. histologically, melanomas are characterized by proliferation of malignant melanocytes arranged along the basal layer, having the potential for tissue invasion and metastasis. these cells show a considerable pleomorphism, with a wide range of shapes, such as round to spindled, plasmacytoid, clear cell and epithelioid, and have a solid, alveolar, organoid or pagetoid pattern. necrosis, lack of an inflammatory reaction, and not always detectable mitotic activity are other histological features of this disease. the purpose of this report is to describe a case of omm and to emphasize the importance of histological examination. an 80-year - old woman was referred to our clinic for evaluation and treatment of a pigmented oral lesion. intraoral examination showed multiple lesions represented by macules and nodules on the hard and soft palate, superior alveolar mucosa and gingiva (fig. a biopsy, which included the free and attached gingiva and alveolar mucosa was performed and submitted to histopathological analysis. 2a, b) showed sheets and nests of melanocytes, some with round, plasmacytoid or epithelioid morphology in the superficial corium extending to the deep tissues. some of them presented round nuclei of various sizes, occupying nearly the entire cell cytoplasm and prominent nucleoli. 3d) and negative for ae1/ae3 (not shown), confirming the diagnosis of melanoma. the patient refused a complete workup examination (i.e. ct scan, mri, etc.) as well as any type of treatment and died 11 months later. we presented a case of omm which was initially misdiagnosed as a probable racial pigmentation elsewhere 2 years earlier. this diagnosis was made by taking only the clinical aspect into account and without performing histological analyses. acquired and small congenital nevi show a high frequency of braf mutations. in cutaneous melanoma, nevertheless, there is no report in the literature about the potential malignant transformation rate of oral melanocytic nevi or other pigmented lesions. in our case, we can not establish if the melanoma arose de novo or from a preexistent benign pigmented lesion or precursor, since a biopsy was not carried out. therefore, all suspicious lesions presenting an asymmetric, irregular form, with variation in color and diameter should be investigated in order to obtain a definitive diagnosis. the differential diagnosis for pigmented lesions includes tattoos, oral melanotic macule, racial pigmentation, nevi, melanoacanthoma, melanoma and systemic diseases such as polyostotic fibrous dysplasia, neurofibromatosis, multiple endocrine neoplasia syndrome, peutz - jeghers syndrome, and addison 's disease. initially, omm appears as an asymptomatic lesion characterized by a flat macule, or a slightly elevated lesion that may later become elevated and develop signs and symptoms such as swelling, ulceration, bleeding, pain and tooth mobility. according to the literature, swelling is considered the main reason for consultation, followed by bleeding, ill - fitting dentures, pain, increased mobility of teeth and delayed healing of extraction sockets. omm occurs in adults of all ages, but it is most common in the elderly. when studying 187 cases, showed an average age of 67.5 years, and an unusual case of a 9-year - old patient was described by loreno.. there is no agreement in the literature concerning the ratio of the patients ' gender, having no sex predilection or presenting a slight predilection for men or sometimes for women. histologically, the tumor cells show a considerable pleomorphism, with a wide range of shapes and patterns that could mimic other tumors such as lymphomas, poorly differentiated carcinomas, neuroendocrine carcinomas, sarcomas, and germ - cell tumors. in addition, more rarely, the melanomas may be nonpigmented, causing difficulties in clinical diagnosis and are typically diagnosed at a later stage. therefore, the use of immunohistochemical staining is an important tool to help distinguish melanomas from other malignancies. the helpful markers of melanoma diagnosis are s-100, hmb-45, melan - a, and tyrosinase. for this case, we used s-100, hmb-45 and melan - a. s-100 is very sensitive (around 97 - 100%) but not very specific (75 - 87%). some carcinomas, sarcomas (such as rhabdomyosarcomas, leiomyosarcomas, and peripheral nerve sheath tumors), and langerhans cell histiocytosis can be positive for s-100. hmb-45 recognizes a type i membrane - bound melanosomal protein called pmel17 or gp100 protein. the analysis of hmb-45 and ki-67 antibodies can be useful for distinguishing primary cutaneous melanomas from some nevi. ki-67 is a marker of tumor cell proliferation, which is commonly used as a complement to distinguish benign nevi from melanoma. with less than 5% of positive cells, the lesion is considered as benign nevi, while with 13 - 30% of positive cells, it is considered as malignant melanoma. in our case the multi - cytokeratin (ae1/ae3) antibody is reported to be characteristic of epidermal cells. many predictors of prognostics have been proposed in the literature on omm such as age, clinical stage, depth of invasion and vascular invasion. however, because of the rarity of this disease, consensus criteria have not yet been established. according to the microstaging classification by prasad., which is based on the invasion of the tissue within the mucosa, level i is defined as melanoma in situ, level ii as melanoma invading up to the lamina propria and level iii as melanoma with deep tissue invasion. our case was classified as level iii because the malignant melanocytes reached the periosteum. however, there was no invasion of blood vessels and nerve fibers. melanoma has an initial phase named radial or horizontal growth, which is characterized by a laterally spreading growth of malignant melanocytes only within the epithelium, representing the in situ stage. later, in the vertical growth phase, there is an invasion of the underlying tissues. omm is normally diagnosed in the vertical growth pattern, which may explain its very poor prognosis. in this case, the patient refused any type of treatment and died 11 months after the diagnosis. it has been shown that early identification of the melanoma and its treatment by radical surgery comprise the most important treatment strategy. an association with adjuvant radiation can also be performed. in cases of metastatic diseases, chemotherapy should be considered with the aim of improving the survival of patients with head and neck melanoma. due to the deadly behaviour of omm, showing a poor prognosis with a 5-year survival rate of 15%, all focally pigmented lesions and most diffusely pigmented lesions require a biopsy for diagnosis. | oral malignant melanoma (omm) is rare, representing less than 0.5% of all oral malignancies. the most affected sites are the palate and the maxillary gingiva. histological examination is important to establish the diagnosis of any suspicious pigmented lesion in the oral cavity, mainly if a precise clinical diagnosis is not possible. we present one case of omm that was initially diagnosed as a racial pigmentation elsewhere 2 years earlier. clinical examination showed multiple macules and nodules located on the hard and soft palate, gingiva and superior alveolar mucosa. these lesions were painless and presented a color variation going from dark blue to black. histological analysis showed sheets and nests of atypical melanocytes displaying a range of shapes such as plasmacytoid, epithelioid, and round cells, located in the superficial corium extending to the deep tissues. a few tumor cells contained variable amounts of melanin. there was no invasion of blood vessels or nerve fibers. immunohistochemical analysis revealed that the neoplastic cells were positive for hmb-45, melan - a, s-100 and negative for ae1/ae3, confirming the diagnosis of melanoma. the ki-67 labeling index was around 25%. the patient refused any treatment and died 11 months later. |
the skin and lung tissues from sencar mice used as part of the environmental protection agency 's (epa 's) carcinogenesis testing matrix were examined. this study included sencar mice used in three different short - term bioassay protocols in which the skin papilloma assay was used to identify initiators, promoters, and complete carcinogens. also included were the pathology findings from sencar mice used in the combined bioassay in which the skin assay and the lung adenoma assay were conducted simultaneously. the gross and microscopic features of treatment - associated and spontaneous lesions of the skin and lung of the sencar mouse used in these studies are defined and the lesions most commonly observed are described. generally, gross observations and microscopic findings in both the skin and lung tissues were poorly correlated. although there are several definite criteria on which gross interpretations of the various skin and lung lesions can be made, with the exception of pedunculated squamous cell papillomas and the classic squamous cell carcinomas, the various lesion types had a wide variety of clinical presentations that severely compromised the accuracy of gross diagnosis. further, in the case of benign skin neoplasms, malignant transformation of these tumors most often occurred at the base of the lesion and was initially hidden from gross observation. as a result, approximately 50% of the neoplasms interpreted clinically as benign tumors (papillomas and keratoacanthomas) were actually malignant neoplasms. moreover, many lesions determined grossly to be nontumorous were in fact found to be neoplastic when examined microscopically. the sencar mouse was found to be more responsive in the lung adenoma assay than other strains examined with exception of the strain a. although accurate interpretation of the lung lesions in the sencar was compromised by nonneoplastic treatment - associated and/or spontaneous lesions, the feasibility of using the sencar skin and lung as target tissues in two - stage combined carcinogenesis studies merits further consideration.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12.figure 13.figure 14.figure 15.figure 16.figure 17. |
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inflammation represents a complex, nonspecific immune response of the body to pathogens, damaged cells, tissue injury, allergens, toxic compounds, or irritant molecules. it may also escape the original tissue and spread via the circulatory and/or the lymphatic system to other parts of the body, producing a systemic inflammatory response syndrome such as sepsis in case of an infection. chronic inflammation is associated with a simultaneous destruction and healing of the tissue from the inflammatory process and has been linked to a number of pathologies, including cancer, chronic asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, and psoriasis, as well as several types of neurological disorders. the inflammatory response is coordinated by a large range of mediators that form complex regulatory networks. it is mediated by several types of chemokines [13 ], which act through their receptors located at the surface of the cytoplasmic membrane of leukocytes. the production of chemokines is induced by inflammatory stimuli such as bacterial lipopolysaccharide (lps), interleukin (il)-1, or tumor necrosis factor (tnf). finely tuned molecular mechanisms exist that ensure that the immune response required for the defense of the body has a limited duration and does not pass a certain maximum activation level, thus avoiding that, it becomes harmful to the organism. epidemiological studies suggest that as many as 25% of all cancers may be due to chronic inflammation [57 ]. the connection between inflammation and cancer consists of an extrinsic pathway, driven by inflammatory conditions that increase cancer risk, and an intrinsic pathway, driven by genetic alterations that cause inflammation and neoplasia. inflammatory mediators released by cancer - related inflammation induce genetic instability, leading to the accumulation of random genetic alterations in cancer cells. the activation of toll - like receptors (tlrs), a group of pattern recognition receptors functioning as sensors of pathogens and tissue damages, leads to the nuclear translocation of nf-b and the production of cytokines such as tnf, il-1, il-1, il-6, and il-8. however, tlr activation has been shown to accelerate the growth of adoptively transferred tumors [811 ]. accordingly, the stimulation of tlrs leads to increased survival and proliferation of several cell lines [12, 13 ], and the intratumoral injection of listeria monocytogenes induces tlr2 signaling in tumor cells, thus promoting their growth. chemokines also affect several tumor progression pathways, such as leukocyte recruitment and function, cellular proliferation, survival, or senescence, as well as invasion and metastasis and are the targets of a number of anticancer agents. tumor microenvironment contains various inflammatory cell types infiltrating the tumor area in response to inflammatory stimuli, such as macrophages, neutrophils, and mast cells [16, 17 ]. tumor - associated macrophages (tams) are thought to play key roles in the production of various growth factors, angiogenic factors, proteinases, chemokines, and cytokines, through crosstalks with cancer cells and other tumor stromal cells [1820 ]. factors secreted by tams stimulate cell migration / motility, proliferation, survival, angiogenesis, and metastasis, resulting in a dynamic environment that favors the progression of cancer, thus affecting the clinical outcome of malignant tumors. tams have thus been described as obligate partners for tumor - cell migration, invasion and metastasis [21, 22 ]. namely, in a genetic model of breast cancer in macrophage - deficient mice, the tumors developed normally but were unable to form pulmonary metastases in the absence of macrophages. as tumor metastasis is responsible for approximately 90% of all cancer - related deaths, a better understanding of inflammation regulatory mechanisms may potentially allow to optimize the use of anticancer drugs that lower tumor - specific inflammatory response. finally, the transforming growth factor (tgf) regulates the immune response as well as the effects of the immune system on tumor progression or regression in vivo. tgf has been shown to suppress the antitumor activity of t cells, natural killer (nk) cells, neutrophils, monocytes, and macrophages, which together are able to promote or repress tumor progression depending on the cellular context [2527 ]. importantly, tgf1, the most abundant and ubiquitously expressed isoform of tgf, is usually considered a tumor - suppressor, due to its cytostatic activity in epithelia. however, on advanced stages of tumors, tgf1 behaves as a tumor promoter, due to its capability to enhance angiogenesis, epithelial - to - mesenchymal transition, cell motility, and metastasis [2830 ]. micrornas (mirnas) are short noncoding rnas which regulate the translation and/or degradation of target messenger rnas [3133 ]. they have been implicated in the regulation of a number of fundamental processes, including muscle, cardiac, neural, and lymphocyte development, or the regulation of both the innate and adaptative immune responses [34, 35 ]. mirnas originate from primary transcripts (pri - mirnas) converted in the nucleus into precursor mirnas (pre - mirnas) by the rnase iii drosha, associated with dgcr8 to form the small microprocessor complex. pre - mirnas are then exported in the cytoplasm where the mirna hairpin is cleaved by the rnase iii dicer within the risc loading complex. the guide strand, which corresponds to the mature mirna, is then incorporated into the risc complex. mirnas and their transcriptional regulators usually form autoregulatory loops aimed at controlling their respective levels. mirnas participate in many gene regulatory networks whose molecular malfunctions are associated with major pathologies such as cancer or auto - mmune diseases [3840 ]. thus the expression of mir-155 is strongly elevated in several human leukemias and lymphomas (and references therein). transgenic mice with b cells overexpressing mir-155 develop b - cell leukemia, and a sustained expression of mir-155 in hematopoietic stem cells causes a myeloproliferative disorder. on the other side, mir-155 has been implicated in the regulation of myelopoiesis and erythropoiesis, th1 differentiation, b - cell maturation, igg1 production, somatic hypermutations, gene conversion, class switch recombination, and b- and t - cell homeostasis as well as in the regulation of the innate immune response. thus, mir-155 levels increase following lps treatment of raw-264 macrophages, and mir-155 transgenic mice show enhanced sensibility to lps - induced endotoxin shock. in contrast, mir-155-knock - out mice are unable to mount a proper t - cell or b - cell immune response. the expression of another mirna, mir-125b, was repressed within 1 hour of lps challenge in raw-264 cells. in topic eczema, mir-125b expression was reduced in regions of the skin that were inflamed, while that of mir-21 was enhanced. similarly, mir-21 expression was increased by inflammation due to ulcerative colitis and also by il-13 and by specific antigens in ova- and aspergillus fumigatus antigens - induced asthma models. the expression of mir-21 changes dynamically during antigen - induced t - cell differentiation, with the highest levels of expression in the effector t cell. mir-21 induction upon t - cell receptor (tcr) stimulation is believed to be involved in a negative feedback loop regulating tcr signaling. mir-663 has drown recent attention due to its role not only as an anti - inflammatory mirna but also as a tumor suppressor mirna. in addition, the expression of this microrna is lost in certain cancers such as gastric or pancreatic cancer, and it induces mitotic catastrophe growth arrest when its expression is restored in these cells. mirnas participate in many gene regulatory networks whose molecular malfunctions are associated with cancers [35, 41 ]. depending on the effects of their downregulation or overexpression, mirnas have been described either as oncogenic (onco - mirs) or tumor suppressors. thus, the mir-17 - 92 cluster on chromosome 13, which contains six mirnas (mir-17, -18a, -19a, -20a, -19b-1, and -92a-1), is amplified and overexpressed in b - cell lymphomas and solid tumors such as breast or small - cell lung cancers, where it may enhance oncogenesis by potentially targeting e2f1, p21/cdkn1a, and bcl2l11/bim. on the other hand, loss of function of mir-17 - 92 mirnas might be advantageous for cancer cells in certain settings. namely, loss of heterozygosity at the 13q31.3 locus has been observed in multiple tumor types, and a genome - wide analysis of copy number alterations in cancer revealed that the mir-17 - 92 cluster was deleted in 16.5% of ovarian cancers, 21.9% of breast cancers, and 20% of melanomas. in contrast, the twelve members of the human let-7 gene family are frequently downregulated in cancers like lung, colon, or other solid tumors [34, 35]s and are, therefore, s considered as tumor - suppressor mirnas in these types of cancers. in particular, let-7 mirnas target oncogenes of the ras family and c - myc, and their expression in colon tumors results in reduced levels of both ras and c - myc. mir-21 is overexpressed in several cancers, including colorectal carcinomas (crcs), gliomas, as well as breast, gastric, prostate, pancreas, lung, thyroid, and cervical cancers [53, 54 ]. mir-21 has been shown to function as an onco - mir, due to its targeting of transcripts encoding key regulators of cell proliferation and apoptosis such as pten and pdcd4. beside mir-21, several mirnas are overexpressed in crcs, including mir-17, mir-25, mir-26a, and mir-181a [54, 55 ]. in human, the levels of both mir-155 and bic transcripts (that is, mir-155 primary rnas) are elevated in diffuse large b - cell lymphoma (dlbcl), hodgkins lymphoma, and primary mediastinal b - cell lymphoma. in contrast, a very weak expression of mir-155 is found in most non - hodgkins lymphoma subtypes, including burkitt lymphoma. in addition, high levels of bic and mir-155 expression were reported in b - cell chronic lymphocytic leukemia (b - cll) and in b - cll proliferation centers. furthermore, mir-155 was also upregulated in a subset of patients with acute myelomonocytic leukemia and acute monocytic leukemia. accordingly, transgenic mice whose b cells overexpress mir-155 developed polyclonal preleukemic pre - b - cell proliferation followed by b - cell malignancy. on the other hand, it was reported that bic cooperates with c - myc in avian lymphomagenesis and erythroleukemogenesis. beside liquid malignancies, high levels of mir-155 expression were found in solid tumors such as breast, colon, and lung cancers. mir-155 was recently shown to induce a mutator phenotype by targeting wee-1, a kinase regulating g2/m phase transition during the cell cycle. furthermore, it was shown that mir-155 increases genomic instability by targeting transcripts encoding components of the dna mismatch repair machinery. as a consequence, the simultaneous mir-155-steered suppression of a number of tumor suppressor genes combined with a mutator phenotype might allow the shortening of steps required for tumorogenesis, and might also explain how chronic inflammation associated with high levels of mir-155 induces cancer. for example, it has been established that the downregulation of both mir-103 - 1 and mir-103 - 2 mirnas induces epithelial - to - mesenchymal transition by targeting dicer1 transcripts. furthermore, several mirnas, including mir-21, have been shown to activate metastasis by acting on multiple signaling pathways and targeting various proteins that are involved in this process. thus, in breast cancer, which represents the most common malignancy among women in the world, mirnas such as mir-9, mir-10b, mir-21, mir-103/107, mir-132, mir-373, and mir-520 stimulate metastasis, while mir-7, mir-30, mir-31, mir-126, mir-145, mir-146, mir-200, mir-205, mir-335, mir-661, and mirnas of the let-7 families in contrast impair the different steps of metastatic process, from epithelial - to - mesenchymal transition to local invasion to colonisation and angiogenesis. numerous reports have provided strong evidence that all of the above mirnas potentially target a myriad of transcripts including those encoding transcription factors, cytokines, enzymes and kinases, implicated in both cancer and inflammation. resveratrol (trans-3,4,5-trihydroxystilbene) is a natural polyphenolic, nonflavonoid antioxidant found in grapes and other berries, produced by plants in response to infection by the pathogen botrytis cinerea. recent studies have documented that resveratrol has various health benefits, such as cardiovascular- and cancer - preventive properties [6365 ], and this compound is currently at the stage of preclinical studies for human cancer prevention [66, 67 ]. resveratrol was first shown to inhibit both tumor promotion and tumor progression in a mouse skin cancer model. resveratrol is also tested for preventing and/or treating obesity and diabetes [69, 70 ]. fortunately, resveratrol toxicity is minimal, and even proliferating tissues such as bone marrow or intestinal tract are not adversely affected. both its m - hydroxyquinone and 4-hydroxystyryl moieties have been shown to be important for the determination of resveratrol inhibitory properties toward various enzymes. this include lipoxygenases and cyclooxygenases that synthesize proinflammatory mediators from arachidonic acid, protein kinases such as pkcs and pkd, and receptor tyrosine kinases, lipid kinases, as well as ikk, an activator of nf-b pathway, which establishes a strong link between inflammation and tumorigenesis. also, resveratrol inhibition of p450/cyp19a1/aromatase, by limiting the amount of available estrogens and consequently the activity of estrogen receptors, has been proposed to contribute to the protection against several types of cancer, including breast cancer [72, 73 ]. of note, resveratrol also inhibits the formation of estrogen - dna adducts, which are elevated in women at high risk for breast cancer. it induces growth arrest followed by apoptotic cell death and interferes with cell survival by upregulating the expression of proapoptotic genes while simultaneously downregulating the expression of antiapoptotic genes. resveratrol induces the redistribution of cd95 and other death receptors in lipid rafts, thus contributing to their sensitization to death receptor agonists. it also causes growth arrest at g1 and g1/s phases of cell cycle by inducing the expression of cdk inhibitors p21/cdkn1a and p27/cdkn1b. in addition, resveratrol directly inhibits dna synthesis by diminishing ribonucleotide reductase and dna polymerase [72, 76, 77 ]. altogether, antiproliferative activities of resveratrol involve the differential regulation of multiple cell - cycle targets in a cell - type - dependent manner [72, 75 ]. one of the possible mechanisms for resveratrol protective activities is by downregulation of the inflammatory responses. that includes the inhibition of synthesis and release of proinflammatory mediators, modifications of eicosanoid synthesis, or inhibiting the enzymes, such as cyclooxygenase-1 (cox-1/ptgs1) or -2 (cox-2/ptgs2), which are responsible for the synthesis of proinflammatory mediators, through the inhibitory effect of resveratrol on transcription factors like nf-b or activator protein-1 (ap-1) [78, 79 ]. of note, constitutive cox-2 expression generally predicts aggressiveness of tumors, therefore, the use of nonsteroidal anti - inflammatory drugs that inhibit cox-2 in cancer treatment. this nuclear relocalization of cox-2 is induced by resveratrol, and exposure of resveratrol - treated cells to a specific cox-2 inhibitor blocked resveratrol - induced apoptosis, indicating that cox-2 displays proapoptotic activity in the nucleus, which may be associated with the generation of complexes of cox-2 and erk1/2 mitogen - activated protein kinases. in mouse macrophages, resveratrol also displays antioxidant activity, decreasing the production of reactive oxygen species and reactive nitrogen species and inhibiting nitric oxygen synthetase (nos)-2 and cox-2 synthesis as well as prostaglandin e2 production. furthermore, in 3t3-l1 adipocytes, resveratrol inhibits the production of the tnf - induced monocyte chemoattractant protein (mcp)-1/ccl2. mcp-1 plays an essential role in the early events during macrophage infiltration into adipose - tissue, which results in chronic low - grade inflammation, a key feature of obesity type 2 diabetes characterized by adipose tissue macrophage infiltration and abnormal cytokine production. finally, it is also well established that some anti - inflammatory effects of resveratrol arise from its capability to upregulate histone deacetylase sirtuin 1 (sirt1) activity, that also presents antitumor and anti - inflammatory capabilities. altogether, it is clear that the key antitumor properties of resveratrol [7782 ] are linked to its anti - inflammatory effects. the fact that resveratrol targets, directly or indirectly, so many different factors, exerts such a wide influence on cell homeostasis, and provides such a range of health benefits, suggested that some of its effects should arise from its capability to modulate the activity of global regulators. furthermore, the ability of each mirna to potentially regulate the levels and, therefore, the activity, of tens to hundreds of target genes, strongly suggested that resveratrol should be able to modify the composition of mirna populations. indeed, it has recently been shown that resveratrol decreases the levels of several proinflammatory and/or oncogenic mirnas and upregulates mirnas with anti - inflammatory and/or antitumor potentials [47, 83 ]. affymetrix microarrays and rnase - protection assays showed that resveratrol treatment of human thp-1 monocytic cells upregulated the expression of loc284801 transcripts, that contain the sequence of pre - mir-663 and thus represent mir-663 primary transcripts. interestingly, in silico analysis using targetscan (http://www.targetscan.org/) suggested that mir-663 may potentially target transcripts encoding factors implicated in (i) the mounting of the immune response, especially junb, jund, and fosb, which encode ap-1 factors known to activate many cytokine genes in partnership with nfat factors, (ii) tlr signaling, such as the kinases ripk1 and irak2, and (iii) the differentiation of monocytes, th1 lymphocytes, and granulocytes. an antisense mir-663 inhibitory rna (663-i) proved capable of increasing global ap-1 activity in unchallenged thp-1 cells, showing that mir-663 indeed target transcripts encoding ap-1 factors in these cells. these effects were in particular directed toward junb and jund transcripts. in agreement with previous results, resveratrol blocked the surge of ap-1 activity that occurs following lps challenge due to the fact that junb transcripts peak within the first hour, leading to the accumulation of junb in the next few hours. this inhibitory effect of resveratrol on ap-1 activity was partly impaired by 663-i, indicating that it arises at least in part from the upregulation of mir-663 by resveratrol. western blots showed that resveratrol impaired junb neosynthesis, while still allowing the phosphorylation, that is, the activation of junb following lps treatment to take place, at least to a certain extent. given that ap-1 factors include c - jun, junb, jund, fosb, fra-1, and fra-2, as well as jun dimerization partners jdp1 and jdp2 or the closely related atf2, lrf1/atf3, and b - atf, so that potentially about 18 different dimeric combinations may be formed, the capability of resveratrol to specifically target a subset of ap-1 dimers through the upregulation of mir-663 might have profound effects on the levels of the transcriptional activity of promoters to whom different ap-1 factors can compete for binding. due to the many roles of ap-1 factors both in inflammation and cancer [86, 87 ], the specific targeting of genes encoding a subset of ap-1 factors, by changing the composition of ap-1 dimers on key promoters, may possibly explain some of the multiple anti - inflammatory and anticancer properties of resveratrol. of note accordingly, mir-663 reduced the upregulation of mir-155 by lps, which may be due to mir-663 targeting of transcripts encoding junb and jund and also possibly fosb and ksrp, an rna binding protein implicated in the lps - induced mir-155 maturation from its primary transcripts bic. this is of primary importance, for mir-155 upregulation is the hallmark of inflammatory response following lps treatment of macrophages / monocytes. resveratrol also dramatically impaired the upregulation of mir-155 by lps, an effect partly inhibited by 663-i. altogether, these results indicate that the anti - inflammatory properties of resveratrol arise, at least in part, from its upregulation of mir-663 and its downregulating effects on mir-155 and that mir-663 might possibly qualify as an anti - inflammatory mirna. the results reported here above also suggested that, due to its targeting of ap-1 factors, known to play a role in tumorigenesis and cell invasion [86, 87 ] and due to its downregulation of mir-155, whose levels increase in solid as well as in liquid tumors, mir-663 may also possibly provide resveratrol with some of its anticancer properties. namely, mir-663 was found to be downregulated in hormone refractory prostate cancer cells, along with mir-146a and mir-146b, further supporting the hypothesis that this mirna is a tumor - suppressor gene whose one of the function is to keep low the expression level of oncogenic mir-155 [47, 48 ]. crc is the third most common malignancy and the fourth biggest cause of cancer mortality worldwide [91, 92 ]. despite the increased use of screening strategies such as fecal occult blood testing, sigmoidoscopy, and colonoscopy, more than one - third of patients with colorectal cancer will ultimately develop metastatic disease. on the other hand, the tgf signaling pathway is one of the most commonly altered cellular signaling pathways in human cancers. among the three tgf isoforms expressed in mammalian epithelia (tgf1, tgf2, and tgf3), tgf signaling is initiated by the binding of tgf ligands to type ii receptors (tgfr2). once bound by tgf, tgfr2 recruits, phosphorylates, and thus activates the type i tgf receptor (tgfr1). tgfr1 then phosphorylates two transcriptional regulators, namely, smad2 and smad3, which subsequently bind to smad4. this results in the nuclear translocation of smad complexes, allowing smads to interact with transcription factors controlling the expression of a multitude of tgf responsive genes. the expression of tgf1 in both tumor and plasma was found to be significantly higher in patients with metastatic colorectal cancer, and increasing colorectal tumor stage was correlated with higher tgf1 expression in tumor tissues. mirna microarrays recently showed that resveratrol treatment of sw480 human colon cancer cells significantly increased the levels of 22 mirnas while decreasing those of 26 others. among the mirnas downregulated by resveratrol, mir-17, mir-21, mir-25, mir-92a-2, mir-103 - 1 and mir-103 - 2 have been shown to behave as onco - mirnas, at least in certain contexts. thus, genomic amplification and overexpression of mir-17 - 92 mirnas is found in b - cell lymphomas as well as in breast and lung cancers [34, 54 ]. mir-21 is overexpressed in several cancers, including crcs, gliomas, as well as breast, gastric, prostate, pancreas, lung, thyroid, and cervical cancers [5355 ]. mir-17, mir-25, mir-26-a, and mir-181a are also overexpressed in crcs [34, 54 ]. in addition, several mirnas, including mir-21, have been shown to activate metastasis by acting on multiple signaling pathways and targeting various proteins that are key players in this process. furthermore, the lower metastatic propensity of sw480 cells as compared with sw620 human colon cancer cells, both derived from the primary tumor and a metastasis of the same patient, respectively, was associated with a lower level of expression of mir-103 - 1 and mir-103 - 2, two mirnas that induce epithelial - to - mesenchymal transition by targeting dicer1 transcripts. in silico analysis using targetscan showed that mirnas downregulated by resveratrol in sw480 cells potentially target transcripts encoding known tumor suppressor factors, such as the two antiproliferation factors pdcd4 and pten, the components of the mismatch repair machinery mlh3, msh2 and msh3, dicer1, the rnase iii producing mature mirnas from their immediate precursors in the cytoplasm, and several effectors and regulators of the tgf signaling pathway. indeed, resveratrol treatment of sw480 cells lead to a greater accumulation of tgfr1, tgfr2, pdcd4, pten, and e - cadherin (a component of adherens junctions implicated in the maintenance of epithelial phenotype). of note, among mirnas upregulated by resveratrol, mir-663 was the only one to target tgf1 transcripts. luciferase assays and western blots showed that resveratrol downregulated tgf1 in both a mir-663-dependent and a mir-663-independent manner. resveratrol treatment also decreased the transcriptional activity of smads under tgf1 signaling, an effect seemingly independent of mir-663. interestingly, it has been recently shown that gam / znf512b, a vertebrate - specific developmental regulator first described in chicken, impairs the upregulation of mirnas of the mir-17 - 92 cluster by tgf1 and that tgf1 in turn downregulates gam, at least in part through the upregulation of mir-17 - 92 mirnas. the facts that gam transcripts contain three consensus target sites for mir-663 and that gam is sensible to resveratrol treatment (tili., unpublished results) raises the question of the possible existence of a gene regulatory network that would allow mir-663 to impair gam repressing activity on tgf1 signaling pathway when tgf1 works as a tumor suppressor, that is, at the early stages of tumorigenesis but not any more when this pathway starts to favor tumorigenesis and metastasis, that is, on advanced stages of cancers. it is important to emphasize that tgf1 has been shown to enhance the maturation of oncogenic mir-21 through the binding of smad3 to mir-21 primary rnas and also to increase the expression of mir-155, which is under the control of tgf/smad activity. thus, by targeting ap-1 factors as well as tgf1 and possibly smad3, mir-663 might inhibit two of the pathways that upregulate mir-155 expression. the tgf signaling pathway present multiple levels of regulation : sequestration of ligands into inactive precursor forms, ligand traps, decoy receptors, and inhibitory smads, not to mention the existence of smad - independent pathways and their interactions with many other critical signaling pathways which also proved to play a role in cancer it is thus not surprising that the short (307-nt long) 3-utr of tgf1 transcripts contains a potential consensus target site for 28 mirnas only. of note, tgf1 3-utr contains two target sites for two of these mirnas and only one target site for 25 of the others. therefore, the fact that mir-663 may potentially target 5 different sites in tgf1 3-utr suggests that this mirna could represent a critical tgf1 regulator which may possibly be called upon action in emergency situations such as those when cells begin to proliferate anarchically or when a stronger immune response is required. the multiplicity of mir-663 targets sites in tgf1 3-utr further suggests that the effects of this mirna might be both dose- and context - dependent, so that resveratrol effects on tgf1 signaling pathway might well be also context - dependent. finally, it is probable that, depending on the cell context, resveratrol might either increase the level of tgf signaling by inhibiting mirnas targeting its main effectors when it is beneficial to the organism, that is, when it works to maintain the integrity of epithelia and impair cell proliferation, or in contrast decrease tgf1 signaling by decreasing its production through the upregulation of mir-663when tgf1 starts to favor epithelial - to - mesenchymal transition and metastasis. for example, the targeting of both tgf1 and smad3 transcripts might possibly allow resveratrol to impair tgf1-induced smad3-dependent promotion of cell motility and invasiveness in advanced stages of gastric cancer [101, 102 ] or when smad2 and smad3 phosphorylated at both linker and cooh - terminal regions transmit malignant tgf1 signal in later stages of human crc. it is notable that striking phenotypes are often driven through small changes in the cellular concentration of key factors. for example, in the b - cell compartment, mir-150 curtails the activity of c - myb transcription factor in a dose - dependent fashion over a narrow range of mirna and c - myb concentrations. thus, even slight effects of resveratrol on a handful of key mirnas might well prove to be critical to its anti - inflammatory, anticancer and antimetastatic properties. in addition, the fact that mir-663, mir-21, mir-155, and tgf1 have all been implicated in the regulation of cell proliferation, tumor apparition and development, metastasis formation, and innate immunity, strongly suggests that the capability of resveratrol to behave at the same time as an antitumor, antimetastatic, antiproliferation and anti - inflammatory agent most probably arises from its effects on the expression of a small set of critical endogenous mirnas having the abilities to impact the cell proteome globally. finally, mirnas have the promise to become biomarkers for different stages of cancer, both for diagnosis and prognosis. furthermore, the discovery that resveratrol can modulate the levels of mirnas targeting proinflammatory and/or protumor factors opens the possibility to optimize resveratrol treatments by manipulating in parallel the levels of expression of a few critical mirnas. for example, from the experiments reported here above, it starts to become clear that the use of resveratrol would be especially beneficiary in the type of cancers where the tgf pathway is implicated. of course, resveratrol use would have to be carefully correlated with the stages of cancers, knowing that tgf can have two faces, that is, anti- and prometastatic. as a last remark, it should be noted that while resveratrol antitumor potential has been linked with data primarily from human cell culture systems, evidence that resveratrol can inhibit carcinogenesis in several organ sites emerged from results of cancer prevention and therapy studies in laboratory animal models. given that mir-663 [47, 83 ] come as a warning that studies in animal may not always allow to predict accurately the molecular effects of resveratrol in human, especially when it comes to mirnas. | micrornas are short noncoding rnas that regulate the expression of many target genes posttranscriptionally and are thus implicated in a wide array of cellular and developmental processes. the expression of mir-155 or mir-21 is upregulated during the course of the inflammatory response, but these micrornas are also considered oncogenes due to their upregulation of expression in several types of tumors. furthermore, it is now well established that inflammation is associated with the induction or the aggravation of nearly 25% of cancers. therefore, the above micrornas are thought to link inflammation and cancer. recently, resveratrol (trans-3,4,5-trihydroxystilbene), a natural polyphenol with antioxidant, anti - inflammatory, and anticancer properties, currently at the stage of preclinical studies for human cancer prevention, has been shown to induce the expression of mir-663, a tumor - suppressor and anti - inflammatory microrna, while downregulating mir-155 and mir-21. in this paper we will discuss how the use of resveratrol in therapeutics may benefit from the preanalyses on the status of expression of mir-155 or mir-21 as well as of tgf1. in addition, we will discuss how resveratrol activity might possibly be enhanced by simultaneously manipulating the levels of its key target micrornas, such as mir-663. |
the principal functions of the mucosal epithelial barrier are to digest and absorb food nutrients, as well as to protect the body from dangerous microorganisms (1, 2). our body is not fully equipped to transform all ingested food into its recyclable constituents, and we need the help of beneficial microorganisms for the digestion of complex macromolecules. these microbes, known collectively as the intestinal flora, have the difficult task of cohabiting and controlling each others ' growth. beneficial microorganisms also partially protect against pathogens by competing for metabolites, producing antimicrobial peptides, occupying epithelial / mucous niches, and preventing host pathogen interactions ; thus, it is our interest to tolerate them. at the same time, however, immunity to dangerous microorganisms has to be initiated. three main models have been proposed to account for how the immune system discriminates between and responds appropriately to commensal and pathogenic microorganisms. first, immune cells could discriminate between the two via engagement of different pattern - recognition receptors (prrs) (3, 4). second, ecs could sense the presence of noninvasive (commensal) versus invasive (pathogenic) microorganisms and transmit this information to antigen - presenting cells (apcs), such as dcs. finally, apcs could be programmed to perform different tasks according to their tissue of origin, such that dcs or macrophages that are resident in the gut mucosa and regularly encounter commensal bacteria are tolerogenic, whereas cells that are recruited to the gut in response to pathogenic insult activate the immune response. there is evidence to support each of these possibilities, and it is likely that all three mechanisms are involved in controlling the immune homeostasis of the gut. as the differential recognition of commensals versus pathogens by immune cells remains to be shown, here we will focus on the last two hypotheses. we will discuss the evidence that the dcs of the gut mucosa are predisposed to be tolorogenic, and that this characteristic is the result of their education by gut ecs. unlike immune cells, there is growing evidence that gut ecs can discriminate between pathogens and commensals. in the intestinal mucosa, dcs are found in two locations, the pps and lamina propria (lp). in pps, two important tasks are performed by dcs : uptake of antigen after its transcytosis across the follicle - associated epithelium (fae), which is mediated by immature dcs located largely in the subepithelial dome, and t and b cell activation by mature dcs, which are found in the interfollicular t cell areas (ifrs) (fig. t and b cells activated in the pps are imprinted to home back to the gut due to the unique ability of pp - dcs to induce lymphocytes to up - regulate expression of the intestinal homing integrin 47 and the chemokine receptor ccr9(58) via a retinoic acid furthermore, pp - dcs promote the differentiation of the b cells into immunoglobulin (ig)a - secreting cells (8, 10). gut mucosal immune cells involved in the steady - state and during infection. noninflammatory resident dcs (including the cx3cr1 subtype), which extend processes into the intestinal lumen. these cells allow sampling and presentation of harmless commensal bacteria and may be involved in tolerance induction under homeostatic conditions or in innate defense in response to mucosal pathogens. these cells are generally noninflammatory and drive the development of th2 or t reg cells and b cell class switch recombination (csr)., invasive pathogens can induce the release of inflammatory mediators, including il-8 and ccl20. to initiate protective immunity, ccr6 dcs in the pps are a reservoir of non - educated dcs that can be rapidly recruited during infection. four different subsets of dcs have been described in mouse pps (11, 12). all of them express the dc - specific cell marker cd11c but can be distinguished by their expression of the surface markers cd11b, cd8, ccr6, and cx3cr1. these cell types have specialized functions : cd11bcd8ccr6 dcs activate primarily t helper (th)2 t cells that produce interleukin (il)-10, cd11bcd8ccr6 dcs are localized in the t cell ifr and activate th1-type responses, and cd11bcd8ccr6 dcs are located both in the subepithelial dome and in the ifr, and can also activate th1-type responses (13). recently, resident dcs expressing cx3cr1 (the receptor of cx3cl1, fraktalkine) have been described. they are scattered in the fae but do not seem to participate in the induction of t cell responses (12). two additional dc subsets are found in mesenteric lymph node and are characterized by the differential expression of cd4 and the lectin dec-205 (14), but their functional significance remains to be elucidated. mesenteric lymph node dcs can also be distinguished by the expression of the integrin cd103, a marker of gut origin. cd103 dcs have the unique ability to confer a ccr947 gut - homing phenotype on cd8 effector t cells (15) and th cells (16), and to drive the development of cd4cd25foxp3 t cells via a retinoic acid the mouse lp contains plasmacytoid dcs (pdcs) and two different subsets of myeloid (cd11ccd11b) dcs, which are distinguished by the expression of cd8 (19). both human and mouse lp dcs respond to inflammatory stimuli but can not initiate inflammatory reactions (19, 20). myeloid lp dcs have been shown to take up luminal bacteria by extending dendrites between ecs, from the lp into the intestinal lumen (21). the extension of these processes relies on the expression of cx3cr1 (22) and in the small intestine depends on the presence of commensal bacteria, as antibiotic treatment markedly reduces the number of extensions (23). further, whereas in the jejenum and proximal ileum dc processes are constitutively present, in the terminal ileum they are induced only after encounter with pathogens, such as salmonella (23). this suggests that two different types of dcs can be found in the gut mucosa : those that are resident, which appear to be noninflammatory and tolerogenic, and those that are recruited after infection, which are likely involved in initiating inflammatory responses as discussed below. the natural propensity of mucosal dcs to drive noninflammatory th2-like immune responses does not seem to be an intrinsic property of all myeloid dcs, as cd11bcd11c dcs isolated from pps, but not from the spleen, drive the differentiation of th2 t cells, even after exposure to strong th1-inducing stimuli (24). thus, the noninflammatory phenotype of mucosal dcs must be imparted by local differentiating factors. one source of such factors could be iecs, which are in close contact with lp dcs and have been shown to release molecules that influence the function of apc. the cytokine thymic stromal lymphopoietin (tslp) is one such molecule. under steady - state conditions, tslp is produced by the intestinal epithelium, and together with other ec - derived factors, it conditions dcs to induce noninflammatory th2 responses by inhibiting the production of il-12, a th1-skewing cytokine (20). it has been proposed that steady - state concentrations of tslp contribute to immune tolerance in the intestine by keeping gut dcs in a noninflammatory mode. in support of this idea, tslp was not detected in the iecs of nearly 70% of crohn 's disease patients, suggesting that lack of tslp - mediated control leads to gut inflammation (20). in addition, uncontrolled expression of tslp production by keratinocytes or airway ecs is associated with th2-driven pathologies, such as atopic dermatitis (25, 26) and allergic asthma (27, 28), suggesting that tslp might also regulate immune homeostasis in other tissues. furthermore, ec - derived tslp is involved in amplifying ig class switch by stimulating dcs to produce more b cell activating factor (29). despite its role in promoting antibody- and th2-associated immunity in the periphery, tslp released by thymic ecs tslp is expressed by ecs of the hassall 's corpuscles and activates myeloid dcs in the thymic medulla. these activated dcs may provide strong survival and proliferation signals to self - reactive thymocytes, positively selecting them into the t reg cell lineage (30). we found that human iec - conditioned dcs are able to induce the generation of t reg cells from naive t cells via a tslp - dependent mechanism (unpublished data). the ability of mucosal dcs to drive t reg cells does not seem to be restricted to conventional myeloid dcs, as bilsborough. (31) have described a population of mucosally derived pdcs able to induce the differentiation of th cells with regulatory functions. tgf- is another regulatory cytokine that has a direct effect on gut apcs. in response to local factors, gut lp mast cells and iecs release abundant latent tgf- that binds to lp stroma (32) and undergoes activation by local proteases (33). once activated, tgf- modulates the activity of intestinal macrophages, which lose their ability to secret inflammatory cytokines in response to bacterial stimuli, but retain phagocytic functions (34). tgf-dependent regulatory mechanisms have also been described in the lung, where alveolar ecs control the inflammatory state of alveolar macrophages under homeostatic conditions (36). in addition to its suppressive effect on mucosa - associated macrophages, tgf- is a potent inhibitor of dc activation. tgf-conditioned myeloid dcs appear be tolorogenic rather than immunogenic, as they do not respond to bacterial stimuli and can protect mice from lethal lipopolysaccharide - induced inflammation (37). and there is evidence that iec - produced tgf- may influence the function of mucosal dcs. ec - conditioned dcs produce more tgf-, which could act in an autocrine fashion to suppress their activity (38). in agreement with this, blockade of tgf- synthesis in ec - conditioned dcs results in increased proinflammatory cytokine production in response to ligands of the toll - like receptors (tlrs) finally, prostaglandins (pgs), and in particular pge-2, are also released by ecs and have been shown to modulate inflammation by influencing several levels of immune cell function (39). pge-2 can inhibit the production of th1 cytokines by t cells (40) and of il-12 by dcs, thus driving the development of th2 t cells (41). collectively, these observations suggest that ec - derived factors play a major role in controlling dc function and driving their tolerogenic phenotype, thus contributing to the homeostasis of the gut. if mucosal apcs are programmed to deliver a tolerogenic signal to t cells, how is an accurate and prompt response mounted during pathogen attack ? the discrimination between harmless and potentially harmful stimuli seems to be performed by the epithelium that first encounters luminal bacteria. specifically, compartmentalization of prrs to the basolateral membrane or into the cytosol of ecs may be a key mechanism to prevent responses to the abundant bacterial components in the gut lumen while preserving the ability to detect and mount responses against invasive pathogens that can cross the epithelium. two different families of prrs are expressed in ecs : the tlrs and the nucleotide - binding oligomerization domain (nod) molecules (nod1, nod2, and the recently described ipaf). nod proteins are located intracellularly, whereas tlrs are typically expressed on the cell surface. recently, however, it has been shown that tlr expression is regulated differently in the intestinal epithelium. although still controversial, it has been reported that tlr2, tlr4, and tlr5, receptors for lipoprotein, lipopolysaccharide, and flagellin, respectively, are mostly localized intracellularly and at the basolateral membrane of iecs (42). in this way, only invasive pathogens, which are able to cross the cell monolayer, could activate an inflammatory response. nod proteins are similarly expressed in the intracellular compartment so they can only be activated by ligands that gain access to the cytosol. pathogenic bacteria such as salmonella typhimurium (43, 44) and helicobacter pylori (45) that are equipped with type iii or iv secretion systems can pump nod ligands directly into the cytosol. some invasive bacteria such as listeria and shigella evade the phagosome and are transmitted into the cytosolic compartment of adjacent cells, thereby making these microbes highly prone to nod recognition. consistent with the hypothesis that the altered location of prrs in iecs allows them to distinguish between pathogenic bacteria and noninvasive commensals is the finding that tlr4 and tlr2 signaling from iecs, but not from dcs, is crucial for the extension of dc processes into the intestinal lumen upon s. typhimurium infection (21). an alternative theory to explain the absence of inflammatory responses to commensal bacteria is that low amounts of tlr2 and tlr4 expressed on the apical surface of iecs could drive the development of so - called endotoxin tolerance, a process that confers nonresponsiveness to subsequent exposure to tlr ligands. in addition, commensals could aberrantly activate the nf-b signaling pathway (46, 47). indeed, tlr signaling by commensals is required to preserve the epithelial barrier (48), most likely via activation of nemo (49) and pge-2 production (50). it is also possible that engagement of tlrs on the apical membrane of ecs without simultaneous engagement of intracellular receptors prevents activation of the inflammatory response. differential recognition of pathogenic versus commensal bacteria by iec results in two distinct outcomes (fig. we found that iec - conditioned dcs are blocked irreversibly in their ability to release il-12 and to activate th1 t cells, and can not produce inflammatory mediators even after encounter with pathogenic bacteria such as salmonella (51). in contrast, when invasive or pathogenic bacteria can deliver their prr ligands intracellularly they initiate an inflammatory response in iecs, which then release inflammatory cytokines and chemokines (51). this culminates in the recruitment of non - educated phagocytes, including dcs, which are absolutely required to initiate anti - salmonella specific t cell responses (12). ccl20 (52) or a still undefined chemokine could be involved in the recruitment of ccr6-expressing immature dcs from the pp. salazar - gonzalez. (12) showed that after bacterial infection, ccr6 dcs are rapidly recruited within the fae a few hours after oral salmonella infection and are critical for the initiation of anti - salmonella immune responses. ccr6 dcs are different from local immature dcs, which express cx3cr1, but not ccr6, and are located in close proximity with m cells. collectively, these findings suggest that in the gut it is possible to distinguish between resident dcs, such as the cx3cr1 cells that are sentinels of the intestinal lumen, and allow sampling and presentation of harmless commensal bacteria. these cells may be involved in tolerance induction under homeostatic conditions or in innate defense against mucosal pathogens, but are generally noninflammatory. to initiate protective immunity, a pool of fully competent dcs able to respond quickly and properly to bacteria is required. ccr6 dcs in the pps are a reservoir of non - educated dcs that can be rapidly recruited should an infection occur. the equivalent of ccr6 cells in the lp still needs to be identified. in conclusion, the homeostasis of the gut is maintained via a continuous control of dc function by ec- and presumably also stromal cell this interaction generates noninflammatory and tolerogenic dcs that promote the development of th2 responses, t reg cells, and b cell class switch recombination. altogether, these responses allow us to tolerate commensal bacteria and food antigens and keep inflammation at bay. deregulation of these mechanisms can lead to chronic inflammation and immune - related gut disorders like inflammatory bowel disease and cancer. | recent work suggests that dendritic cells (dcs) in mucosal tissues are educated by intestinal epithelial cells (iecs) to suppress inflammation and promote immunological tolerance. after attack by pathogenic microorganisms, however, non - educated dcs are recruited from nearby areas, such as the dome of peyer 's patches (pps) and the blood, to initiate inflammation and the ensuing immune response to the invader. differential epithelial cell (ec) responses to commensals and pathogens may control these two tolorogenic and immunogenic functions of dcs. |
impressions play a vital role in the success of prosthesis because a prosthesis can be no more accurate than the impression from which it has been made. one of the most critical factors in the production of an accurate impression is the use of an appropriate impression material. there is a continuous effort to improve materials used to achieve dimensional stability, precision and fine tissue details. the dimensional stability and surface detail obtained with elastomeric impression materials has made them the material of choice for recording impressions that require a high degree of accuracy. it is also necessary for the impression materials to allow easy flow of the stone as is being poured. silicone impression materials, however are recognized as having poor surface wettability.1 clinically, the topical use of a surface wetting agent has been demonstrated to improve wettability and significantly reduce the number of voids in dental casts poured in silicone impressions.2 the impressions are contaminated with saliva, plaque and sometimes blood, all of which may carry pathogenic micro - organisms. mere washing of the impression as it is practiced does not clear away all the micro - organisms.3 these contaminated impressions may cause cross - infection from the patient to operator or dental assistant or laboratory technician.4 hence, it is necessary that the disinfection of dental impressions be adopted as a mandatory procedure in dental practice. in trying to achieve the goal of disinfection for the protection of the dental team, it is absolutely necessary that accuracy and surface details of impressions, not be compromised. moreover currently available impression materials were never originally designed to undergo disinfection or sterilization and it is quite conceivable that available disinfecting solutions may adversely affect the impressions.5 commonly used disinfecting agents like glutaraldehyde and sodium hypochlorite may alter the surface characteristics of impression materials, which may lead to dimensional changes in the impressions, along with changes in surface tension and wettability of the impression material. thus, the need arises to critically evaluate how these disinfecting solutions could affect the physical properties of elastomeric impression materials in order to ensure efficient, ethical and a consistent modern day prosthodontic practice. hence, this study was designed to evaluate the effect of two commercially available chemical disinfectants namely sodium hypochlorite and glutaraldehyde and a surface wetting agent on the physical property of three high precision elastomeric impression materials, viz. the study was conducted : to determine the contact angle and the wettability of various elastomeric impression materials before and after immersion disinfectionto determine the contact angle and the wettability of various elastomeric impression materials before and after treatment with a surface wetting agent. to determine the contact angle and the wettability of various elastomeric impression materials before and after immersion disinfection to determine the contact angle and the wettability of various elastomeric impression materials before and after treatment with a surface wetting agent. for the purpose of this study, three different types of elastomeric impression materials commonly used in prosthodontic practice were selected. addition silicone impression material : express stdputty and light body syringeable (3 m espe dental products u.s.a.) condensation silicone impression material : zetaplusputty and light body (zhermack italy) polyether impression material : impregum fhydrophilic medium consistency (3 m espe dental products u.s.a.) addition silicone impression material : express stdputty and light body syringeable (3 m espe dental products u.s.a.) putty and light body syringeable (3 m espe dental products u.s.a.) condensation silicone impression material : zetaplusputty and light body (zhermack italy) putty and light body (zhermack italy) polyether impression material : impregum fhydrophilic medium consistency (3 m espe dental products u.s.a.) hydrophilic medium consistency (3 m espe dental products u.s.a.) the glutaraldehyde and sodium hypochloride solutions were employed to disinfect the impressions made with the above mentioned elastomeric impression materials. true blue surface wetting agent was selected. glutaril2% glutaraldehyde (glaxo india ltd.)sodium hypochlorite sodium u.s.p.4 - 6% sodium hypochlorite (snow chem industries, mumbai)true bluesurface wetting agent (georg taub products, u.s.a.) 2% glutaraldehyde (glaxo india ltd.) sodium hypochlorite sodium u.s.p. 4 - 6% sodium hypochlorite (snow chem industries, mumbai) surface wetting agent (georg taub products, u.s.a.) aluminum model die, glass slide.gbx contact angle analyzer to measure advancing and receding contact angle. three different types of elastomeric impression materials were selected as representative of the materials currently used. specimens 20 mm 10 mm 2 mm were prepared by compressing the elastomeric material in a mold and glass slide. the test conditions were as follows : drysurface wetting agent (spray twice)2% glutaraldehyde (10 min immersion)2% glutaraldehyde (10 min immersion) and surface wetting agent4% sodium hypochlorite (10 min immersion)4% sodium hypochlorite (10 min immersion) and surface wetting agent. surface wetting agent (spray twice) 2% glutaraldehyde (10 min immersion) 2% glutaraldehyde (10 min immersion) and surface wetting agent 4% sodium hypochlorite (10 min immersion) 4% sodium hypochlorite (10 min immersion) and surface wetting agent. care was taken not to permit contamination of the surfaces. a contact angle analyzer linked to a compatible computer as used to measure the advancing and receding contact angles. used, the material was suspended in an electro balance while the wetting medium scans along at a constant speed via a computer - controlled stage. the meniscus at the interface is characterized by the dynamic contact angle (). both the advancing and receding contact angles are measured as the stage moves up (the specimen) and then down. a simple equation relates the cosine of the contact angle () to the magnitude of the wetting force recorded by the balance, the surface tension of the liquid, and the wetted perimeter of the solid specimen : v = surface tension (dyne / cm) three different types of elastomeric impression materials were selected as representative of the materials currently used. specimens 20 mm 10 mm 2 mm were prepared by compressing the elastomeric material in a mold and glass slide. the test conditions were as follows : drysurface wetting agent (spray twice)2% glutaraldehyde (10 min immersion)2% glutaraldehyde (10 min immersion) and surface wetting agent4% sodium hypochlorite (10 min immersion)4% sodium hypochlorite (10 min immersion) and surface wetting agent. surface wetting agent (spray twice) 2% glutaraldehyde (10 min immersion) 2% glutaraldehyde (10 min immersion) and surface wetting agent 4% sodium hypochlorite (10 min immersion) 4% sodium hypochlorite (10 min immersion) and surface wetting agent. a contact angle analyzer linked to a compatible computer as used to measure the advancing and receding contact angles. used, the material was suspended in an electro balance while the wetting medium scans along at a constant speed via a computer - controlled stage. the meniscus at the interface is characterized by the dynamic contact angle (). both the advancing and receding contact angles are measured as the stage moves up (the specimen) and then down. the difference between these two contact angles a simple equation relates the cosine of the contact angle () to the magnitude of the wetting force recorded by the balance, the surface tension of the liquid, and the wetted perimeter of the solid specimen : v = surface tension (dyne / cm) contact angle of express 3 m, zetaplus and impregum f (addition silicone, condensation silicone and polyether respectively) were evaluated after 10 min immersion disinfection in one of the two disinfectants namely sodium hypochlorite and glutaraldehyde and also after treatment with surface wetting agent. for the evaluation of contact angle of impression materials, gbx contact angle analyzer instrument was used. the contact angle measurements were conducted using each sample and each test solution only once before discarding. the results of this study have been presented in tables 1 - 5 with their standard deviations. comparison of advancing contact angle between three elestomeric impression materials (dry sample) : control group. impressions play a vital role in the success of a prosthesis because a prosthesis can be no more accurate than the impression from which it has been made. the impressions are contaminated with saliva, plaque and sometimes blood all of which may carry pathogenic microorganisms. these contaminated impressions may cause cross - infection from the patient to operator or dental assistant. this study was designed to evaluate the wettability of the three commercially available elastomeric impression materials viz. addition silicone (express 3 m), condensation silicone (zetaplus) and polyether (impregum f) after immersion disinfection in two commercially available chemical disinfectants, 2% glutaraldehyde (glutaril), and 4% sodium hypochlorite for 10 min and after spraying with surface wetting agent (true blue). wettability was assessed by measuring the contact angle of elastomeric impression materials with the help of gbx contact angle analyzer.the data collected was tabulated and subjected to statistical analysis.considering the results of the study, following conclusions can be drawn. the polyether impression material was the most hydrophilic of all the materials, followed by hydrophilic addition silicone. condensation silicone was least hydrophilic of the three materials.all the three elastomeric impression materials showed improvement in the wettability when a topical surfactant was used.the short term disinfection of the three elastomeric impression materials does not affect the wettability of these impression materials.the treatment with a topical surface wetting agent after short term disinfection resulted in an improvement in the wettability of the impression materials. thus it is recommended that elastomeric impression materials be treated with a surface wetting agent after disinfection to obtain accurate and void free casts and dies.thus the evaluated impression materials could be safely disinfected with any of the given chemical disinfectants for a short duration of 10 min, without compromising wettability. from the results, it was observed that none of the disinfectants affect the physical properties of the impression materials beyond the acceptable limits.furthermore, it was observed that treatment with a surface wetting agent improved the wettability of elastomeric impression materials.however, statistically significant differences were found in the wettability of the three types of impression materials used.it should be kept in mind that the current in vitro study has certain limitations. further long term in vivo studies with respect to these impression materials and disinfectants are suggested to enhance the conclusion obtained from the current study. wettability was assessed by measuring the contact angle of elastomeric impression materials with the help of gbx contact angle analyzer. the data collected was tabulated and subjected to statistical analysis. considering the results of the study, following conclusions can be drawn. the polyether impression material was the most hydrophilic of all the materials, followed by hydrophilic addition silicone. condensation silicone was least hydrophilic of the three materials.all the three elastomeric impression materials showed improvement in the wettability when a topical surfactant was used.the short term disinfection of the three elastomeric impression materials does not affect the wettability of these impression materials.the treatment with a topical surface wetting agent after short term disinfection resulted in an improvement in the wettability of the impression materials. thus it is recommended that elastomeric impression materials be treated with a surface wetting agent after disinfection to obtain accurate and void free casts and dies.thus the evaluated impression materials could be safely disinfected with any of the given chemical disinfectants for a short duration of 10 min, without compromising wettability. the polyether impression material was the most hydrophilic of all the materials, followed by hydrophilic addition silicone. all the three elastomeric impression materials showed improvement in the wettability when a topical surfactant was used. the short term disinfection of the three elastomeric impression materials does not affect the wettability of these impression materials. the treatment with a topical surface wetting agent after short term disinfection resulted in an improvement in the wettability of the impression materials. thus it is recommended that elastomeric impression materials be treated with a surface wetting agent after disinfection to obtain accurate and void free casts and dies. thus the evaluated impression materials could be safely disinfected with any of the given chemical disinfectants for a short duration of 10 min, without compromising wettability. from the results, it was observed that none of the disinfectants affect the physical properties of the impression materials beyond the acceptable limits. furthermore, it was observed that treatment with a surface wetting agent improved the wettability of elastomeric impression materials. however, statistically significant differences were found in the wettability of the three types of impression materials used. it should be kept in mind that the current in vitro study has certain limitations. further long term in vivo studies with respect to these impression materials and disinfectants are suggested to enhance the conclusion obtained from the current study. | background : this study was carried out to evaluate the effect of two commercially available chemical disinfectants namely sodium hypochlorite and glutaraldehyde and a surface wetting agent on the wettability of three high precision elastomeric impression materials, addition silicone, condensation silicone and polyether.materials and methods : three different types of elastomeric impression materials commonly used in prosthodontic practice were selected. the glutaraldehyde and sodium hypochloride solutions were employed to disinfect the impressions made with the above - mentioned elastomeric impression materials.true blue surface wetting agent was selected.gbx contact angle analyzer was used to measure advancing and receding contact angle.results:the results of this study have demonstrated that the polyether impression material was the most hydrophilic of all the materials, followed by hydrophilic addition silicone. condensation silicone was least hydrophilic. all materials showed improvement in the wettability when a topical surfactant was used.conclusion:the short term disinfection of the three elastomeric impression materials does not affect the wettability of these impression materials. |
early genetic studies suggest that a lack of decorin is permissive for tumor development. more specifically, crossing decorin - null mice with the p53-null mice caused early lethality of the double mutant animals because of massive organ infiltration by t - cell lymphoma. a more recent publication from our group demonstrates that decorin can indeed act as a tumor suppressor gene in the intestinal epithelium. we found that approximately 30% of decorin - null mice developed spontaneous intestinal tumors, and this process was accelerated and amplified by subjecting the decorin - null mice to a western diet enriched in fat and low in calcium and vitamin d. tumor development involved down - regulation of p21 and p27 and up - regulation of -catenin signaling. notably, decorin binds directly to epidermal growth factor receptor (egfr) and down - regulates its activity, as well as the activity of other members of the erbb family,. these receptors are overexpressed and/or mutated in many cancers, driving tumor progression. decorin competes with egf for receptor binding on the surface of tumor cells. after binding decorin, the receptor dimerizes and subsequently undergoes caveolin - mediated internalization and degradation in the lysosomes. decorin inhibits tumor cell proliferation by evoking a signaling cascade distinct from egf, possibly by inducing a different egfr confirmation and selectively activating phosphotyrosines in the receptor autophosphorylation domain. in addition, decorin suppresses the activity of the erbb2 and erbb4 receptors via degradation,. decorin also interacts with met, the hepatocyte growth factor receptor, and induces transient receptor activation, recruitment of the e3 ubiquitin ligase c - cbl, and rapid intracellular degradation of the receptor. furthermore, decorin suppresses intracellular levels of -catenin, a key downstream effector of met, and inhibits cell migration and growth. thus, by antagonistically targeting multiple tyrosine kinase receptors, decorin reduces primary tumor development and progression. another important step in cancer progression is angiogenesis, the process whereby new vessels grow from preexisting blood vessels. notably, by binding transforming growth factor- (tgf-), decorin interacts with tgf-, preventing tgf- from binding to its receptor, and therefore plays a significant role in tumor progression and angiogenesis. decorin can also play a proangiogenic role by facilitating endothelial cell adhesion and migration on type i collagen. specifically, decorin mediates adhesion by binding to 21 integrin and promoting the integrin - collagen interaction. interestingly, decorin mediates endothelial cell migration by modulating insulin - like growth factor (igf)/insulin receptor 1 (ir1) signaling, confirming that the decorin / igf-1r interaction plays a role in angiogenesis. two potential mechanisms may underlie these antiangiogenic activities : interfering with thrombospondin-1, or suppressing the production of endogenous vascular endothelial growth factor (vegf) in tumor cells, which in turn attenuates migration and capillary - like formation of endothelial cells. a more recent study showed that decorin antagonizes the angiogenic network in breast cancer cells via inhibition of met, hypoxia inducible factor-1 (hif-1), and vegfa, as well as induction of the angiostatic proteins thrombospordin-1 and tissue inhibitor of metallo - proteinases-3 (timp3). thus, one of decorin 's roles in the microenvironment is the modulation of angiogenesis, albeit in a cell - specific context, upon the physiological or pathological stimulation. the involvement of decorin in metastasis is still open to investigation and is far from being resolved. however, because of its functions in the microenvironment and extracellular matrix, we postulate that decorin plays a significant role in tumor metastasis. loss of e - cadherin promotes metastasis by inducing cancer cell disaggregation, activating specific downstream signal transduction pathways, and causing epithelial - mesenchymal transition (emt), which facilitates metastasis. our recent studies in decorin - null mice and increasing decorin expression in human colon cancer cells in vitro have demonstrated an interaction between decorin and e - cadherin, which may underlie another mechanism of the antimigratory and antimetastatic actions of decorin in colorectal carcinoma,,,. interestingly, decorin has also been implicated in down - regulating the e - cadherin binding partner -catenin in vitro, in vivo, and in xenograft models,,. accumulating evidence suggests that the stromal - epithelial interaction is important for tumor progression and metastasis. tumor stroma represents the surrounding normal ecm or the microenvironment of the tumor cell through which stromal and tumor cells talk. reduced decorin levels in cancer tissues might alter several signal pathways due to the well known interactions between decorin and the tyrosine kinase receptors described above. alternatively, one could postulate that abundant expression of decorin may lead to an organized indeed, there is a regulation among decorin and stromal components including matrix metalloproteinases (mmps). an earlier study also indicated decorin may affect signaling via chemokine receptor cxcr4, possibly affecting metastasis. decorin syngerizes with carboplatin to inhibit ovarian cancer cell growth but antagonizes the effects of carboplatin and gemcitabine on pancreatic cancer cells. considering that decorin can be administered alone or concomitantly with a variety of compounds, we anticipate that decorin will attract more interest in the future as an anticancer therapeutic agent. administration of decorin core protein in an a431 squamous carcinoma model resulted in decorin localizing specifically within the tumor, antagonizing egfr activity, and inducing apoptosis. systemic injection of decorin reduced breast tumor growth and metabolism and halted metastatic spread to the lungs. earlier studies also show that decorin delivered by adenovirus slowed the growth of lung, squamous, and colon carcinoma tumor xenografts in immune - compromised mice,. adenovirus - delivered decorin also slowed the growth of mammary adenocarcinoma and prevented metastatic spreading to the lungs by down - regulating erbb2 receptor levels. notably, the size of the tumors in that model was directly proportional to how early and how much decorin was expressed. the finding that decorin antagonizes primary tumor growth and metastasis in vivo raises hope for clinical application. decorin might be utilized in the near future as an adjunct protein therapeutic for solid tumors in which receptor tyrosine kinases play a key role. slrps, including decorin, are characterized by a protein core containing leucine repeats with a glycosaminoglycan chain, consisting of either chondroitin sulfate or dermatan sulfate. in particular, highly metastatic cell lines show the prevalence of chondroitin sulfate, but not dermatan sulfate. future studies should also be undertaken to elucidate the functional interaction between decorin and existing anticancer chemotherapeutics to evaluate potential limitations. because decorin expression is altered in several types of cancer, it is considered a possible prognostic marker in cancer patients. reduced decorin levels were associated with poor prognosis in node - negative, invasive breast cancer and some soft - tissue tumors. specifically, low decorin levels correlated with large primary breast tumor burden, high risk of early recurrence, and overall poor prognosis. breast cancer patients with tumors high in egfr and low in decorin had an even worse outcome. this reinforces the notion that decorin is a key player in egfr signal modulation in vivo. low decorin levels in liposarcomas and malignant peripheral nerve sheath tumors were also associated with low disease - free survival and overall survival rates. a recent study suggested that stromal decorin expression provides additional prognostic value in breast cancer when used with established primary markers. we foresee that decorin will attract more interest in the future as a promising prognostic marker for cancer patients. decorin 's biological activity is rather complex ; it regulates multiple processes in the extracellular matrix and in tumor cells (figure 1). studies in mouse models and cancer patients suggest that decorin plays an important role throughout the tumor growth and development process and may be a promising antitumor agent. in vivo studies with more tumor models decorin interacts with egfr, erbb, c - met, igf - ir, tgfr, and 21, respectively, leads to induction of p21waf1, signaling modulation, proteasome deregulation, activation of pi3k / akt / mtor and pi3k / smad signaling, finally causes degradation of endocytosis, inhibition of tumor cell growth, migration, angiogenesis, endothelial cell proliferation, and motility, exerting tumor suppressor. egfr, epidermal growth factor receptor ; igf - ir, insulin - like growth factor - insulin receptor ; tgf-, transforming growth factor- ; 21, integrin alpha2-beta1 ; pi3k, phosphatidylinositol 3-kinase ; mtor, mammalian target of rapamycin. | decorin is a member of the extracellular matrix small leucine - rich proteoglycans family that exists and functions in stromal and epithelial cells. accumulating evidence suggests that decorin affects the biology of various types of cancer by directly or indirectly targeting the signaling molecules involved in cell growth, survival, metastasis, and angiogenesis. more recent studies show that decorin plays important roles during tumor development and progression and is a potential cancer therapeutic agent. in this article, we summarize recent studies of decorin in cancer and discuss decorin 's therapeutic and prognostic value. |
there is general consensus that radiosurgery is a preferred option to surgical removal for the treatment of an arteriovenous malformation in the basal ganglia, thalamus, and other deep brain areas. this is because surgical removal of a deep seated avm is especially difficult, producing high mortality and morbidity rates.1)2)6)8) consequently, gamma - knife radiosurgery (gkrs) has been employed for patients with a deep seated avm. although rare, radiation - induced vasculopathy is one of the most serious complications after gkrs.3)10) the basal ganglia and thalamus are extremely sensitive to radiation, therefore, gkrs for a deep seated avm has increased risk of side effects during the latency period.9)14) complication of large intracranial arteries related to gkrs has occasionally been reported.4)11) however, to the best of our knowledge, radiation induced ischemic injury to small arteries has never been reported. we report two cases of basal ganglia infarction after gkrs for deep seated avm, which were located adjacent to the avm. we suggested that radiation injury to the lenticulostriate artery could be one possible cause of the infarctions. a 70-year - old male presented with an atypical headache for several months. there was no de finite history of underlying disease or medication such as hypertension and/or diabetes. magnetic resonance imaging (mri) and cerebral angiography showed a spetzler - martin grade 2 avm, and the radiosurgery - based avm score (rbas) was 1.53, located in the right deep temporal lobe, adjacent to the basal ganglia (fig 1a, b). it was also drained to the basal vein of rosenthal and straight sinus (fig. gkrs was performed with a 9.6cc lesion volume and a 20 gy marginal dose (fig. one year later, a post - radiosurgical mri follow - up showed nidus regression. seventeen months after the radiosurgery procedure, the patient presented with acute - onset left hemiparesis (4/5) and sensory change. mri showed an acute cerebral infarction at the right basal ganglia in the lenticulostriate artery territory, in an area adjacent to the gkrs (fig. the patient was administered 100 mg of aspirin from the stroke onset, and suffered no additional ischemic attack. three months later, he recovered without prominent deficit (modified rankin scale, mrs = 0). it is interesting to note that complete obliteration of the avm was confirmed at the 2-year follow - up angiography (fig. a 54-year - old male presented with an incidentally - detected avm during his health examination. mri and cerebral angiography showed a spetzler - martin grade 3 avm, and rbas score was 1.78 in the right basal ganglia (fig. it was mainly supplied by the right m1 segment, and was drained into the internal cerebral vein (fig. gkrs was performed with a 2.4 ml lesion volume and a 18 gy marginal dose (fig. eleven months after gkrs, the patient presented with sudden - onset left hemiparesis (2/5). subsequent mri showed an acute cerebral infarction in the right putamen, which probably originated by occlusion of the lateral lenticulostriate arteries (fig. the patient suffered no additional stroke attack, he then recovered with slight disability (mrs = 2) at 3 months from onset. remarkably, the draining vein had decreased in diameter and most of the arteriovenous shunt including the nidus was no longer visible (fig. a 70-year - old male presented with an atypical headache for several months. there was no de finite history of underlying disease or medication such as hypertension and/or diabetes. magnetic resonance imaging (mri) and cerebral angiography showed a spetzler - martin grade 2 avm, and the radiosurgery - based avm score (rbas) was 1.53, located in the right deep temporal lobe, adjacent to the basal ganglia (fig 1a, b). it was also drained to the basal vein of rosenthal and straight sinus (fig. gkrs was performed with a 9.6cc lesion volume and a 20 gy marginal dose (fig. one year later, a post - radiosurgical mri follow - up showed nidus regression. seventeen months after the radiosurgery procedure, the patient presented with acute - onset left hemiparesis (4/5) and sensory change. mri showed an acute cerebral infarction at the right basal ganglia in the lenticulostriate artery territory, in an area adjacent to the gkrs (fig. the patient was administered 100 mg of aspirin from the stroke onset, and suffered no additional ischemic attack. three months later, he recovered without prominent deficit (modified rankin scale, mrs = 0). it is interesting to note that complete obliteration of the avm was confirmed at the 2-year follow - up angiography (fig. a 54-year - old male presented with an incidentally - detected avm during his health examination. mri and cerebral angiography showed a spetzler - martin grade 3 avm, and rbas score was 1.78 in the right basal ganglia (fig. it was mainly supplied by the right m1 segment, and was drained into the internal cerebral vein (fig. gkrs was performed with a 2.4 ml lesion volume and a 18 gy marginal dose (fig. eleven months after gkrs, the patient presented with sudden - onset left hemiparesis (2/5). subsequent mri showed an acute cerebral infarction in the right putamen, which probably originated by occlusion of the lateral lenticulostriate arteries (fig. the patient suffered no additional stroke attack, he then recovered with slight disability (mrs = 2) at 3 months from onset. remarkably, the draining vein had decreased in diameter and most of the arteriovenous shunt including the nidus was no longer visible (fig. gkrs is an important therapeutic modality for a deeply located cerebral avm.6)8) at the expense of a latency period to cure of 2 - 3 years, successful angiographic obliteration of avms is generally seen in approximately 70% of cases following radiosurgical treatment.3)5) although gkrs is a widely accepted treatment modality, progressive intracranial artery occlusion due to radiation induced vasculopathy is a serious side effect of conventional radiation therapy for intracranial disease. as the follow - up lengthens in modern series, details of radiation - induced injury have become more clearly elucidated. because the basal ganglia and thalamus are extremely sensitive to radiation side effects and hemorrhage during the latency period, deep avms have increased risks associated with radiosurgical therapy.9)12) in addition, one autopsy study reported that a normal surrounding blood vessel may also be affected by irradiation of 10 gy or more, and remarkable intimal hypertrophy with fragmentation of the elastic laminae, or even complete occlusion, can occur in these arteries.13) vascular complication associated with gkrs for deep avm has been noted in several case reports, including de novo aneurysm formation, venous varix formation or stenosis of major intracranial arteries. there have been occasional reports of stenosis or occlusion of major cerebral arteries occurring several years after stereotactic radiosurgery for cerebral arteriovenous malformations.4)7)11) exposure of a major cerebral vessel to radiation doses of 15 to 25 gy can potentially result in postradiosurgical stenosis or occlusion, although the incidence occurs in fewer than 1% of patients.11) some literature studies have reported that steno - occlusive complication could occur even at larger intracranial arteries after gkrs, in spite of low - dose radiation exposure.7)12) however, in the current study, in both cases the ischemic injury involved small perforating arteries (lateral lenticulostriate arteries), and those events occurred at 11 and 17 months after the gkrs. a limitation to this case report is that there might be some debate about the etiology, because we present only 2 case reports and both patients had other risk factors for acute stroke. the patient in case 1 was old (70-year - old), and the patient in case 2 had underlying hypertension. therefore, an alternative explanation of the underlying cause in each case may be a small vessel occlusion due to atherosclerosis. in addition, evidence of radiation induced vasculopathy has not been confirmed in our two cases. in this regard, future prospective studies and long - term follow - up are necessary to prove the relationship between delayed perilesional ischemic injury and gkrs for deep seated avms. the authors present two cases of delayed perilesional ischemic events after gkrs for a deep seated avm, which occurred at 11 and 17 months after the treatment and may have been caused by radiation injury to the lateral lenticulostriate arteries. although alternative causes of the ischemic events are possible, awareness of the possibility of radiation - induced vasculopathy of small arteries will be helpful in establishing early and accurate diagnoses along with the appropriate therapy. larger studies with longer, formal angiographic follow - up will be necessary to clarify these events and their clinical significance. | radiation - induced vasculopathy is a rare occurrence, however, it is one of the most serious complications that can occur after gamma - knife radiosurgery (gkrs). the authors present two cases of incidentally found deep cerebral arteriovenous malformation (avm), which were treated by gkrs, where subsequently there occurred delayed - onset cerebral infarction (11 and 17 months after gkrs) at an area adjacent to the avm. in both cases, perforators of the m1 segment of the middle cerebral artery were included in the radiation field and delayed injury to these is suggested to be the mechanism of the ischemic event. |
alzheimer 's disease (ad) is a pernicious neurodegenerative disease which is incurable with remedies developed up to date. the number of patients increases every year worldwide and 5.2 million people in the united states are suffering from ad. this number is expected to expand to 36 million to 115 million in worldwide around the year of 2050 and the estimated economic cost and suffering is increasing greatly (1, 2). therefore, ad is seemingly insurmountable disease and the increasing numbers of patients produce diverse societal concerns in different aspects. senile plaques of amyloid (a) in the brain parenchyma have been regarded as not only the main pathological phenomena (3, 4) but also the culprit of this disease according to amyloid cascade hypothesis based on molecular information found in ad study (5). abnormal production and accumulation of a in brain parenchyma result in ad pathologies through sequential events by aggregated forms of this protein and the amyloid plaque. a is generated as a consequence of sequential cleavages of amyloid precursor protein (app) by - and -secretases (6, 7, 8). app is first cleaved by either - or -secretase, and then, the remaining remnants of c83 or c99, respectively, are vulnerable to intramembrane proteolysis by -secretase. amyloidogenic process of -secretase cleavage followed by -secretase produces aggregation - prone a which are in the center of ad etiology (4, 9). disrupted synaptic plasticity, reduced dendritic spine density and memory impairment were proven in rodent model by extraction of a oligomers from human patients (10). along with a, microtubule - associated protein tau is another major factor of ad pathogenesis as a component of neurofibrillary tangles (nfts) (11). tau stabilizes microtubule protein and microtubule - associated processes in normal condition. during ad pathogenesis, tau becomes hyperphosphorylated, aggregated and finally accumulated as neurofibrillary tangles (12). tau hyperphosphorylation and nft formation is tightly related to the existence of excessive a and plaques, proving the tau pathology in ad (13, 14). not only as axonal protein but also as regulator of dendritic function, tau plays a pivotal role, especially mediating early a toxicity during ad progress (15). many clinical trials aiming these two proteins have been performed whereas several lines of targets are still under investigations. up to date, only a few ad medications have been proved as improving ad symptoms, but none of them modify disease progress or pathological cascades (16). researchers and clinicians suspect that the reason for many drug target candidates to fail in their clinical trials reside in the improper time of drug treatment, in fairly late stage of ad progression where irreversible damages have already occurred, including excessive a deposits, neuronal impairment, death and blood brain barrier (bbb) disruption (17). therefore, finding a diagnostic biomarker, especially for the early stages of ad pathology, is desperately needed for developing a valuable therapeutic target at the early stage and preventing progression of the disease. diverse approaches for ad therapeutic strategy have arisen along with better understanding of cellular and molecular mechanism of ad pathogenesis. in fig. 1 and table 1, we listed possible strategies of drug development target based on accumulated scientific findings. in this review, we summarize the recent status of some ad drug targets using different strategies among them from published reports and ongoing clinical studies. in each category in spite of better understanding for molecular mechanism during ad pathogenesis, the available medications for ad up to date provide only symptomatic benefit, not regulate or delay the progression of disease pathology. the us food and drug administration (fda) has approved only five medications for ad, including acetylcholinesterase inhibitors (acheis : donepezil, rivastigmine, galantamine and tacrine) and n - methyl - d - aspartate (nmda) receptor antagonist, such as memantine (18, 19, 20, 21).. combined administration of these two medications accelerates symptomatic improvement, representing slow progress of cognitive and functional impairment and delayed time for nursing home admission (22, 23). besides, several lines of regulators for neurotransmission were suggested for symptomatic therapies in ad, including neuronal nicotinic acetylcholine receptor activation (abt-418), gabab receptor antagonism (sgs-742) and serotonergic modulation (lu ae58054). however, they were insufficient to show significant efficacies of symptomatic improvement in clinical trials (24, 25, 26, 27). recently, high affinity 5-hydroxytryptamine (ht) 6 receptor antagonist lu ae58054 ([2-(6-fluoro-1h - indol-3-yl)-ethyl]-[3-(2,2,3,3,-tetrafluoropropoxy)-benzyl]-amine) was reported to improve novel object recognition task in a rat model with cognitive impairment induced by phencyclidine (28). the combined treatment of lu ae58054 and donepezil is under phase iii clinical trial (clinicaltrials.gov identifier : nct01955161). additionally, even drugs with uncertain mechanism were reported to be effective in symptomatic improvement and protection against neurotoxicity by a, including ethanolic extract of angelica gigas (inb-176) and ginkgo biloba (egb761) respectively, however, none of which showed successful effectiveness in their preclinical and clinical trials (29, 30, 31). abnormal a production and accumulation in brain parenchyma have been regarded as the central etiological hypothesis in ad pathogenesis (5, 10, 32). therefore, the first line of strategy was inhibition of a generation processes to prevent or cure the disease. the tight relevance of -, - and -secretases to a production made researchers to discover modulating drugs for these enzyme activities in order to reduce intracellular and extracellular a level. whereas effective -secretase activator was rarely identified, several types of -secretase inhibitors were discovered and tested, starting with first - generation potent inhibitor om99 - 2, om00 - 3 (33, 34). since then, numerous reports and patents of -secretase inhibition were published, however, finding drug candidate with desirable potencies and efficacy has been fairly challenging (35). recently discovered mk-8931 (merck) is a promising -secretase inhibitor whose result of phase i clinical trial was released in april, 2012. mk-8931 is now under phase ii / iii trial which was initiated in 2012 (clinicaltrials. gamma - secretase plays the critical role in a generation, in charge of the rate - limiting cleavage of app into a. however, modulating this enzyme activity may cause diverse side effects because of its multiple cleavage actions on diverse substrates which are physiologically important, including notch receptor signaling. for this reason, modulating -secretase activity seems to be greatly complicated, requiring restricted substrate specificity for app to reduce a only, not affecting other substrate processing such as notch signaling (36, 37). consequently, substrate specificity is the critical issue in the development of ad therapy using -secretase inhibition. semagacestat (ly450139, eli lilly) was a promising drug candidate targeting -secretase inhibition (38), tested in two phase iii clinical trials. even though both trials finished with a disappointing result of insufficient efficacy it showed a breakthrough for possible utilization of -secretase modulation in ad therapeutic development. mostly, a elicits its toxicity by aggregated forms (10, 39, 40). therefore, the inhibition of a aggregation is one of the most effective strategies in order to inhibit a toxicity. curcumin and -sheet breaker such as rs-0406 were discovered to inhibit polymerization of a into oliogmer and fibril forms (41, 42). compound d737 showed the most effective inhibition of a aggregation among a collection of 65,000 small molecule candidates and elicited increased lifespan in a drosophila melanogaster model of ad as well as reduction of a toxicity in cell culture system (43). indirect inhibition of a aggregation was suggested by metal hypothesis of ad (44). cupper / zinc ionophore, pbt2, which target the copper and zinc ions that mediate a aggregation was proven to facilitate the aggregated a clearance in the cortex, to lower a level of cerebrospinal fluid (csf) and to restore the cognitive impairment in ad patients (44, 45, 46). pbt2 completed phase ii clinical trial (clinicaltrials.gov identifier : nct00471211) and are now under phase ii clinical trial for huntington disease as well. additional large - scale clinical tests and high throughput screening for candidates of a aggregation inhibitor are strongly encouraged in further investigation. especially, pyroglutamyl modification in n - terminus of a is critical alteration because pyroglutamated a (pglu - a) species readily accumulated into senile plaque and vasculature deposit due to increased stability and aggregation velocity (47, 48, 49). glutaminyl cyclase (qc) was demonstrated as the main catalytic enzyme responsible for this pyroglutamyl modification of a and intracortical microinjection of qc inhibitor, pbd150, significantly decreased pglu - a formation (50, 51). since inflammation response and activation of phagocytic cells such as microglia and astrocytes had been appreciated as a pivotal contributor to ad pathogenesis, immune system became one of the most prominent targets in the aspect of ad therapeutic invention (52). cytokines and other neurotoxic adducts secreted by immune - related cells were suspected as possible mediators of neuronal degeneration and cell death (53, 54). furthermore, data analysis using genome wide association study (gwas) supported this idea by proving that specific over - representation of genes related to immune pathway linked to ad risk (55). the protection effect of non - steroidal anti - inflammatory drugs (nsaids), especially ibuprofen, against ad proved that the suppression of immune response should be beneficial in ad (56). many factors seemed to be tightly related to the protective effect of nsaids against ad, including age of cohort, apolipoprotein e (apoe) genotype, the duration of nsaids usage and nasaids types, showing significant effect in apoe 4 allele carrier (56, 57, 58, 59). unfortunately, diverse clinical trials with different types of nsaids concluded not only beneficial effects but also insufficient efficacies and negative results (57, 60, 61, 62, 63, 64, 65). narrowing down the with amyloid hypothesis, immunotherapy against a peptide attracted a great deal of attention because it is direct resolution of the seemingly main cause of pathogenesis and progression of disease in ad. several strategies for a peptide immunotherapy has been tested, including passive immunization with monoclonal antibody against different regions of a42 as well as active immunization using synthetic a42 (66). because a peptide, the major component of senile plaques in ad brain, is regarded as the critical contributor in ad pathogenesis, enhanced clearance of a via the administration of anti - a monoclonal antibody, including bapineuzumab and solanezumab (passive vaccination), or a antigen with adjuvant such as an1792 (active vaccination) seemed fairly promising (67, 68). preclinical trials for both active and passive immunotherapies against a represented diverse beneficial effects of ameliorated brain a burden, prevention of memory loss and improved cognitive function in different animal models of ad (67, 69). preliminary test on human patients exerted promising outcomes of reduced plaque burden and cognitive benefit (70, 71), suggesting multiple mechanisms of actions including modulation of a equilibrium balance between the central nervous system and plasma (72) or improved peripheral clearance and sequestration of brain a (73). however, unavoidable side effects found in clinical trials hindered the further clinical development into ad therapeutic treatment on human, including meningoencephalitis, microhemorrhages and vasogenic edema (68, 74). also, clinical evaluation in human ad patients failed to replicate the identical results as in the animal ad model, showing unsynchronized phenomena between reduced a plaque and rescue of neurodegeneration during ad progression (75). immunotherapy is regarded as one of the most promising therapeutic strategies in ad and some immunotherapeutic drug candidates are still under clinical trials, including the first monoclonal antibody for a protofibril (ban2401, clinicaltrials.gov identifier : nct01767311) and immunoglobulins combined with albumin by means of diverse application methods (clinicaltrials. intracellular neurofibrillary tangle (nft) is another hallmark in ad pathogenesis, cytoskeletal inclusions consisted of hyperphosphorylated microtubule associated protein tau with paired helical filament structure (14). especially, modulating endogenous tau level in app - overexpressing mice halted a-induced behavioral deficit in spite of maintaining high a level, suggesting the relevance of tau during ad pathogenesis and implying the possibility for tau - targeting immunotherapy in ad (76, 77). moreover, tight relationship between a and tau pathologies during ad strengthened the rationale for tau - aiming therapeutic strategy in ad. it was proven by that a immunotherapy reduced not only the extracellular a plaques but also intracellular a accumulation which resulted in the absence of early tau pathology (78). different mechanistic approaches of tau - targeting therapies were tried, including reducing tau level itself, preventing tau hyperphosphorylation and inhibiting the aggregation (26). many researches have focused on preventing hyperphosphorylation of tau, earlier event that cause detachment of tau protein from microtubule. kinases responsible for tau phosphorylation (glycogen synthase kinase, gsk-3, cyclin - dependent kinase-5, cdk5 and microtubule affinity - regulating kinase) and phosphatase (protein phosphatase 2a, pp2a) are the possible targets to achieve tau - aiming therapeutics, altering tau phosphorylation by modulating activity of the enzymes (79, 80, 81). especially, gsk-3 inhibition is implicated in both a and tau pathway concomitantly and is greatly appreciated in ad therapeutic development. gsk-3 inhibition by lithium, valproate, caffeine were also tested in preclinical and clinical studies and their efficacies were needed to be confirmed in further study because of inconsistent outcomes in different studies (82, 83, 84, 85). azd 1080 (astrazeneca) and np-12/tideglusib (noscria) were the most promising gsk-3 inhibitor, however, azd 1080 was withdrawn from ad therapeutic development due to the nephrotoxic side effect in phase i clinical trials (68, 86, 87). since then, np-12/tideglusib has been recognized as an effective gsk-3 inhibitor and completed not only pilot clinical study using small sample with the result of positive trends in mini - mental state examination (mmse), alzheimer 's disease assessment scale - cognitive subscale (adas - cog), global deterioration scale (gds) and global cortical atrophy (gca) (88) but also phase ii clinical trial (clinicaltrials.gov identifier : nct01350362). in addition to tau phosphorylation, several agents were also suggested to prevent tau aggregation. methylthioninium chloride (methylene blue, mtc) was the first tau aggregation inhibitor discovered and reduced version of mtc, trx0237, is now in the process of phase iii clinical trial (89) (clinicaltrials.gov identifier : nct01689246). also, diverse possible candidates were suggested as tau aggregation inhibitor, including anthraquinones, aminothienopyridazines, polyphenols and phenothiazines (68, 90, 91, 92). these compounds, however, need more verification because they failed to show consistent efficacies in in vivo studies. since type 2 diabetes mellitus (dm2) was found to be related to ad, glucose metabolism has emerged as a new interest in ad research. it was widely known that glucose metabolism and insulin signaling are impaired in ad brain (93, 94, 95). insulin - degrading enzyme (ide) was revealed to be responsible for a degradation as well, more efficiently intracellular a than extracellular form (96, 97, 98). even though ad and dm2 share ide as the key metabolic enzyme for their main etiological proteins, a for ad and insulin for dm2, it is not enough to explain all the ad - mimic pathological phenomena found in diverse mechanism - driven diabetes mouse model with insulin resistance (98, 99, 100). insulin has effect on cerebral function per se and is also tightly involved in inflammation and oxidative stress, representing enhanced inflammatory response and markers of oxidative stress by hyperinsulinemia (101). in other studies, increased autophagosome was suggested to accelerate the amyloidogenic app processing in insulin - resistant condition (102). thereby, insulin itself represented tight relevance to ad and became a new therapeutic target in ad. metformin, a peripheral insulin sensitizer drug approved by the fda, was reported to sensitize brain insulin action and prevent ad - associated pathological alteration in in vitro ad model (103). also, other diverse insulin sensitizers are needed to be tested because they may have possibility to show valuable efficacy in ad as well. msdc-0160 (mtot modulator, metabolic solution development company, msdc) was recently performed successful phase iia clinical trial for type 2 diabetes (104). msdc-0160 was demonstrated to elicit its insulin - sensitizing effect through newly discovered mitochondrial target of thiazolinedione (mtot) located in the mitochondrial inner membrane (104, 105). mitochondria are one of the most devastated organelle in the process of ad development. during ad pathogenesis, mitochondrial impairment occurs in various brain areas, representing not only morphological alteration but also physiological dysfunction (106). a was detected inside mitochondria compartment of ad mouse model expressing human mutant amyloid precursor proteins (app), mostly within membrane and membrane - associated region (107, 108). study using mitochondria - specific targeting a proved that mitochondrial accumulation of a induced not only morphological alteration but also physiological dysfunction which was fatal enough to induce neuronal apoptosis (109, 110). other findings also showed morphological alteration of mitochondria and physiological dysfunction, especially electron transport chain through cytochrome c oxidase and increased oxidative stress in in vitro and in vivo ad - mimic system and patients (110, 111, 112). therefore, mitochondrial dysfunction by a is a critical contributor during ad pathogenesis, and in the same line of thought, mitochondrial protection becomes a new strategy in ad treatment. also, mitochondrial dysfunction and oxidative stress is more evidently the common linker between ad and abnormal glucose metabolism (113, 114). therefore, mitochondrial recovery drug was proposed as a new concept for ad therapy and treatment of insulin - resistance. dimebon (pfizer), originally allergy - treating drug used in russia, was known to improve atp generation and energy metabolism in mitochondria and was tested its effectiveness in clinical trial for ad therapeutics (115). unexpectedly, phase iii trial performed with 598 patients ended up as failure with the lack of improvement. this failure leads researcher to suspect the novel mitochondrial mechanism of action of dimebon, rather than promiscuous clinical effects, including inhibition of histamine h1 and serotonin receptors. in extended thoughts, other medicines with the effect of mitochondrial rejuvenation have been investigated as probable candidates for ad therapeutics. piracetam is a nootropic drug and has effect on cognitive impairment during aging and dementia (116). the mechanism of action for piracetam was controversial, including effects on glutamate receptors, gaba - mimetic action and activation of calcium influx into neuronal cells (116). recently, mitochondrial relevance of this drug has been found to enhance the membrane fluidity in brain mitochondria and consequently improve membrane potential, atp generation and decrease apoptotic vulnerability in aging and ad model (117). these findings suggested the possibility that this drug exerted therapeutic effect through mitochondrial recovery in ad. in addition to mitochondrial function itself, axonal transport of mitochondria through microtubule protein was observed to be impaired in ad (118). the acetylation status of -tubulin by histone deacetylation (hdac6) is highly related to cargo transport along microtubules (119). restored -tubulin acetylation by hdac6 inhibitor improves both anterograde and retrograde motility of mitochondria and, furthermore, rescued mitochondrial morphology in hippocampal neurons under ad - mimic condition of a-induced impairment of mitochondria function (120). these diverse mitochondria - targeting mechanisms of action are all likely to be probable therapeutic mechanism for ad treatment and deserve to be evaluated. besides the relevance to mitochondrial dysfunction, disturbed glucose metabolism could be directly connected to a generation and accumulation by modulating the enzyme activity of a-generating enzyme through post - translational modification. addition of o - linked -n - acetylglucosamine (o - glcnac) to protein is a glucose level - dependent post - translational modification and the specific inhibition of o - glcnacase, 1,2-dideoxy-2-propyl--d - glucopyranoso-[2,1-d]-2-thiazoline (nbutgt), ameliorated a generation by modulating nicastrin activity, a component of -secretase, through s708 site o - glcnacylation (121). different from other o - glcnacylation modulating drugs, such as o-(2-acetamido-2-deoxy - d - glucopyranosylidene) amino n - phenyl carbamate (pubnac) and streptozotocin (stz), nbutgt was specific to o - glcnacase and showed the lack of cellular toxicity nor insulin resistance (122). additionally, o - glcnacase inhibitor was reported to reduce the tau phosphorylation and improve long - term potentiation (ltp) as well, which are possibly beneficial in ad (123, 124). o - glcnacase inhibitor seems to be effective not only in a generation but also in memory impairment and taupathies, which is needed to be further verified in future clinical studies. in addition to o - glcnacylation, diverse post - translational modification could be another valuable target for ad therapeutics, especially specific modification for ad - related proteins, including -secretase and -secretase. recent findings suggest that a accumulation is fairly slow and time - consuming process, likely to require more than two decades (125). during this long process, more than one physiological system seems to be linked each other to harmonize in order to induce pernicious ad pathology. taken information together, it is unlikely that a single remedy could cure ad because of its complexity and intricate relationship among the multitude of pathological components during pathogenesis. for this reason, therapeutic targets for ad should include multiple strategies and combinational remedy, not single, for the maximum effectiveness and better consequences. also, not only direct therapeutic treatment for pathological intervention but also delaying this long pathogenic process would contribute to reduce the number of ad patients and increase prognostic benefit (26). up to date, researchers are desperate to find new ways for ad treatment and tune the drug candidates for the maximum efficacies. for instance, in developing a synthesis modulator as ad therapeutic target, researchers has to consider diverse questions and concerns, including the right margins of decreased a production level, maintaining proper physiological level and off - target effects by influencing other substrates besides a. because it is hard to tune the enzyme activity within the right physiological catalytic range, the successful development of ad therapeutics with enzyme modulator is dependent upon its efficacy on aiming action, specificity and selectivity to target substrate. also, as everyone agrees that the right timing of drug treatment is essential for the evaluation of efficacies for therapeutic targets, improvement in early diagnostic tool for ad have to be pursued along with ad therapeutic development. | alzheimer 's disease (ad) is the most common form of dementia caused by neurodegenerative process and is tightly related to amyloid (a) and neurofibrillary tangles. the lack of early diagnostic biomarker and therapeutic remedy hinders the prevention of increasing population of ad patients every year. in spite of accumulated scientific information, numerous clinical trials for candidate drug targets have failed to be preceded into therapeutic development, therefore, ad - related sufferers including patients and caregivers, are desperate to seek the solution. also, effective ad intervention is desperately needed to reduce ad - related societal threats to public health. in this review, we summarize various drug targets and strategies in recent preclinical studies and clinical trials for ad therapy : allopathic treatment, immunotherapy, a production / aggregation modulator, tau - targeting therapy and metabolic targeting. some has already failed in their clinical trials and the others are still in various stages of investigations, both of which give us valuable information for future research in ad therapeutic development. |
a 10-year - old boy came with a complaint of anterior cross bite to our department. the patient had no relevant past dental or medical history and no habits. on extra - oral examination [figure 1 ], patient had a dolico - cephalic head shape, lepto - prosobic facial form, concave profile, anterior divergence and acute nasolabial angle. pre - treatment extraoral photographs on intraoral examination [figure 2 ], all soft - tissues and hard tissues were normal. molar, canine and incisor relationship were class iii on the both right and left side and there was a reverse overjet of 2 mm and overbite of 5 mm and anterior cross bite. the patient had class iii skeletal base with retrognathic maxilla and prognathic mandible having average growth pattern with labially tipped mandibular incisors. the objective of the treatment was (1) to intrude and proclined the upper incisors, (2) to retroclined the lower incisors. (3) obtaining class i canine relation. (4) obtaining ideal overjet and overbite. (5) obtaining ideal esthetics. pre - treatment intraoral photographs patient was treated with a combination of face mask and rme [figures 3 - 5 ] until 2 mm positive overjet was achieved. the expansion screw was activated one to three turns (0.25 mm / turn) at weekly visits until the desired amount of expansion had been achieved. the face mask was adjusted to rest on the forehead and the chin of the patient. elastics (5/16 inch by 14 ounces) were worn from hooks located 2 - 3 cm in front of the lips to the intraoral attachments located on the expansion appliance, approximately at the gingival level of the canine. rapid maxillary expansion appliance rapid maxillary expansion appliance the face mask applaince after duration of 9 months of orthopedic correction, then the pre - adjusted edgewise appliance was fixed with extraction of lower 1 premolar and treatment was continued for 12 months. maxillomandibular relations [figures 6 and 7 ] showed significant improvements during the treatment period, with changes due primarily to the increase in the sella nasion- point a angle (sna) angle. the sella - nasion - point b (snb) angle showed no significant changes during protraction. the dental measurements showed a tendency for the upper incisors to flare during treatment ; the lower incisors were uprighted significantly. patient was treated with a combination of face mask and rme [figures 3 - 5 ] until 2 mm positive overjet was achieved. the expansion screw was activated one to three turns (0.25 mm / turn) at weekly visits until the desired amount of expansion had been achieved. the face mask was adjusted to rest on the forehead and the chin of the patient. elastics (5/16 inch by 14 ounces) were worn from hooks located 2 - 3 cm in front of the lips to the intraoral attachments located on the expansion appliance, approximately at the gingival level of the canine. rapid maxillary expansion appliance rapid maxillary expansion appliance the face mask applaince after duration of 9 months of orthopedic correction, then the pre - adjusted edgewise appliance was fixed with extraction of lower 1 premolar and treatment was continued for 12 months. maxillomandibular relations [figures 6 and 7 ] showed significant improvements during the treatment period, with changes due primarily to the increase in the sella nasion- point a angle (sna) angle. the sella - nasion - point b (snb) angle showed no significant changes during protraction. the dental measurements showed a tendency for the upper incisors to flare during treatment ; the lower incisors were uprighted significantly. this case report showed the results of the treatment of patients with class iii malocclusion at an early stage with an efficient orthodontic therapy of rme plus maxillary protraction. class iii combination therapy is a comprehensive non - surgical treatment strategy designed for developing skeletal class iii malocclusions by incorporating orthodontic and orthopedic mechanics to effectively improve the patient 's occlusion and profile. in properly selected cases, this modality of treatment can be a successful alternative that satisfies a patient 's request to avoid surgery or premolar extraction. | a case report is presented of a class iii malocclusion with a class iii skeletal pattern and maxillary retrusion. patient, a 10-year - old boy was treated with an orthopedic face mask in conjunction with rapid maxillary expansion and standard pre - adjusted edgewise appliance. treatment was completed after 3 years and proved to be stable following the active treatment. |
a 44 year old single male, presented to our outpatient department with complaints of day time excessive sleepiness, lethargy, low mood and disturbed sleep at night for the past 3 months. history revealed that these symptoms were triggered by the death of his father. on detailed evaluation, it was found that he had been on treatment for his mental illness from 2005. the course of illness was continuous characterized by withdrawn behavior, preoccupation, fear, auditory hallucinations, referential delusions and decline in social and occupational functioning. he was evaluated by a psychiatrist and started on psychotropics. during follow - up, the patient complained of episodes of depressed mood, anxiety and sleep disturbance, lethargy and sleepiness that affected his shift work, for which he was prescribed modafinil 200 mg, along with the antipsychotics risperidone 4 mg and amisulpride 400 mg. the patient himself gradually increased the dose to overcome the drowsiness that interrupted his shift work. he started with 100 mg every 3 - 4 h over a shift work of 12 h. for the last 6 months he was unable to overcome his sleepiness during work without modafinil 100 mg / h thus making a total of 1200 mg / day of modafinil which he used to obtain over the counter. he claimed to have symptoms of worsening of lethargy, tremors of hands, anxiety and erratic sleep hours when he skipped modafinil, patient reported a sense of well - being only with the drug and with the above dose. 12.4 gm% ;, wbc count 6300 cells / mm ; platelet count 2.4 lakhs / mm ;, random blood sugar 122 mg / dl. his metabolic parameters including renal function, liver function, lipid profile were within normal limits. an additional diagnosis of modafinil dependence syndrome (dependence criteria as per dsm v) was made in view of the tolerance, withdrawal, inability to cut down, progressive increase in the amount of drug over a longer period of time and craving. the dose was tapered slowly over a period of 1 month with 100 mg every 2 - 3 days and started on bupropion. he reported sleep disturbance, increased sense of body warmth, lethargy and low mood during the process of tapering the drug. in literature, only one case study has been reported on modafinil dependence at higher doses. in our case report, the patient reported himself started consuming more tablets to increase the effects derived from modafinil and landed with dependence phenomenon. unlike amphetamine and its derivatives, modafinil is said to act by mechanisms independent of dopminergic system which is involved in dependence phenomenon of sympathomimetics and opioids and hence is devoid of addiction potential. however there are few recent studies reporting that modafinil also binds to dopamine receptors and also affects dopamine uptake by dopamine transporters in the neurons. increasing doses of modafinil another study conducted to know about modfinil 's subjective and behavioral effects showed that modafinil increases rating scale of the addiction research center inventory (arci) as much as amphetamine. modafinil dependence can be attributed to its dopamine uptake blockade thereby increasing its concentration in dopaminergic areas of brain. overdose of modafinil is not safe ; as a central nervous system stimulant it is prone to cause insomnia, agitation, tachycardia, rise in blood pressure etc. there are no controlled trials or reports available for the treatment of modafinil addiction. in this patient, we cross - tapered modafinil with bupropion because of its action on dopamine and norepinephrine reuptake inhibiton, thus producing an effect similar to that of modafinil with antidepressant activity. modafinil is listed as a schedule iv drug in the united states for its restricted sale. there is no regulation on modafinil sale which has to be revised to prevent its abuse. it is widely purchased over the counter by night shift workers to increase work the efficiency without sleep. though abuse potential is claimed to be less, it should be considered serious due to increased use of modafinil among youngsters. our case report is one among the very few reports of modafinil dependence, it gives a call for the need of regulations on the sale of modafinil. | modafinil, a non - amphetamine psychostimulant, is indicated for narcolepsy, shift work sleep disorder and severe obstructive sleep apnea syndrome. modafinil is prescribed at the dose of 100 mg once in a day or as two doses, 12 h apart in a day. it has also been found that it reduces cocaine dependence and withdrawal phenomenon. modafinil is claimed to have very low liability for abuse and dependence. here we report a rare case of modafinil dependence. |
the carcinogenic and mutagenic effects of the aromatic amines are believed to depend on their covalent modification of dna, primarily through the formation of adducts at c8 of guanine. the actual biologic and biochemical responses to these adducts can be envisioned as the consequence of the abilities of the cell to repair the lesions, with or without fidelity, and the introduction of errors through bypass of the adducts by polymerases. a key question is whether changes in dna sequence arise through the participation of common repair processes that cause mutations independent of adduct structure. alternatively, do mutations arise through miscoding during polymerase bypass at the site of the adducts and are, therefore, more likely to produce sequence changes that are more characteristic of adduct structure ? this question has been approached using single, site - specific, or randomly introduced aromatic amine dna adducts in bacterial cells, and in vitro studies with dna polymerases that employ site - specifically modified templates. the results of both approaches demonstrate that these adducts are distinguished readily by virtue of their structures, thus supporting the conclusion that mutagenic effects of the aromatic amines arise from their structures rather than from their triggering a common inaccurate repair response.imagesfigure 1. |
|
atrial fibrillation (af), whether paroxysmal (paf) or persistent, is a chronic disorder, and recurrence is likely at some point in most patients with af. stroke as well as functional impairments and cardiac failure are the well - known consequences of af. however, interventions such as antiarrhythmic drugs, electrical cardioversion, or catheter ablation for terminating or suppressing af have not been shown to prevent stroke or reduce mortality. on the other hand, patients with af also experience a broad range of symptoms which adversely affect their quality of life (qol). the impact of af on qol is strongly influenced by the segment of the population that is concerned, as some patients are entirely asymptomatic. one study shows that the majority of patients with paf consider the dysrhythmia disruptive to their life ; however, qol literature specific to paf patients is sparse. in patients with recurrent arrhythmias, radiofrequency catheter ablation of the atrioventricular (av) node and pacemaker insertion improved qol as determined by the sf-36 health status questionnaire and the disease - specific symptom checklist frequency and severity scale. however, the relative impact of af among patients who have received little prior treatment to restore or maintain sinus rhythm is not clear. in a study of patients with symptomatic af, qol improved after pharmacological treatment of their ailment, and patients in whom therapy prevented af recurrence experienced the greatest benefit. the latter study also suggests that measures of subjective well - being are important adjunct measures, in addition to objective measures of disease severity (e.g. frequency or duration of af attacks). other studies have confirmed a meaningful improvement of qol after both pharmacological and non - pharmacological af therapies. thus, one of the primary goals of rhythm control interventions should be to control symptoms and improve qol. flecainide acetate, a class ic antiarrhythmic used as a preventive treatment, has been shown to significantly decrease the incidence of paf episodes, with a good safety profile. the first is an immediate - release formulation (flec ir) to be taken twice a day, the second is a once - a - day controlled - release formulation (flec cr). the primary objective of the present study was to evaluate the impact of the control of symptomatic paf on qol. a composite efficacy and safety criterion of clinical success was also evaluated as a secondary endpoint. this study was conducted in compliance with the ethical principles of the revised declaration of helsinki (somerset west, republic of south africa, 1996). local independent ethics committees approved the study protocol and the patients provided informed consent prior to study entry. patients presenting with documented symptomatic paf (arrhythmia terminating spontaneously) either controlled (defined a priori in the protocol as no more than one symptomatic paf episode per 6 months) or uncontrolled (two or more symptomatic paf episodes per 6 months) were considered for study eligibility. during the study, an issue with the above definition was identified : patients undergoing a treatment modification within a few months of inclusion and experiencing no symptomatic paf episodes since the modification were being classified as uncontrolled at inclusion based on symptomatic paf episodes in the last 6 months but prior to the treatment modification. in particular, patients having undergone a paf treatment modification in the last 16 months prior to inclusion were defined as controlled if they had no symptomatic paf episodes occurring under the last therapeutic strategy or uncontrolled if they had at least one symptomatic paf episode. since it was considered that sites would more easily recruit controlled patients and in order to ensure more than one - third of recruited patients were uncontrolled at inclusion, patients of either sex were included in the study if they met the following criteria : aged 18 to < 80 years ; in sinus rhythm at the time of inclusion ; who had experienced symptomatic af episodes of 1 min to 72 h, based on the history of at least two symptomatic paf episodes, at least one of which was documented by electrocardiography (ecg) and/or holter ; requiring antiarrhythmic therapy in the investigator 's judgement ; with left ventricular ejection fraction (lvef) of at least 40% documented by ultrasonography ; and women of childbearing potential had to use a reliable method of contraception. those patients exhibiting the following criteria were not included : intolerance and/or failure of previous therapy with flec ir ; severe symptoms (syncope and ischaemic angina) during episodes of arrhythmia ; coronary heart disease and/or history of myocardial infarction ; congestive heart failure [new york heart association classes ii, iii, and iv ] ; history of arrhythmia other than paf ; paroxysmal supraventricular tachycardia or atrial flutter unless ablated ; sinus dysfunction or atrial disease (bradytachycardia syndrome) ; heart rate < 45 b.p.m. ; second- or third - degree av block ; right bundle branch block associated with left hemiblock or complete left bundle branch block ; implanted pacemaker ; renal failure ; decompensated cirrhosis ; significant extracardiac or systemic disease susceptible of interfering with assessment of qol. this international, open - label study was conducted from september 2003 until september 2005 by 49 cardiologists from 47 cardiology practices (8 hospitals, 4 private hospitals, 1 cardiology centre, and 34 private practices) in france, belgium, and italy. depending on their previous antiarrhythmic treatment, patients were managed as follows : (i) patients not exposed to flec ir at inclusion entered a 2-week titration period starting with flec cr 100 mg once a day that was increased to 200 mg from day 8 onwards provided that the qrs duration was < 140 ms and had not increased by 25% from baseline and that there were no tolerability issues ; (ii) patients under flec ir at inclusion were switched to the equivalent daily dose of flec cr for 48 weeks without titration. follow - up visits were scheduled for all patients at week 12 (w12), w24, and w48. study data were reported in paper case report forms designed according to the study protocol. on the basis of pre - defined safety and efficacy criteria, doses of flec cr could be increased to a maximum of 200 mg per day or reduced by 50 mg per day to a minimum of 100 mg per day, where applicable. amiodarone as a preventive treatment of paf had to have been interrupted for at least 4.5 months before inclusion in the study. previous use of amiodarone for the purpose of cardioversion was allowed, provided treatment duration was limited to a maximum of 8 days. the ongoing use of beta - blockers, digoxin, or calcium antagonists was permitted, provided daily doses used before the start of flec cr were kept constant throughout study treatment. a generic and a disease - specific self - administered qol questionnaires were filled in by the patients in their own language (french for france, french or dutch for belgium, and italian for italy) at baseline, w12, w24, and w48. to assess the primary endpoint of qol, the generic medical outcomes study 36-item short - form health survey (mos sf-36) was used. the mos sf-36 provides eight subscale measures of qol based on 35 of the 36 items in the survey : physical functioning (10 items scored 13), role limitations due to physical problems (role physical, 4 items scored 12), bodily pain (2 items scored 15 and 16, respectively), general health (5 items scored 15), vitality (4 items scored 16), role limitations due to emotional problems (role emotional, 3 items scored 12), social functioning (2 items scored 15), and mental health (5 items scored 15). two summary scores, mental component scale and physical component scale, were computed by linear combinations of the eight subscale measures. the atrial fibrillation severity scale (afss, parts a, b, and c) was also used to assess qol. at baseline, w12, w24, and w48, patients completed the disease - specific self - administered questionnaire to assess well - being and bothering symptom frequency via question 4 of afss part a and questions 17 of afss part c. at baseline, patients were also asked afss part a questions 58 pertaining to af burden. afss part b was completed by the investigator at baseline to further characterize paf history. the clinical success composite safety and efficacy criterion was defined as follows : patient alive ; without pharmacological or electrical cardioversion ; with sinus rhythm maintained (not more than one documented af per 24-week period) ; still receiving flec cr ; with qrs duration < 140 ms and change from baseline < 25% ; and with lvef of at least 35%. patients not fulfilling all of these criteria for clinical success were considered as clinical failures. other efficacy endpoints were the time to first paf recurrence objectively documented by ecg or subjectively documented by inquiry and comments recorded on the patient 's diary. cardiac safety was assessed based on the incidence of proarrhythmic effects (worsening paf and/or occurrence of atrial flutter or of paroxysmal supraventricular tachycardia not previously diagnosed and/or occurrence of a clinically significant ventricular arrhythmia in the absence of the known history of ventricular arrhythmia), ecg changes (mostly qrs and qtc changes), and signs of cardiac failure (clinical examination and left ventricular function by ultrasonography). the safety data set included all patients who took at least one capsule of controlled - release flecainide acetate after study inclusion. the intention - to - treat (itt) efficacy data set included all patients from the safety data set except for those treated with flec ir who were not controlled at inclusion (exclusion criterion). the qol baseline and post - baseline data sets excluded patients with missing baseline sf-36 or control status data. any missing post - baseline qol data for patients in the qol post - baseline data set were extrapolated using the last observation carried forward (locf) method for analysis purposes. the score of each subscale of the sf-36 was calculated as the mean of the items, except in the case where more than half of the items were missing, in which case, the subscore was considered as missing. the physical component and mental component summary scores were calculated as the linear combination of the eight standardized [mean = 0, standard deviation (sd) = 1 ] subscores. each summary score was then transformed by multiplying by 10 and adding 50 in order to be compared with scores issued from a reference population with mean 50 and sd 10. the reference population was derived from national surveys representative in terms of age and gender. the process matched each treated study patient with a random sample of country / language-, gender-, and age - matched subjects from a general reference population. controlled and uncontrolled patients were compared with the reference population for sf-36 scores at baseline, w24, and w48 by paired t - tests. complementary analyses were stratified by country / language, with age and gender as covariates. the robustness of the results was assessed using a non - parametric analysis of covariance (ancova). afss scores [part a (4) and part c (17) ] were analysed and compared between the pre - defined analysis subgroups using a wilcoxon test. clinical failure was analysed globally and in terms of time to clinical failure using the kaplan cox 's proportional hazards models were used to assess the relationship between the difference from baseline to end of study for each qol score and (i) clinical failure and (ii) first paf recurrence. it was considered necessary to enrol 240 patients (assuming 15% of the patients would be non - evaluable) in order to achieve 95% power at a two - sided type i error rate of 5% for comparisons of sf-36 physical and mental health summary measures between patient subgroups and the reference population. it was considered that half of the sd of the reference population (sd = 10) would be a perceptible variation (medium effect size). this study was conducted in compliance with the ethical principles of the revised declaration of helsinki (somerset west, republic of south africa, 1996). local independent ethics committees approved the study protocol and the patients provided informed consent prior to study entry. patients presenting with documented symptomatic paf (arrhythmia terminating spontaneously) either controlled (defined a priori in the protocol as no more than one symptomatic paf episode per 6 months) or uncontrolled (two or more symptomatic paf episodes per 6 months) were considered for study eligibility. during the study, an issue with the above definition was identified : patients undergoing a treatment modification within a few months of inclusion and experiencing no symptomatic paf episodes since the modification were being classified as uncontrolled at inclusion based on symptomatic paf episodes in the last 6 months but prior to the treatment modification. in particular, patients having undergone a paf treatment modification in the last 16 months prior to inclusion were defined as controlled if they had no symptomatic paf episodes occurring under the last therapeutic strategy or uncontrolled if they had at least one symptomatic paf episode. since it was considered that sites would more easily recruit controlled patients and in order to ensure more than one - third of recruited patients were uncontrolled at inclusion, each investigator was encouraged to recruit one uncontrolled patient for one controlled patient. patients of either sex were included in the study if they met the following criteria : aged 18 to < 80 years ; in sinus rhythm at the time of inclusion ; who had experienced symptomatic af episodes of 1 min to 72 h, based on the history of at least two symptomatic paf episodes, at least one of which was documented by electrocardiography (ecg) and/or holter ; requiring antiarrhythmic therapy in the investigator 's judgement ; with left ventricular ejection fraction (lvef) of at least 40% documented by ultrasonography ; and women of childbearing potential had to use a reliable method of contraception. those patients exhibiting the following criteria were not included : intolerance and/or failure of previous therapy with flec ir ; severe symptoms (syncope and ischaemic angina) during episodes of arrhythmia ; coronary heart disease and/or history of myocardial infarction ; congestive heart failure [new york heart association classes ii, iii, and iv ] ; history of arrhythmia other than paf ; paroxysmal supraventricular tachycardia or atrial flutter unless ablated ; sinus dysfunction or atrial disease (bradytachycardia syndrome) ; heart rate < 45 b.p.m. ; second- or third - degree av block ; right bundle branch block associated with left hemiblock or complete left bundle branch block ; implanted pacemaker ; renal failure ; decompensated cirrhosis ; significant extracardiac or systemic disease susceptible of interfering with assessment of qol. this international, open - label study was conducted from september 2003 until september 2005 by 49 cardiologists from 47 cardiology practices (8 hospitals, 4 private hospitals, 1 cardiology centre, and 34 private practices) in france, belgium, and italy. depending on their previous antiarrhythmic treatment, patients were managed as follows : (i) patients not exposed to flec ir at inclusion entered a 2-week titration period starting with flec cr 100 mg once a day that was increased to 200 mg from day 8 onwards provided that the qrs duration was < 140 ms and had not increased by 25% from baseline and that there were no tolerability issues ; (ii) patients under flec ir at inclusion were switched to the equivalent daily dose of flec cr for 48 weeks without titration. follow - up visits were scheduled for all patients at week 12 (w12), w24, and w48. study data were reported in paper case report forms designed according to the study protocol. on the basis of pre - defined safety and efficacy criteria, doses of flec cr could be increased to a maximum of 200 mg per day or reduced by 50 mg per day to a minimum of 100 mg per day, where applicable. amiodarone as a preventive treatment of paf had to have been interrupted for at least 4.5 months before inclusion in the study. previous use of amiodarone for the purpose of cardioversion was allowed, provided treatment duration was limited to a maximum of 8 days. the ongoing use of beta - blockers, digoxin, or calcium antagonists was permitted, provided daily doses used before the start of flec cr were kept constant throughout study treatment. a generic and a disease - specific self - administered qol questionnaires were filled in by the patients in their own language (french for france, french or dutch for belgium, and italian for italy) at baseline, w12, w24, and w48. to assess the primary endpoint of qol, the generic medical outcomes study 36-item short - form health survey (mos sf-36) was used. the mos sf-36 provides eight subscale measures of qol based on 35 of the 36 items in the survey : physical functioning (10 items scored 13), role limitations due to physical problems (role physical, 4 items scored 12), bodily pain (2 items scored 15 and 16, respectively), general health (5 items scored 15), vitality (4 items scored 16), role limitations due to emotional problems (role emotional, 3 items scored 12), social functioning (2 items scored 15), and mental health (5 items scored 15). two summary scores, mental component scale and physical component scale, were computed by linear combinations of the eight subscale measures. the atrial fibrillation severity scale (afss, parts a, b, and c) was also used to assess qol. at baseline, w12, w24, and w48, patients completed the disease - specific self - administered questionnaire to assess well - being and bothering symptom frequency via question 4 of afss part a and questions 17 of afss part c. at baseline, patients were also asked afss part a questions 58 pertaining to af burden. afss part b was completed by the investigator at baseline to further characterize paf history. the clinical success composite safety and efficacy criterion was defined as follows : patient alive ; without pharmacological or electrical cardioversion ; with sinus rhythm maintained (not more than one documented af per 24-week period) ; still receiving flec cr ; with qrs duration < 140 ms and change from baseline < 25% ; and with lvef of at least 35%. patients not fulfilling all of these criteria for clinical success were considered as clinical failures. other efficacy endpoints were the time to first paf recurrence objectively documented by ecg or subjectively documented by inquiry and comments recorded on the patient 's diary. cardiac safety was assessed based on the incidence of proarrhythmic effects (worsening paf and/or occurrence of atrial flutter or of paroxysmal supraventricular tachycardia not previously diagnosed and/or occurrence of a clinically significant ventricular arrhythmia in the absence of the known history of ventricular arrhythmia), ecg changes (mostly qrs and qtc changes), and signs of cardiac failure (clinical examination and left ventricular function by ultrasonography). the safety data set included all patients who took at least one capsule of controlled - release flecainide acetate after study inclusion. the intention - to - treat (itt) efficacy data set included all patients from the safety data set except for those treated with flec ir who were not controlled at inclusion (exclusion criterion). the qol baseline and post - baseline data sets excluded patients with missing baseline sf-36 or control status data. any missing post - baseline qol data for patients in the qol post - baseline data set were extrapolated using the last observation carried forward (locf) method for analysis purposes. the score of each subscale of the sf-36 was calculated as the mean of the items, except in the case where more than half of the items were missing, in which case, the subscore was considered as missing. the physical component and mental component summary scores were calculated as the linear combination of the eight standardized [mean = 0, standard deviation (sd) = 1 ] subscores. each summary score was then transformed by multiplying by 10 and adding 50 in order to be compared with scores issued from a reference population with mean 50 and sd 10. the reference population was derived from national surveys representative in terms of age and gender. the process matched each treated study patient with a random sample of country / language-, gender-, and age - matched subjects from a general reference population. controlled and uncontrolled patients were compared with the reference population for sf-36 scores at baseline, w24, and w48 by paired t - tests. complementary analyses were stratified by country / language, with age and gender as covariates. the robustness of the results was assessed using a non - parametric analysis of covariance (ancova). afss scores [part a (4) and part c (17) ] were analysed and compared between the pre - defined analysis subgroups using a wilcoxon test. clinical failure was analysed globally and in terms of time to clinical failure using the kaplan cox 's proportional hazards models were used to assess the relationship between the difference from baseline to end of study for each qol score and (i) clinical failure and (ii) first paf recurrence. it was considered necessary to enrol 240 patients (assuming 15% of the patients would be non - evaluable) in order to achieve 95% power at a two - sided type i error rate of 5% for comparisons of sf-36 physical and mental health summary measures between patient subgroups and the reference population. it was considered that half of the sd of the reference population (sd = 10) would be a perceptible variation (medium effect size). of the 230 patients enrolled in the study, 229 were treated (the patient excluded from the safety data set had no treatment data and no adverse reactions reported). the baseline control status of five patients was missing and five other patients had missing sf-36 data at baseline ; these patients were excluded from the qol baseline data set (219 patients : 155 french, 22 french - speaking belgians, 20 dutch - speaking belgians, and 22 italians). two patients were excluded from post - baseline analyses due to their failure to satisfy the major entry criterion of control under flec ir at inclusion. in particular, the itt efficacy data set was comprised of 227 patients : 126 controlled (100 of whom were switched from flec ir at inclusion) ; 96 uncontrolled ; and 5 unknown status (1 of whom was switched from flec ir at inclusion). the qol post - baseline data set was comprised of 217 patients : 123 controlled and 94 uncontrolled. demographic and main baseline characteristics of the safety data set according to symptomatic paf control at inclusion are summarized in table 1. of the five patients with missing control status, three were male and two were female. they had a median time since paf diagnosis of 0.1 months and four of the patients had a median time since first symptomatic paf episode of 1.7 months. demographic and other baseline characteristics of the patients (safety data set) five unknown control status ; sd, standard deviation ; paf, paroxysmal atrial fibrillation. differences in age and gender were not statistically different between the two subgroups. time since first symptomatic paf episode was much shorter in uncontrolled compared with controlled patients, as shown in particular by the percentages of patients with a time shorter than 6 months (54.2 vs. 5.8%, respectively) and conversely by the percentages of patients with time between 12 and 60 months (17.7 vs. 46.7%) or at least 60 months (18.8 vs. 35.0%). sixteen patients in the controlled group were diagnosed with paf (according to the study requirement of two symptomatic pafs with at least one documented by ecg and/or holter) in the last 6 months prior to inclusion. however, only seven of them had experienced their first symptomatic paf episode within the last 6 months. these seven patients were included in the controlled group because they had started treatment for paf following several symptomatic paf episodes at least 1 month prior to inclusion and had experienced no symptomatic paf episodes since treatment instauration. overall, the most frequent symptoms accompanying paf were palpitations (85.2% of patients), fatigue (31.0%), respiratory disorders (20.5%), and chest pain (11.8%). a comparison of the baseline sf-36 scores of controlled patients to their matched reference population showed that their qol scores were significantly better for the physical functioning subscale, the bodily pain subscale, and the physical component score. the other sf-36 scores showed non - statistically significant differences that favoured the controlled group for all scores except for the role emotional and mental health scores, and consequently the global mental component score, which favoured the reference population. for the uncontrolled group, all of the 10 qol scores were worse compared with the matched reference population, of which 7 differences were statistically significant. the relationship between uncontrolled symptomatic paf and inferior qol impacted the mental scores (significantly worse for role emotional, social functioning, mental health, and mental component) more than the physical scores, although the role physical score, which reflects work and daily activities, and the mixed general health and vitality scores were also significantly worse for uncontrolled patients compared with the reference population. the difference of 4.82 for the global mental component score in the uncontrolled group corresponds to an influence of non - control of af on qol close to half the sd (4.82/10.91 = 0.44), which can be considered as a medium effect size. mean differences in baseline sf-36 scores are presented by control group in table 2. mean differences (reference study subgroup) in baseline sf-36 scores for controlled and uncontrolled patients (qol baseline data set) higher subscale scores indicated better qol. n, total number of patients ; ci, confidence interval ; sd, standard deviation. the analysis of the differences between the controlled and the uncontrolled groups in baseline sf-36 scores and changes from baseline at w12, w24, and w48 are presented in table 3. the differences were mostly positive at baseline (indicating worse qol in the uncontrolled group, and reaching statistical significance for the general health and vitality scores and for the mental component score), then mostly negative for changes from baseline at the subsequent time points, indicating a greater improvement of qol under flec cr treatment in the uncontrolled group. the most significant results were obtained at w24, and the effect was maintained at w48. patients entering the trial under flec ir, who represented 80% of the controlled patients, had their qol maintained when switched to flec cr. summary of controlled and uncontrolled subgroup scores for baseline, w12, w24, and w48 in sf-36 scores (qol post - baseline data set) results are expressed in least square means (p - value) adjusted for age, country, and language in country. p - values for post - baseline differences are adjusted for multiplicity using a step - down permutation procedure taking into account correlations among hypothesis tests. the mean well - being score (sd) at baseline as assessed from question 4 of afss part a was 6.7 (2.0) in the uncontrolled group compared with 7.2 (1.5) in the controlled group. the total afss score, calculated as the sum of scores for questions 17 of afss part c was assessed at baseline, w12, w24, w48, and at end of study. as expected, the mean score at baseline was higher for uncontrolled patients compared with controlled patients. still higher in the uncontrolled group at w12, it then became similar to that of the controlled group at w24 and was maintained at the same level in both groups thereafter (table 4). evolution of total afss [part c (17) ] (qol baseline data set) the clinical success rate was similar in the controlled and uncontrolled groups with, respectively, 89 of 126 (70.6%) and 66 of 96 (68.8%) patients classified as successes. the other 67 patients failed for one or more of the following reasons : pharmacological / electrical cardioversion, 7 of 37 and 4 of 30 for the controlled and uncontrolled groups, respectively ; two or more documented paf per 24-week period, 1 of 37 and 2 of 30 ; withdrawn for treatment inefficacy / safety reasons, for non - compliance or for death, 24 of 37 and 22 of 30 ; and finally withdrawn due to delta qrs 25 or duration 140, 11 of 37 and 12 of 30, respectively. lvef < 35%. of the 158 patients (69.8%) who were clinical successes, 153 (67.4%) completed the study and 5 (2.2%) were withdrawn from the study for reasons not related to efficacy, safety, or non - compliance. the latter five patients were right censored at the end of treatment (or end of study if end of treatment was missing) for time - to - event analyses. the cox proportional hazards analysis of the relationship between clinical failure and each sf-36 score difference from baseline to end of study showed that physical functioning [p = 0.01, hazard ratio (hr) = 0.981 (0.966, 0.996) ], bodily pain [p = 0.001, hr = 0.981 (0.970, 0.993) ], and physical component [p = 0.0003, hr = 0.946 (0.917, 0.974) ] scores were strongly related to clinical failure. (an hr of < 1 means there is a lower risk of clinical failure for every one point reduction in the sf-36 component score.) of the 227 patients in the itt data set, 87 (38.3%) experienced at least one paf episode on study treatment [41 (32.5%) controlled and 45 (46.9%) uncontrolled patients ]. twenty - three (10.1%) patients experienced at least one documented paf episode on study treatment. three (1.3%) patients experienced two or more documented paf episodes per 24-week period. time to first paf episode after the start of study treatment was significantly shorter (log - rank test, p = 0.015 ; wilcoxon test, p = 0.003) in the uncontrolled group [mean (sd) time of 42.0 (63.0) days ; median of 7.0 days ] compared with the controlled group [mean time (sd) of 94.2 (93.6) days ; median of 81.0 days ]. the analysis of the relationship between first paf recurrence and each qol score difference from baseline to end of study showed that physical functioning [p = 0.005, hr = 0.981 (0.969, 0.994) ] and physical component [p = 0.02, hr = 0.967 (0.942, 0.994) ] scores were strongly related to first paf recurrence. (an hr of < 1 means there is a lower risk of clinical failure for every one point reduction in the sf-36 component score.) similar trends were seen in the 23 patients (12 controlled, 10 uncontrolled, and 1 unknown) who experienced at least one documented paf recurrence. fifteen events related to proarrhythmic effects were observed in 14 of 229 (6.1%) patients. they included atrial flutter (six patients), bradycardia (three patients), right bundle branch block (two patients), and sudden death (one patient). the other three events were not reported by the investigator but were documented as adverse events related to proarrhythmic effects by the sponsor after a post - study review of the ecg data. the sudden death of a 77-year - old male with uncontrolled symptomatic paf at inclusion, who had a 10-year history of high blood pressure and venous insufficiency and a recent aortic regurgitation, was considered probably not related to the study drug by the investigator. in the absence of sufficient information to definitely exclude a proarrhythmic effect of flec cr, the serious adverse event was documented by the sponsor as possibly related to the study drug. a qrs duration 140 ms or an increase in qrs by 25% or more was noted for 2 of 109 (1.8%) patients by w12 in the controlled group vs. 7 of 83 (8.4%) in the uncontrolled group. the mean (sd, median) qrs change at the end of the study was 1.1% (17.9, 0.0%) for the controlled group compared with 4.7% (15.1, 2.6%) for the uncontrolled group. at the end of the study, qtc was prolonged to over 440 ms in 15 of 106 (14.2%) controlled patients with a baseline qtc interval of no more than 440 ms compared with 15 of 80 (18.8%) uncontrolled patients. comparing patients based on flec ir status at inclusion (flec ir vs. no flec ir), at the end of the study, qtc was prolonged to over 440 ms in 10 of 88 (11.4%) flec ir patients with a baseline qtc interval of no more than 440 ms compared with 20 of 101 (19.8%) no flec ir patients. a total of 146 of 229 (63.8%) patients experienced at least one treatment emergent adverse event during the study and 23 serious adverse events were reported in 20 (8.7%) patients (including one death described earlier). gastrointestinal disorders (20.5% of the patients), nervous system disorders (19.7%), musculoskeletal disorders (18.8%), infections and infestations (16.2%), general disorders (13.1%), cardiac disorders (12.7%), and vascular disorders (11.4%) were the most common system organ classes (meddra) of adverse events in the study. thirty - five (15.3%) patients experienced events which were thought by the investigator to be possibly or probably related to study treatment and 7.9% of the patients were withdrawn from the study due to the occurrence of an adverse event. demographic and main baseline characteristics of the safety data set according to symptomatic paf control at inclusion are summarized in table 1. of the five patients with missing control status, they had a median time since paf diagnosis of 0.1 months and four of the patients had a median time since first symptomatic paf episode of 1.7 months. demographic and other baseline characteristics of the patients (safety data set) five unknown control status ; sd, standard deviation ; paf, paroxysmal atrial fibrillation. time since first symptomatic paf episode was much shorter in uncontrolled compared with controlled patients, as shown in particular by the percentages of patients with a time shorter than 6 months (54.2 vs. 5.8%, respectively) and conversely by the percentages of patients with time between 12 and 60 months (17.7 vs. 46.7%) or at least 60 months (18.8 vs. 35.0%). sixteen patients in the controlled group were diagnosed with paf (according to the study requirement of two symptomatic pafs with at least one documented by ecg and/or holter) in the last 6 months prior to inclusion. however, only seven of them had experienced their first symptomatic paf episode within the last 6 months. these seven patients were included in the controlled group because they had started treatment for paf following several symptomatic paf episodes at least 1 month prior to inclusion and had experienced no symptomatic paf episodes since treatment instauration. overall, the most frequent symptoms accompanying paf were palpitations (85.2% of patients), fatigue (31.0%), respiratory disorders (20.5%), and chest pain (11.8%). a comparison of the baseline sf-36 scores of controlled patients to their matched reference population showed that their qol scores were significantly better for the physical functioning subscale, the bodily pain subscale, and the physical component score. the other sf-36 scores showed non - statistically significant differences that favoured the controlled group for all scores except for the role emotional and mental health scores, and consequently the global mental component score, which favoured the reference population. for the uncontrolled group, all of the 10 qol scores were worse compared with the matched reference population, of which 7 differences were statistically significant. the relationship between uncontrolled symptomatic paf and inferior qol impacted the mental scores (significantly worse for role emotional, social functioning, mental health, and mental component) more than the physical scores, although the role physical score, which reflects work and daily activities, and the mixed general health and vitality scores were also significantly worse for uncontrolled patients compared with the reference population. the difference of 4.82 for the global mental component score in the uncontrolled group corresponds to an influence of non - control of af on qol close to half the sd (4.82/10.91 = 0.44), which can be considered as a medium effect size. mean differences in baseline sf-36 scores are presented by control group in table 2. mean differences (reference study subgroup) in baseline sf-36 scores for controlled and uncontrolled patients (qol baseline data set) higher subscale scores indicated better qol. n, total number of patients ; ci, confidence interval ; sd, standard deviation. statistically significant at the 0.05 level. the analysis of the differences between the controlled and the uncontrolled groups in baseline sf-36 scores and changes from baseline at w12, w24, and w48 are presented in table 3. the differences were mostly positive at baseline (indicating worse qol in the uncontrolled group, and reaching statistical significance for the general health and vitality scores and for the mental component score), then mostly negative for changes from baseline at the subsequent time points, indicating a greater improvement of qol under flec cr treatment in the uncontrolled group. the most significant results were obtained at w24, and the effect was maintained at w48. patients entering the trial under flec ir, who represented 80% of the controlled patients, had their qol maintained when switched to flec cr. summary of controlled and uncontrolled subgroup scores for baseline, w12, w24, and w48 in sf-36 scores (qol post - baseline data set) results are expressed in least square means (p - value) adjusted for age, country, and language in country. p - values for post - baseline differences are adjusted for multiplicity using a step - down permutation procedure taking into account correlations among hypothesis tests. the mean well - being score (sd) at baseline as assessed from question 4 of afss part a was 6.7 (2.0) in the uncontrolled group compared with 7.2 (1.5) in the controlled group. the total afss score, calculated as the sum of scores for questions 17 of afss part c was assessed at baseline, w12, w24, w48, and at end of study. as expected, the mean score at baseline was higher for uncontrolled patients compared with controlled patients. still higher in the uncontrolled group at w12, it then became similar to that of the controlled group at w24 and was maintained at the same level in both groups thereafter (table 4). these results are consistent with those obtained with the sf-36 questionnaire. evolution of total afss [part c (17) ] (qol baseline data set) the clinical success rate was similar in the controlled and uncontrolled groups with, respectively, 89 of 126 (70.6%) and 66 of 96 (68.8%) patients classified as successes. the other 67 patients failed for one or more of the following reasons : pharmacological / electrical cardioversion, 7 of 37 and 4 of 30 for the controlled and uncontrolled groups, respectively ; two or more documented paf per 24-week period, 1 of 37 and 2 of 30 ; withdrawn for treatment inefficacy / safety reasons, for non - compliance or for death, 24 of 37 and 22 of 30 ; and finally withdrawn due to delta qrs 25 or duration 140, 11 of 37 and 12 of 30, respectively. < 35%. of the 158 patients (69.8%) who were clinical successes, 153 (67.4%) completed the study and 5 (2.2%) were withdrawn from the study for reasons not related to efficacy, safety, or non - compliance. the latter five patients were right censored at the end of treatment (or end of study if end of treatment was missing) for time - to - event analyses. the cox proportional hazards analysis of the relationship between clinical failure and each sf-36 score difference from baseline to end of study showed that physical functioning [p = 0.01, hazard ratio (hr) = 0.981 (0.966, 0.996) ], bodily pain [p = 0.001, hr = 0.981 (0.970, 0.993) ], and physical component [p = 0.0003, hr = 0.946 (0.917, 0.974) ] scores were strongly related to clinical failure. (an hr of < 1 means there is a lower risk of clinical failure for every one point reduction in the sf-36 component score.) of the 227 patients in the itt data set, 87 (38.3%) experienced at least one paf episode on study treatment [41 (32.5%) controlled and 45 (46.9%) uncontrolled patients ]. twenty - three (10.1%) patients experienced at least one documented paf episode on study treatment. three (1.3%) patients experienced two or more documented paf episodes per 24-week period. time to first paf episode after the start of study treatment was significantly shorter (log - rank test, p = 0.015 ; wilcoxon test, p = 0.003) in the uncontrolled group [mean (sd) time of 42.0 (63.0) days ; median of 7.0 days ] compared with the controlled group [mean time (sd) of 94.2 (93.6) days ; median of 81.0 days ]. the analysis of the relationship between first paf recurrence and each qol score difference from baseline to end of study showed that physical functioning [p = 0.005, hr = 0.981 (0.969, 0.994) ] and physical component [p = 0.02, hr = 0.967 (0.942, 0.994) ] scores were strongly related to first paf recurrence. (an hr of < 1 means there is a lower risk of clinical failure for every one point reduction in the sf-36 component score.) similar trends were seen in the 23 patients (12 controlled, 10 uncontrolled, and 1 unknown) who experienced at least one documented paf recurrence. fifteen events related to proarrhythmic effects were observed in 14 of 229 (6.1%) patients. they included atrial flutter (six patients), bradycardia (three patients), right bundle branch block (two patients), and sudden death (one patient). the other three events were not reported by the investigator but were documented as adverse events related to proarrhythmic effects by the sponsor after a post - study review of the ecg data. the sudden death of a 77-year - old male with uncontrolled symptomatic paf at inclusion, who had a 10-year history of high blood pressure and venous insufficiency and a recent aortic regurgitation, was considered probably not related to the study drug by the investigator. in the absence of sufficient information to definitely exclude a proarrhythmic effect of flec cr, the serious adverse event was documented by the sponsor as possibly related to the study drug. a qrs duration 140 ms or an increase in qrs by 25% or more was noted for 2 of 109 (1.8%) patients by w12 in the controlled group vs. 7 of 83 (8.4%) in the uncontrolled group. the mean (sd, median) qrs change at the end of the study was 1.1% (17.9, 0.0%) for the controlled group compared with 4.7% (15.1, 2.6%) for the uncontrolled group. at the end of the study, qtc was prolonged to over 440 ms in 15 of 106 (14.2%) controlled patients with a baseline qtc interval of no more than 440 ms compared with 15 of 80 (18.8%) uncontrolled patients. comparing patients based on flec ir status at inclusion (flec ir vs. no flec ir), at the end of the study, qtc was prolonged to over 440 ms in 10 of 88 (11.4%) flec ir patients with a baseline qtc interval of no more than 440 ms compared with 20 of 101 (19.8%) no flec ir patients. a total of 146 of 229 (63.8%) patients experienced at least one treatment emergent adverse event during the study and 23 serious adverse events were reported in 20 (8.7%) patients (including one death described earlier). gastrointestinal disorders (20.5% of the patients), nervous system disorders (19.7%), musculoskeletal disorders (18.8%), infections and infestations (16.2%), general disorders (13.1%), cardiac disorders (12.7%), and vascular disorders (11.4%) were the most common system organ classes (meddra) of adverse events in the study. thirty - five (15.3%) patients experienced events which were thought by the investigator to be possibly or probably related to study treatment and 7.9% of the patients were withdrawn from the study due to the occurrence of an adverse event. in this study, patients with uncontrolled symptomatic paf, unlike patients with controlled symptomatic paf, had an inferior perceived health - related qol when compared with a country / language-, gender-, and age - matched general reference population. these patients showed an inferior qol in all eight sf-36 subscales, with significantly worse scores for role physical, general health, vitality, role emotional, social functioning, and mental health and a significantly worse summary score for mental health. the sf-36 is the most widely validated generic instrument available to measure perceived health - related qol and has been used in a number of studies. the present results are in agreement with several published studies including an international study of 152 patients with paf (60.5%) or persistent (39.5%) af and a large inspection cohort of 963 newly diagnosed patients in north america with paf (4.0%) or persistent (96.0%) af. the distinctive feature of the present study is the focus on paf which may disable qol in a different way to persistent or permanent af. indeed paf can concern a different population regarding associated cardiac disease and can be perceived differently due to the paroxysms. in this study, extracardiac co - morbidity could have impacted the sf-36 result as it is a generic instrument ; however, the af - specific scale confirms the difference at baseline between controlled and uncontrolled patients. furthermore, the study population had few co - morbid health conditions. following treatment with controlled - release flecainide acetate, sf-36 scores showed an improvement in the qol of uncontrolled patients, with maximum effect obtained at w24 and maintenance of improvement at w48. results of a sensitivity analysis using a country / language-, gender- and age - adjusted non - parametric ancova on sf-36 scores validate these findings. previous studies have shown that qol improves with a variety of af control therapies, whether pharmacological for rate or rhythm control in persistent af and for rhythm control in studies investigating amiodarone, propafenone, or sotalol in persistent af, or non - pharmacological, such as catheter ablation. total afss scores illustrated a marked improvement of symptoms in the uncontrolled group at w12. scores then continued to improve between w12 and w24 and were maintained at this level between w24 and w48. flecainide acetate is known to significantly decrease the incidence of paf episodes and the relationship between afss improvement and decrease / disappearance of af recurrence has been previously reported in the ctaf. clinical success, based on a composite safety and efficacy endpoint, was observed in 70% of the patients with no difference between the uncontrolled and the controlled groups. there was a significant relationship between clinical failure and the sf-36 scores physical functioning (p = 0.01), bodily pain (p = 0.001), and physical component (p = 0.0003). the hrs presented above indicate a 1.9% reduction in risk is associated with a decrease of one point in both the bodily pain and physical functioning subscales of the sf-36 ; and a 5.4% reduction in risk per single point in the physical component score. physical functioning and physical component scores were also significantly related to the time to first paf recurrence (p = 0.005 and p = 0.02, respectively). the time - to - first - paf - recurrence hrs presented above indicate a 1.9% reduction in risk is associated with a decrease of one point in the physical functioning subscale ; and a 3.3% reduction in risk per single point in the physical component score. since one of the main goals of maintenance therapy is suppression of symptoms and the most beneficial therapies for patients prevent af recurrence, this study was designed to investigate the impact of the control of symptomatic paf on qol. post - baseline analyses investigating the relationship between af burden and qol are beyond the scope of this study. caution should be taken when interpreting study results due to the fact that patient control status is defined based on symptoms reported by the patient. furthermore, there is a significant difference between the two control status subgroups in the duration of illness at inclusion (54.2% of uncontrolled patients experienced their first symptomatic paf episode < 6 months prior to inclusion compared with 5.8% of controlled patients). as such, improvements in qol in the uncontrolled group may be related to patients adjusting to their af. it is beyond the scope of this study to directly attribute improvements in qol to a specific drug effect. another key limitation of the study is the inability to assess all symptomatic paf episodes objectively (ecg). in an attempt to collect information on all symptomatic paf episodes, inquiry and patient diaries were also used to record symptomatic paf based on subjective evaluations without the objective confirmation of an ecg. some analyses combined objective and subjective assessments (as indicated in the description of the analyses) and this should be taken into consideration when interpreting results. in this study, patients with uncontrolled symptomatic paf at baseline had an inferior qol to patients with controlled symptomatic paf. following treatment with controlled - release flecainide acetate, their qol improved to a level comparable to controlled patients. the use of qol questionnaires to assist with therapeutic decision - making and follow - up evaluation for patients with symptomatic paf could prove valuable. funding to pay the open access publication charges for this article was provided by meda pharma sas. france : b. agraou, valenciennes ; a. attal, la seyne sur mer ; a. bendada, maisons laffitte ; a. da costa, st priest en jar ; p. duron, chaville ; y. frances, marseille ; p. giraud, cavaillon ; p. hallali, grenoble ; g. hannebicque, arras ; a. lagrange, limoges ; e. laine, amiens ; r. latour, brive la gaillarde ; r. luccioni, marseille ; j.p. arniaud, marseille ; m. arnoux, marseille ; a. bourdon, marseille ; j.c. pascariello, marseille ; r.- d., cohen, marseille ; b. sorensen, marseille ; b. gindre, ecully ; z. mesli, montpellier ; e. page, grenoble ; f. pellerin, bourges ; o. piot, st denis ; p. poncelet, henin beaumont ; p. remplon, blois ; p.y. tilmant, cambrai ; p. dambrine, freyming merlebach ; r. luccioni, aubagne ; a. abdoullahi, six fours les plages ; j.l. corbelli, la seyne sur mer ; g. lavabre, six fours les plages ; m. proult, sanary sur mer. belgium : a. hita, apt ; p. geelen, aalst ; y. vandekerckhove, brugge ; m. goethals, roeselare ; p. evrard, lige ; j.p. italy : g. boriani, bologna ; f. mascia, caserta ; a. puglisi, roma ; g.l. | aimspatients with atrial fibrillation (af) consider the related symptoms disruptive to their quality of life (qol). this study aimed to evaluate the impact of the control of symptomatic paroxysmal af (paf) on qol.methods and resultspatients with symptomatic paf were treated for 48 weeks with open - label flecainide acetate controlled release (flec cr). quality of life was assessed by sf-36 and atrial fibrillation severity scale scores at baseline, week 12 (w12), w24, and w48. of the 229 treated patients, 217 were analysed for qol (123 with controlled and 94 with uncontrolled symptomatic paf at inclusion). the controlled group had a similar or better qol (sf-36) at baseline compared with a reference population (significantly better for : physical functioning, bodily pain, and physical component). the uncontrolled group had an inferior qol (significantly worse for : role physical, general health, vitality, role emotional, social functioning, mental health, and mental component). when treated with flec cr, the controlled group baseline qol scores were maintained and the uncontrolled group scores were improved to a level comparable to the controlled group scores. safety findings reflect the known clinical safety profile of flecainide acetate.conclusionin this study, patients with uncontrolled symptomatic paf at baseline had an inferior qol to those with controlled symptomatic paf. following treatment with controlled - release flecainide acetate, their qol improved to a level comparable to controlled patients. |
t regulatory cells (tregs) are important mediators of immune suppression, and their presence prevents reactions against self by inducing regulatory signals to antigen presenting cells (apcs) and/or effector t cells [1, 2 ]. their ablation increases the risk of autoimmunity whilst, on the contrary, their signals could also affect nonautoreactive clones, leading to inhibition of antineoplastic, antimicrobial, antiparasitic, and antiviral immune responses [1, 4, 5 ]. accumulating evidence indicates that patients with chronic viral hepatitis display increased numbers of tregs (both natural and inducible) in peripheral blood [68 ] or liver [911 ], which in turn exert a suppressive function against specific hepatitis c virus- (hcv-) or hepatitis b virus- (hbv-) t effector clones in vitro [610 ]. thus, it has been suggested that the expansion of tregs during viral hepatitis may contribute to an inadequate immune response, causing persistent viral infection. however, the precise role of tregs in the pathogenesis of chronic hepatitis is the subject of intense debate, since it has been demonstrated that tregs suppress the function and the expansion of virus - specific t effector cells ex vivo, irrespective of the patients having chronic or resolved virus infection [11, 12 ]. herein, we describe an mrna expression study in biopsy material scheduled to further elucidate the role of tregs in the pathogenesis of liver damage in chronic viral hbv and hcv hepatitis. bearing in mind that recent structural and molecular studies suggest apoptosis, rather than necrosis, being the mechanism of liver cell death in chronic viral hepatitis [13, 14 ], the expressions of forkhead box p3 (foxp3) gene, characterizing naturally occurring cd4 tregs (ntregs), as well as that of inteleukin-10 (il-10) and transforming growth factor-1 (tgf-1), characterizing tr1 and th3 inducible tregs (itregs), respectively, were examined in relation to the expression of major apoptosis mediators, namely, fas, fas ligand (fasl), tumor necrosis factor - alfa (tnf-), and tumor necrosis factor - related apoptosis - inducing ligand (trail). furthermore, in order to clarify whether the expansion of tregs is a characteristic finding of chronic viral hepatitis, we examined foxp3 expression in liver biopsies of patients with other hepatic diseases, including nonalcoholic fatty liver disease (nafld), autoimmune hepatitis, primary biliary cirrhosis, and liver - toxicity induced by methotrexate. correlations with the grade of inflammation and fibrosis were also investigated in order to identify its possible interaction with the above factors towards the progression of liver damage. liver biopsy specimens obtained from 26 patients with chronic hbv hepatitis (chb) (4 with cirrhosis) and 14 with chronic hcv hepatitis (chc) were examined. amongst chb patients, 19 were newly diagnosed and 7 were responders under treatment for 48 weeks with peg - ifna2a or antivirals and relapse after treatment withdrawal for 24 weeks. no patient presented with coinfection with other hepatitis viruses (types a, d, and e) or superinfection with hiv. results were compared with those obtained from the examination of biopsies from 11 patients with nonalcoholic fatty liver disease (nafld). liver biopsies from 8 patients with autoimmune hepatic diseases (4 with autoimmune hepatitis and cirrhosis and 4 with primary biliary cirrhosis, pbc) and 2 patients with rheumatoid arthritis and methotrexate- (mtx-) related hepatotoxicity were also analyzed. none of the patients were receiving antiviral or immunomodulatory treatment during the last 3 months prior to liver sampling, except for the 2 patients with rheumatoid arthritis who were receiving mtx. eight individuals submitted to liver biopsy due to a mild increase of aminotransferases but without liver necroinflammatory and architecture changes (histology negative for disease) served as controls. hbv dna and hcv rna quantification was performed by the use of the bdna assay v2.0 (bayer, siemens) and the cobas amplicor system (roche molecular systems), respectively. demographic, clinicopathologic and serologic data of the 69 analyzed subjects are summarized in table 1. one of them was immediately fixed in 10% formalin solution for diagnostic histological examination, and the other was snap frozen and stored at 80c until further use. two independent pathologists assessed and scored each biopsy, and discrepancies were further evaluated by an expert pathologist. in patients with viral hepatitis, the histological activity index (hai) and the staging of fibrosis (06) in formalin - fixed tissues were assessed according to the modified hai scoring by ishak.. in patients with nafld, the necroinflammatory grade and the fibrosis score (04) were assessed according to the scoring by brunt.. according to the intensity of liver inflammation biopsies, patients were classified as i-0 (absent inflammation), i-1 (minimal, hai score 14 and mild grade inflammation for nafld), i-2 (mild, hai score 58 and moderate - i grade with minimal portal inflammation for nafld), i-3 (moderate, hai score 912 and moderate - ii grade with marked portal inflammation for nafld), and i-4 (marked, hai score 1318 and severe grade for nafld) (table 1). for patients with autoimmune hepatitis, the inflammation grade and fibrosis stage were estimated by morphological criteria similar with chb and chc patients and stratified accordingly, while, for patients with mtx - related hepatotoxicity, the inflammation grade and fibrosis stage were assessed and stratified similar to the patients with nafld (table 1). however, patients with pbc, two of them with stage 1 and two with stage 3, according to ludwig. and scheuer, were not estimated in the above stratification grades, because of the distinct pathological findings of the disease. informed consent was obtained by all participants, and the study was approved by the institutional review board. total rna was isolated from stored liver samples after homogenization, using tri (ambion, austin, usa), according to manufacturer 's instructions. cdna was reversed transcribed from 1 g of the rna, using a random 6-mer oligonucleotide primer (50 pmol/l) (roche, usa) and m - mlv reverse transcriptase (invitrogen, uk), according to manufacturer 's instructions. the mrna levels of ten genes, namely, foxp3, fas, fasl, trail, caspase-3, il-10, tgf-1, tnf-, interferon - gamma (ifn-), and interleukin-1beta (il-1), were determined in a qrt - pcr reaction using platinum - sybr - green pcr supermix (invitrogen, uk), in the automated thermocycler rotorgene 6000 (corbett life science, sydney, australia). the beta-2-microglobulin (b2 m) gene was used as an endogenous control for sample normalization (reference gene). an 1/20 aliquot of the cdna reaction product was used in duplicate qrt - pcr reactions, and all measurements were averaged. thermocycler conditions for the foxp3, tgf-1, caspase-3, tnf-, and b2 m genes included an initial holding at 50c for 2 min and subsequently at 95c for 2 min, followed by 40 cycles at 95c for 15 sec and 60c for 60 sec. for the fas, fasl, il-10, and ifn- genes, three - step pcr was performed with annealing at 55c (for fas, fasl, and il-10) or 58c (for ifn-) for 15 sec and extension at 72c for 30 sec (denaturation and cycles were similar as for the other genes). the efficiency of each qrt - pcr reaction ranged between 0.9 and 1.05. in order to verify the specificity of the pcr products, melting curve analysis was performed from 65c to 95c with 0.1c / sec intervals and stepwise fluorescence acquisition. relative quantification and calculation of the range of confidence were performed using the comparative method, as described in. the relative expression of each gene is presented as a multiple of the respective gene expression in the sample of a normal control, who presented with the lowest levels of aminotransferases. expression data of this patient (expression = 1, for all genes) were, therefore, excluded from the statistical analysis. the foxp3 protein expression was determined in randomly selected samples from 7 patients with hepatic diseases (3 with chb, 2 with chc, and 2 with nafld) and 2 normal controls, using an anti - foxp3 mouse monoclonal antibody (ab22510) from abcam (cambridge, uk). gapdh (rabbit polyclonal, code : 2275-pc-100 ; trevigen, gaithersburg, md, usa) served as a loading control, using an ultraviolet detection system (westerndot 625 goat anti - rabbit western blot kit ; invitrogen), according to manufacturer 's recommendations. for basic statistical calculations, foxp3, il-10, tgf-1, fas, fasl, trail, caspase-3, tnf-, differences of gene expression between disease groups were analyzed by the nonparametric mann - whitney u test. the association of the above parameters with inflammation and fibrosis grade was tested with kruskal - wallis h test. sprearman 's rank correlation coefficient was used to estimate the correlations of the expression among the aforementioned genes, as well as the correlations of gene expressions with aminotransferases levels or viral load. all statistical calculations were performed by the use of spss (version 16.0, chicago, il, usa). differences were considered statistically significant when the p value (two sided) was.05). furthermore, a significant positive correlation between the expression levels of il-1 and those of tnf- and caspase-3 was also observed (p <.001 and p <.001, resp.). finally, as expected, hai score was positively correlated with alt levels (p =.038), in patients with chb and chc. our study provides clear evidence that irrespective of the cause of liver damage, foxp3 expression, and not il-10 and tgf-1, appeared with a dramatic increase that relates to the intensity of liver inflammation. moreover, apoptosis - induced inflammation is observed in a wide range of liver diseases. it is well known that viruses sensitize hepatocytes to apoptosis whilst they are also capable of inhibiting it, in order to allow the survival of the infected host cells, as well as that increased expression of fas / fasl appears in chronic viral hepatitis [2227 ]. however, contradictory results exist in the literature regarding their relation with inflammation (hai score) and alt levels [2628 ]. according to our results, a significant increase of fas and trail appears at the early stages of liver inflammation that could contribute to its induction. as the inflammation exacerbates and striking fibrosis (cirrhosis) is established, the expression of these apoptosis mediators declines. this finding can be attributed to the evolution of liver damage followed by destruction of hepatocytes and accumulation of lymphocytic infiltrate. as such, the elevated fasl expression, mainly expressed by ctls, can also be explained, despite the fact that it is not followed by a parallel increase of trail expression, as expected. most interestingly, however, our findings indicate that not only apoptosis is taking place in nafld, but also it can equally be responsible for the induction of inflammation, considered till now to be associated with lipid - related nf-b activation [28, 29 ]. notwithstanding the limited cases examined with autoimmune hepatitis, pbc and mtx - hepatotoxicity, the same phenomenon was observed. till now, there is only scarce evidence [3033 ] that an increased expression of fas / fasl accompanies nafld. the covariation of apoptosis mediators with inflammation that was observed in our nafld patients represents a line of supporting evidence to the results by feldstein., correlating inflammation with tunel - positive cells in this disease. as mentioned above, a high intrahepatic expression of another death ligand activating the caspase cascade and apoptosis, namely, trail, was demonstrated. although trail induces apoptosis mostly in transformed cells, recent in vitro studies showed that it triggers steatosis and massive apoptosis in fresh liver explants from patients with viral hepatitis or fatty liver [3437 ]. the increased intrahepatic expression of trail that we found followed the same pattern of expression of fas, indicating the significant role that this molecule might play in liver inflammation, irrespective of the cause. the nature (soluble or transmembrane) and the source (immune cells or hepatocytes) of trail as well as the downstream molecules (receptors and mediators) activated during its signalling in hepatocytes or stellate cells remain to be determined. we demonstrated that the intensity of chronic liver inflammation was not associated with il-1 expression, and its mrna levels were significantly lower in patients with chb and chc compared to normal controls. moreover, a strong positive correlation of the expression of proinflammatory cytokines il-1 and tnf- was also observed. interestingly, our findings are in accordance with those of bortolami., whereas a lower expression of il-1 in patients with chb and chc compared to normal controls was also reported. a plausible explanation could be the accumulation of lymphocyte infiltrate in chronic viral hepatitis, resulting in altered cellularity with a low proportion of cells producing the aforementioned cytokines. on the other hand, a low production of the above proinflammatory cytokines in chronic viral inflammation can not be excluded. il-1 and tnf- are prototypic cytokines that exert pleiotropic effects on a variety of cells, playing a fundamental role in acute and chronic inflammatory conditions [39, 40 ]. their role at the initial phases of local and systemic inflammation, triggering a complex network of signalling molecules, is indisputable and well characterized [39, 40 ]. moreover, a sustained increased expression of il-1 and tnf- has been reported in several cases of chronic local inflammation, as in rheumatoid arthritis and inflammatory bowel diseases, where the anticytokine therapy is very effective, reducing symptoms and slowing or arresting tissue damage. however, in cases of severe systemic inflammation, a downregulation of mrna levels of the proinflammatory cytokines il-1 and tnf-, produced by liver and blood cells, has been reported [41, 42 ]. as a result, our findings may display that a similar phenomenon is observed in chronic liver inflammation, especially in cases of chronic viral hepatitis. thus, considering the sustained low levels of the proinflammatory cytokines in patients with chronic viral hepatitis as well as the fact that the anticytokine therapy for rheumatoid arthritis and inflammatory bowel diseases can also result in reactivation of hbv or hcv infections, the contribution of an inappropriate inflammatory reaction as the causative of the chronicity of viral infections should be taken into account and remains to be determined. our results, regarding foxp3, il-10, and tgf-1 expression, imply that ntregs, and not itregs, could contribute by processes unknown as yet to inflammatory liver disease. most intriguing is the finding that increased foxp3 expression, and therefore ntregs intrahepatic accumulation, characterizes not only viral hepatitis but also to an equal degree, nafld as well as autoimmune hepatitis, pbc and mtx - related hepatotoxicity. till now, the increased frequency of ntregs in the peripheral blood and/or their accumulation in the liver of patients with chb or chc has been attributed to the expansion of tregs modulating the function of virus - specific t cells [811, 13 ]. moreover, it has been postulated that viruses contribute to the production of virus - specific tregs, suppressing the virus - specific t cell clones and thus allowing the persistence of viral infections [68, 11 ]., it has been described that in mice, the depletion of ntregs leads to immunopathology and deterioration of infectious diseases induced by various pathogens [44, 45 ]. indeed, wei. demonstrated that the depletion of foxp3 tregs results in fulminant hepatitis in a mouse model of immune - mediated liver damage (induced by concanavalin a). according to our results, persistent liver inflammation, regardless of its cause, seems to represent a main factor that contributes to the expansion of ntregs. should this be the case, then the described suppression of virus - specific t cells could be considered as a bystander effect of the ntregs that have been expanded due to the persistent apoptosis - induced inflammation. taken collectively, our results are in line with the attractive view of zheng and rudensky claiming that tregs have a vital role in preventing autoimmunity and pathology inflicted by uncontrolled immune responses to infections. as such, the evidence provided by our work can be integrated in a comprehensive protective model as detailed below. the excessive apoptosis induced by various tissue insults may overcome the capability of macrophages to safely clear the apoptotic cells. consequently, the emergence of inflammation can not be prevented by the secretion of anti - inflammatory cytokines from macrophages. in parallel, excessive autoantigen presentation is taking place leading to the activation of autoreactive t cells. pre existing ntreg clones are therefore expanded, in order to prevent the self - tissue damage and to avoid catastrophic pathology. recent animal and human studies demonstrated that intrahepatic tregs were increased in autoimmune liver diseases [47, 48 ], considering also that in aih they were found fewer than in pbc, estimated by immunocytochemistry. despite the fact that our work provides also limited, yet consistent, data with regard to the autoimmune hepatitis / cirrhosis and pbc, it can be postulated that, in these cases, apoptosis - induced inflammation might be the result, rather than the cause, of the autoimmune damage that might have initiated due to a defective ntreg function. this being the case, the above initiates and perpetuates a vicious cycle leading to the destruction of self - tissues. in other words, our model indicates that tregs could be the missing link to the waste disposal should a similar protective role of ntregs be uncovered in other types of apoptosis - induced inflammation, then the proposed targeting of apoptosis, rather than of ntregs, could prove to be a more promising therapeutic modality. our results and the pathophysiological model we propose confirm that, possibly with the exception of autoimmunity, ntregs represent a protective mechanism whose manipulation should be carefully considered. however, further research towards the elucidation of the underlying casual relationships is required, in order to clarify whether our findings signify the existence of a uniform treg - mediated regulatory mechanism of apoptosis - induced inflammation. | patients with chronic viral hepatitis display increased expression of foxp3 in liver, suggesting that tregs expansion contributes to persistent infection. the purpose of this study was to elucidate whether the expression of foxp3 relates not to the viral infection but to the resulting liver inflammation. liver biopsies obtained from 69 individuals (26 chronic hbv hepatitis, 14 chronic hcv hepatitis, 11 nonalcoholic fatty liver disease, 8 autoimmune diseases, 2 methotrexate - related toxicity, and 8 controls) were examined, by qrt - pcr, for the mrna expression of foxp3, il-10, tgf-1, fas, fasl, trail, caspase-3, tnf-, ifn-, and il-1. significant increase of foxp3 was observed in all disease groups compared to controls, which was positively correlated with the intensity of inflammation. the expression of the apoptosis mediators fas, fasl, and trail, but not of il-10 and tgf-1, was also significantly elevated. our findings indicate that, independently of the initial inducer, liver inflammation is correlated with elevated expression of apoptosis mediators and is followed by local treg accumulation. further research towards the elucidation of the underlying casual relationships is required, in order to clarify whether our results signify the existence of a uniform treg - mediated regulatory mechanism of apoptosis - induced inflammation. |
pneumatosis intestinalis (pi) is a rare and unexpected disorder defined as accumulation of gas in the submucosa or subserosa of the large or small intestine. it is associated with a variety of diseases, and its pathogenesis is still obscure., it has been reported in 12 cases after pediatric orthotopic lt [1 - 5 ], although nearly none has been reported in adult liver recipients. we report three cases of adult liver transplant recipients in whom the diagnosis of pi was made by computer tomography (ct) and simple x - ray (table 1). the patients in case 1 and 2 were unremarkable during post - operative period, and they were discharged home. the patient in case 1 experienced high fever and watery diarrhea after two months and the patient in case 2 complained of watery diarrhea after four months. the patient in case 3 experienced high fever and watery diarrhea one month after the transplantation without discharge. at admission, all patients were stable in blood pressure, pulse rate, and respiratory rate, but in case 1 and 3, patients had high body temperature (38.8 and 38.5). the physical examination showed severely distended, non - tender abdomen, with hypoactive bowel sound and tympanic percussion in case 1. the laboratory findings of all cases did not reveal abnormal findings except mildly elevated c - reactive protein, ranging 0.38 to 1.59 mg / dl (normal range, < 0.3 mg / dl). serial blood tests of case 1 were positive twice for cytomegalovirus (cmv) antigenemia, but other cases showed all negative results. the peak titer of cmv antigenemia was 10/400,000 and colonoscopic finding did not show abnormal finding in case 1. the cytotoxicity assay of the all patient 's stool was negative for clostridium difficile toxin. stool cultures and special stains for micro - organisms were all negative in all patients. abdomen simple x - rays and ct in abdomen revealed diffuse pneumatosis intestinalis of the colon, ileus, and free air under diaphragm, but no air in the portal vein in case 1 and 2 (fig. 1). an abdominal ct of case 3 demonstrated pneumopericardium, diffuse linear pneumatosis intestinalis of the colon and extraluminal air within retroperitoneum and mesentery but no free intraperitoneal air (figs. 2, 3). all patients were conservatively managed with bowel rest, parenteral nutritional support, intravenous antibiotics (cefotaxime, ampicillin / sulbactam, and metronidazole) because of no signs of peritonitis. the patients in case 1 was treated with additional antiviral agent (ganciclovir) for positive cmv antigenemia and nasogastric tube decompression for free air, but other patients did not receive antiviral agent and did not have nasogastric tube decompression. the patients in case 1 and 3 did not receive immunosuppressive agent for 1 week, but the patient in case 2 received reduced immunosuppressive treatment with fk506 (2 mg daily), methylprednisolone (2 mg daily), and mycophenolate mofetil (500 mg daily). he remained in excellent general condition from the admission and had a sense of well - being during hospitalization. the abdominal distension of case 1 resolved and diarrhea improved within a week and antibiotics were discontinued. all patients were given a diet, and were discharged home after the pi resolved clinically and radiographically. the common factor linking pi to organ transplant recipients is steroid treatment. in this report, one patient did not receive steroid treatment and the other two had steroid treatment but the amount was only 4 mg / day. drug concentrations of fk506 and mycophenolate mofetil (mmf) in all patients have low levels as well. it might be possible that immunosuppression including fk506, steroid, and mmf did not cause of pi in our cases. symptoms that have been attributed to pi are in decreasing order of frequency : diarrhea, bloody stool, abdominal pain, abdominal distension, constipation, weight loss, and tenesmus. in this report, three recipients had watery diarrhea, and two had high fever. the physical examination of recipients did not show specific findings except abdominal distension in one case. the patients did not have abdominal pain, tenderness, rebound tenderness, and rigidity of abdominal wall. there was no clear evidence of peritonitis despite of pneumoperitoneum and air density in mesentery and retroperitoneum in simple x - rays and ct scan of abdomen. we assumed that there was no direct communication between lumen and peritoneum, therefore conservative treatment should be performed rather than surgical exploration. however, patients with white blood cells of more than 12,000/mm with or without the symptoms of clinical obstruction such as emesis, vomiting, and pain, age over 60 years were considered to be candidates for surgical intervention. based on this report, pi should be considered as the differential diagnosis of adult after lt who suffers from watery diarrhea and fever. pneumoperitoneum, air - density in mesentery and retroperitoneum in patients with pneumatosis intestinalis without signs of peritonitis improved with conservative management. | pneumatosis intestinalis is an uncommon disorder characterized by an accumulation of gas in the bowel wall. we described three cases undertaking liver transplantation. the patients developed diarrhea in three cases and high fever in two. an abdominal x - ray and computed tomography scan demonstrated extensive pneumatosis intestinalis in the colon with pneumoperitoneum mimicking hollow organ perforation. however, the patients had no abdominal symptoms and there was no evidence of peritonitis. the infection work - up was negative except one case with cytomegalovirus antigenemia. after one week of conservative management including bowel rest and antibiotic therapy, their pneumoperitoneum resolved spontaneously without any complication. pneumatosis intestinalis should be considered as a differential diagnosis after adult liver transplantation with patients suffering from watery diarrhea and fever. pneumoperitoneum, air - density in mesentery and retroperitoneum in patients with pneumatosis intestinalis without signs of peritonitis improved with conservative management, which included bowel rest and antibiotic therapy. |
facial and smile esthetics is a major reason for many patients to seek orthodontic and dental treatment. smile esthetics is affected by several factors (such as the occlusion, gingival display, lip position, dentolabial harmony, or dental anomalies like congenitally missing teeth) and is critical for enhancing one 's psychological well - being and self - perceived psychological impact. moreover, esthetics is an intricate and subjective matter, affected by various factors such as the prejudiced judgment of attractiveness depending on the observer 's psychosocial and cultural condition or the properties of the observed object or the medium on which the scene is visible. a photograph of low quality might reduce the patient 's satisfaction by affecting their esthetic judgment. therefore, it would be more desirable to use equipments that can better reflect the reality. nevertheless, it is not known whether the quality improvement by means of better equipment (e.g., a macro lens) can contribute to the esthetics of the smile photographed, as there is no study in this regard. in digital devices, many image properties are affected by the type of the lens. there are brochures suggesting that a macro lens is necessary for close up dental imaging. nonetheless, there is no study of any kind on the beauty or subjective quality of dental images taken with macro lenses compared to usual lenses. another factor potentially affecting one 's sense of beauty might be a set of psychosocial factors including beauty standards installed by media, population anthropometric norms, age, occupational or artistic interests of the evaluator, or education. it has been shown that dentists, orthodontists, surgeons, and laypeople might notice different aspects of the smile. therefore, it would be of interest to know whether dentists from three esthetic - oriented specialties orthodontics, prosthodontics, and restorative dentistry might have similar smile esthetic tastes. in this study, it was hypothesized that an image taken with a normal lens and then digitally enlarged is similar in terms of beauty with a close - up smile image taken using a macro lens. in addition, it was hypothesized that being educated in different dental specialties might affect the beauty judgment of the specialists. in the first phase of this study, 40 female dental students with balanced faces and angle class i molar relationship were enrolled after evaluating female students of 1824 years old. the included students needed to be healthy with balanced faces, looking subjectively normal (but not necessarily attractive), with no obvious disharmony between its facial features, or no excessive departures from the population norms subjectively determined by two experienced faculty members of the orthodontic department (a dentist and an orthodontist).. posed smiles of the subjects were photographed in similar conditions (no makeup, natural head position, a white background, focal spot of 100 mm, distance = 60 mm, f/8, no flashlight, standardized fluorescent light, brightness set at white balance), using a 18.0-megapixel digital single - lens reflex (dslr) camera (eos 550d, canon, japan) installed on a tripod (canon) and equipped with a macro lens and a grid visor. six orthodontists, three prosthodontists, and three specialists in restorative dentistry independently ranked the smile esthetics. each referee evaluated each image for 20 s, without any rewind. a 100 mm visual analog scale (vas) the images were handed again to the same referees, who rated the images, as stated above. the ranks were calculated by summing up all the scores given by all referees in both sessions. digital photographs were taken in standardized situations (no make ups, natural head position, focal spot of 100 mm, distance = 60 mm, standardized fluorescent light) from posed natural smiles of the 20 students, using a camera (eos 550d, canon) with a macro lens (100 mm focal spot) and a regular lens. the images taken with the regular lens were then digitally magnified and cropped to enclose the smile [figures 1 and 2 ]. smile photographs of two students using a regular lens smile photographs of the same two students shown in figure 1 in the same order, taken using a macro lens the images were first sorted in a random order. then they were shown to each referee (the same order of images were given to all judges). each image was seen for 20 s (by the automatic slide view feature, set at 20 s) without a rewind or without skipping any images. each referee in the same panel of experts evaluated the beauty of the smiles on a vas. the vas was converted to 11 equal ranks (0 definitely not pleasing, 10 the same procedure was repeated 2 weeks later, however, in another (randomly chosen) order. the average score of each referee given to each image was calculated between two sessions. the referees were blinded of the type of lenses, and all images were coded. the scores were calculated by a statistical expert blinded of the groups and camera types. the mean score of all referees for each image was considered as the final score of that image. the coded groups were compared using two - way analysis of variance (anova), kruskal if the result of kruskal wallis test comparing specialties became significant, pairwise comparisons would be conducted between specialties, using mann the level of significance for this particular test would be adjusted to 0.0167, according to the bonferroni method. posed smiles of the subjects were photographed in similar conditions (no makeup, natural head position, a white background, focal spot of 100 mm, distance = 60 mm, f/8, no flashlight, standardized fluorescent light, brightness set at white balance), using a 18.0-megapixel digital single - lens reflex (dslr) camera (eos 550d, canon, japan) installed on a tripod (canon) and equipped with a macro lens and a grid visor. six orthodontists, three prosthodontists, and three specialists in restorative dentistry independently ranked the smile esthetics. a 100 mm visual analog scale (vas) was used to rate the beauty of each smile. the images were handed again to the same referees, who rated the images, as stated above. the ranks were calculated by summing up all the scores given by all referees in both sessions. digital photographs were taken in standardized situations (no make ups, natural head position, focal spot of 100 mm, distance = 60 mm, standardized fluorescent light) from posed natural smiles of the 20 students, using a camera (eos 550d, canon) with a macro lens (100 mm focal spot) and a regular lens. the images taken with the regular lens were then digitally magnified and cropped to enclose the smile [figures 1 and 2 ]. smile photographs of two students using a regular lens smile photographs of the same two students shown in figure 1 in the same order, taken using a macro lens the images were first sorted in a random order. then they were shown to each referee (the same order of images were given to all judges). each image was seen for 20 s (by the automatic slide view feature, set at 20 s) without a rewind or without skipping any images. each referee in the same panel of experts evaluated the beauty of the smiles on a vas. the vas was converted to 11 equal ranks (0 definitely not pleasing, 10 extremely beautiful). the same procedure was repeated 2 weeks later, however, in another (randomly chosen) order. the average score of each referee given to each image was calculated between two sessions. the referees were blinded of the type of lenses, and all images were coded. the scores were calculated by a statistical expert blinded of the groups and camera types. the mean score of all referees for each image was considered as the final score of that image. the coded groups were compared using two - way analysis of variance (anova), kruskal if the result of kruskal wallis test comparing specialties became significant, pairwise comparisons would be conducted between specialties, using mann the level of significance for this particular test would be adjusted to 0.0167, according to the bonferroni method. there was a significant intra - rater agreement between the smile esthetics scores given by the observers at both sessions (cronbach alpha = 0.72, p < 0.05). the regular lens resulted in an average beauty score of 5.24 (out of 10), while the average score was 5.73 in the macro lens group [table 1 ]. the difference between these scores of beauty perception was not statistically significant according to the mann the vas scores for each lens the average scores of smile beauty given by orthodontists, prosthodontists, and specialists in restorative dentistry (both lenses combined, n = 40 observations per field of specialty) were 5.560, 5.545, and 5.285, respectively. there was no statistically significant difference between subjective beauty perception of specialties, according to the kruskal the two - way anova indicated no significant differences between the results pertaining to different lenses (p = 0.1750) or specialties (p = 0.7677). it also did not show any significant interaction between lenses and specialties [p = 0.7852, figure 3 and table 2 ]. the visual analog scale scores for dental specialists perception of smile beauty according to the specialty and lens the vas scores given by each specialty to images taken using each lens various factors contribute to the beauty of smile, such as the buccal corridor, shape of the teeth, asymmetries, arch widths, and gingival display. however, not only the smile itself, but also the evaluation methods matter as well. some authors find static records (photography) insufficient for esthetic and diagnostic purposes, but some others consider photographs appropriate methods for these purposes. therefore, much of clinicians ' professional time is spent in judging and discussing pictures and photographs. high - quality photographs are fundamental for pre and postoperative documentation as well as for scientific development and staff training. the dslr camera system uses a single lens for both image capture and composition, which allows direct viewing and focusing without parallax error. a lens selected for dental purposes must be able to capture diagnostic views of oral structures while the clinician is positioned at a comfortable and convenient working distance from the patient. it is necessary to equip the camera with a lens that enables recording from close distance and with flash, and having a minimum of f/22 aperture. it has been suggested to use a fixed portrait focal length (90105 mm) high - quality macro lens for both facial and intraoral pictures, to ensure a maximum field depth, with the smallest possible distortion and minimal alteration of colors. macro lenses with a fixed focal length designation of 100 mm to 105 mm might provide the ideal combination of working distance convenience and magnification ability for dental photography. the quality of the lens has a significant influence on the sharpness, clarity, and ultimate quality of the final image. this focal length allows the reproduction of the natural anatomy without the bulging that happens with wide - angle lenses. a dual purpose (portrait / close up) lens is the best option for dental applications. the ideal choice is a macro telephoto lens capable of producing 1:2 or 1:1 (actual size) magnifications, which can provide both functions. despite numerous guidelines suggesting that macro lenses are necessary for obtaining high quality close - up images, it is not supported by scientific evidence. since advanced camera sensors are high resolution, we believed that by taking the smile image from a far distance and then zooming in digitally, a similar quality of smile might be obtained. the findings of this study indicated that the lenses did not affect the beauty of smile perceived by the specialists. this indicates that with the advent of new high - resolution sensors, more economic regular lenses can be used to document and judge the beauty of smile. in addition, the expertise in the three assessed fields of specialties did not affect the judgment of smile attractiveness. since there is no similar study in this regard, this study was limited by some factors. both digital photography and the notion of beauty moreover, it was very difficult, if not impossible, to obtain reproducible photographs from a single participant. the only way to eliminate the inevitable inconsistencies between participants ' smiles or head positions was to take a single photograph from each participant, and then take pictures from that single photograph, using different cameras. nevertheless, this method would have its own limitations : as the qualities of the second - hand images would be all overshadowed by the quality of the original image. moreover, taking pictures from two - dimensional images could not represent three dimensional clinical conditions. the generalizability of our findings was favored by the participation of specialists from three different fields of dentistry. future studies should predetermine the sample size based on power calculations, and use scores given by general dentists, visual artists, and laypeople, etc., in order to improve the generalizability. on the other hand, the results obtained by a single brand and type of camera can not be generalized to all other cameras and lenses of the same type. the results of this study might be generalizable also to taking films (instead of photographs) with the same lenses because, in essence, a movie is a fast slideshow of pictures. however, future studies should compare the perception of beauty in digital movies, which are becoming popular with the advent of teleconferences, and can relate to modern treatment planning. as clinical implications, it was understood that the digital zoom in a photograph taken by a regular lens might serve as similar as a macro lens in the perception of close up image beauty, which is an important part even in dental documentation in multidisciplinary practice. since there was no similar study on this subject, we could not compare our results and discuss them further. unless more research is implemented in this regard, making any recommendation seems out of place. clinicians should take the advantages versus limitations of each lens (and camera) while purchasing a camera. given the very close results of the three specialties, it might be concluded that different dental fields have similar esthetic standards, which is favorable in multidisciplinary tasks. nevertheless, future studies with larger groups of specialists are necessary to verify our preliminary results in this matter. the images created by regular lenses, then cropped, and digitally zoomed are as appealing as photographs taken by macro lenses. therefore, as long as this factor is the major concern of the clinician or patient, there might be no need to replace the regular lens with a more expensive macro lens. experts in different specialties (orthodontics, prosthodontics, and restorative dentistry) showed similar subjective judgments of smile beauty. | background and aim : the purpose of this study was to investigate whether different camera lenses and dental specialties can affect the perception of smile esthetics.methods:in the first phase of this study, 40 female smile photographs (taken from dental students) were evaluated by six orthodontists, three specialists in restorative dentistry, and three prosthodontists to select the most beautiful smiles. the 20 students with the best smile ranks were again photographed in standard conditions, but this time with two different lenses : regular and then macro lenses. each referee evaluated the beauty of the smiles on a visual analog scale. the referees were blinded of the type of lenses, and the images were all coded. the data were analyzed using two - way analysis of variance (anova), kruskal wallis and mann whitney u - tests (alpha = 0.05, alpha = 0.0167).results : the lenses led to similar scores of beauty perception (mann whitney p = 0.8). there was no difference between subjective beauty perception of specialties (kruskal wallis p = 0.6). two - way anova indicated no significant role for lenses (p = 0.1750), specialties (p = 0.7677), or their interaction (p = 0.7852).conclusion : the photographs taken by a regular lens and then digitally magnified can be as appealing as close - up photographs taken by a macro lens. experts in different specialties (orthodontics, prosthodontics, and restorative dentistry) showed similar subjective judgments of smile beauty. |
bioaerosols and particulate matter of indoor and outdoor environment have a direct effect on the human health. bacteria have the diameter of about 28 m but these remain rarely free in the air. generally, they tend to aggregate or attach to nonviable particles to form large clumps. the total bacterial count is also significantly correlated with the number and size of particles [3, 4 ]. staphylococci are quite hardy, nonspore forming, and relatively heat resistant commensal bacteria that can survive longer on dry and inanimate surface in every environment in which humans coexist. methicillin resistant staphylococci (mrs) including staphylococcus aureus (mrsa) and coagulase negative staphylococci (mr - cns) can cause superficial to deep life - threatening diseases even in healthy or immune - compromised individuals. initially, mrsa infections were generally hospital - acquired (ha - mrsa) affecting the persons directly associated with healthcare facilities due to high risk or weak immune systems. subsequently, mrsa infection spread to healthy people who have not been hospitalized and such strains were called community - associated mrsa (ca - mrsa). these are genetically distinguished from the ha - mrsa and may cause rapidly progressive and fatal disease like necrotizing pneumonia, severe sepsis, and necrotizing fasciitis. mrsa is evolving continuously and now the distinction between ca - mrsa and traditional ha - mrsa is blurring. in previous studies in india, carriage rate of mrs and involvement of mrsa in nosocomial infection was found higher than usa [810 ]. the studies revealed that more than 50% of people were nasal carriers for s. aureus alone and out of them 3.89% were positive for mrsa [11, 12 ]. airborne mrsa may cause infection but their transmission frequency is lower than transmission via direct contact. however, the airborne transmission has been implicated in a number of nosocomial outbreaks of mrsa [14, 15 ]. in a previous study, we have detected mrs in bioaerosols during a trade fair at gwalior, india. thus, a method for direct isolation and identification of airborne mrs is desirable for molecular surveillance of mrs in countries having higher prevalence of these bacteria. the present study aimed to optimize a protocol for direct isolation of airborne mrs and to study their distribution in atmosphere of residential houses at gwalior, central india. several studies have used the methods of impaction on agar media. however, in this study, we have used the antibiotics for the direct selection of methicillin resistant staphylococci (mrs) for the first time. this procedure requires one additional day to confirm whether it is methicillin resistant or sensitive. a total of 14 residential houses located in 7 different colonies (2 houses from each colony) were selected for indoor and outdoor bacterial aerosol sampling from gwalior, central india (longitude 7813e, latitude 2613n). those houses were selected from the city that had no adverse health issues and the inhabitants were neither healthcare worker nor hospitalized within the past 1 year. air samples for microbiological analysis were collected using reuter centrifugal sampler (biotest, germany) at a height of 1.5 meters from the surface to simulate human breathing zone. the head of air sampler was disinfected with alcohol swabs before each air sampling and the sampler was turned on for 2 min prior to sampling to allow the alcohol to evaporate. sterile media strips containing microbial content test agar (mca) supplemented with cycloheximide (100 g / ml), mannitol salt agar (msa), and msa containing 6 g / ml methicillin were loaded into the disinfected air sampler. air samples were collected for 2 min in duplicate from inside and outside of each house at a flow rate of 280 l / min (the separation volume of the instrument was 40 l / min). mca, msa, and msa containing methicillin were used for enumeration of total aerobic bacteria, staphylococci, and methicillin resistant staphylococci, respectively. the media strips were incubated at 35c for 24 and 48 h to determine if the strips were overgrown. air sampling results for cultivable bacteria were reported as colony forming units per cubic meter of air (cfu / m) using the following formula:(1)cfu / m3=number of colonies on agar strip1000separation volumesampling time in minutes.thus, a maximum of 1062 colonies were present in the strip. each strip contains 34 compartments or wells, which means each well contains 31 colonies (1062/34 = 31). airborne particulate matter concentrations of six different sizes (aerodynamic diameter 0.3 m, 0.5 m, 1 m, 5 m, 10 m, and 25 m) were measured with commercial aerosol particle number counter (lasair ll particle counter ; particle measuring systems, usa). particles were counted in 4 sets (each set of 2 min) with 2 min delay after every set. five representative colonies of mannitol fermenting and nonfermenting bacteria from msa containing methicillin from each house (indoor and outdoor) were cultured on brain heart infusion (bhi) agar. a part of colony was emulsified in normal saline then mixed with rabbit plasma ; clumping indicated positive result. colonies grown on bhi agar were further incubated for 2 h at 60c and overlaid with thermonuclease agar and further incubated at 37c till the development of pink zone around the positive control. the bacterial colonies were grown in lb broth for 18 h. one ml of broth was centrifuged and the pellet was processed for dna extraction using genomic dna extraction kit as per the manufacturer instructions (mbi fermentas, vilnius, lithuania). the amount and purity of the dna were measured by spectrophotometer (nanodrop-1000, australia). a multiplex pcr was performed as described elsewhere, using the primers targeting a staphylococcus specific region of the 16s rdna, s. aureus specific clf gene encoding a surface - associated fibrinogen - binding protein, and meca gene, a primary determinant of methicillin - resistance in both s. aureus (mrsa) and coagulase negative staphylococci (mr - cns) species [19, 20 ]. all the mrs isolates were subjected to antibiotic susceptibility testing by disc diffusion method according to clinical laboratory standards institute guidelines. fresh cultures from tryptic soy agar plate were picked up and suspended in pbs and the turbidity of the tube was adjusted as the 0.5 macfarland standards. a 100 l of the suspension was swabbed evenly onto each mueller - hinton agar (mha) plate (difco laboratories, sparks, md). the agar surface was dried and the antibiotic discs were placed on the mha surface using a sterile forceps. the disks were allowed to settle and the plates were incubated in inverted position at 37c for 1824 h. macrolide, lincosamide, and streptogramin b (mls) resistance phenotype were determined by placing erythromycin disc 20 mm away from clindamycin disc. the test was conducted in duplicate for each isolate and the organisms giving the same resistance profile in both the plates were included in the study. indoor and outdoor total bacterial count and staphylococcal and mrs count were compared using paired t - test. the total bacterial concentrations inside the residential homes varied in the range of 5650 to 12425 cfu / m the average indoor / outdoor (i / o) ratio was found to be 1.35 (table 1). the difference between mean of indoor and that of outdoor total bacteria was nonsignificant (p > 0.05). staphylococci concentration for indoor residential homes was found in the range of 1225 to 4125 cfu / m (2454.9 880.96). the difference between mean concentration of indoor and that of outdoor staphylococci was nonsignificant (p > 0.05). mrs concentration for indoor residential homes was found in the range of 162.5 cfu / m to 1175 cfu / m outdoor total bacterial concentration ranged from 87.5 cfu / m to 806 cfu / m (338.03 208.01) which was 4.6% of the total bacterial concentration. the difference between mean indoor and outdoor concentration of mrs was statistically significant (p 7.5 m correlated with exhaled carbon dioxide indicating nasal carriers. bacteria of size between 3 and 7.5 m (in tracheal and bronchial regions) remain in clumps whereas bacteria in the size range of 12 m (terminal bronchial region) remain free. our previous studies demonstrated a significant rise in airborne microbes including mrs during human gathering or anthropogenic activities in urban environment. the presumptive mrs strains selected from methicillin containing msa strips were found to be pcr positive for staphylococci specific 16s rdna gene. out of them, 94% strains were found positive for meca gene (figure 1) and the remaining strains were observed to be pcr negative for meca gene cassettes but were able to grow on methicillin concentration more than mic. among mrs, 9.5% of strains harbored clf gene specific for s. aureus. a total of 87.4% of meca positive (mrs) strains were found to be multidrug resistant (resistance for more than two different classes of antibiotics). mls resistance phenotypes were found in 27.4% of strains including 16.7% of inducible expression (imls) and 10.7% of constitutive (cmls) expression phenotype. the m resistance phenotype (resistance to erythromycin but not to lincosamides or streptogramins) was found only in 6 (7.1%) strains. among the other antibiotics, 47.6% of strains were observed to be resistant to sulfamethoxazole - trimethoprim, 42.9% to quinolones, 8.3% to gentamicin, and 7.1% to mupirocin and 4.8% of strains were found to have intermediate resistance to tetracycline. mrs infections, including mrsa, occur most frequently among persons in hospitals and healthcare facilities who are at high risk or have weakened immune systems. now ca - mrsa (usa300) has started replacing traditional mrsa (healthcare associated) in hospitals on a large scale and become dangerous epidemic strain worldwide. the main objectives of this study were to optimize a methodology for direct isolation of airborne mrs and to determine their distribution pattern. isolation of bioaerosols followed by their identification to genus (staphylococci) or species level and screening for the presence of antibiotic resistant determinant (meca genotype) is a time consuming activity. in our previous report we did isolation of bacterial bioaerosols followed by their identification and characterization for meca genotype which was labor intensive and required additional time. therefore, a direct method for isolation of airborne mrs is utmost important to complete a timely surveillance program. mrs are well known as nasal colonizer in normal community and the colonization of virulent mrsa is significantly high [28, 29 ]. airborne mrs may infect a person in two ways either by inhalation or by settling directly onto susceptible area, such as a wound. mrs infections are important concern for health authorities and researchers across the world as it caused an approximate threefold increase in direct cost and prolonged hospital stay in comparison to infections due to methicillin sensitive staphylococci. infection of multidrug resistant mrs makes treatment very difficult because the drug of choice relies on newer generations of medicine which may be neither cost effective nor easily available to local market. inappropriate and inadequate practices like misuse, abuse, and overprescription of antibiotics also result in the development of multidrug resistance. penicillin, cephalosporin, cotrimoxazole, and quinolones cover most of the antimicrobials prescribed in india. penicillin, cephalosporin, and quinolones are dna damaging agents and can stimulate the drug resistance via sos independent manner and through the induction of reca - mediated repair, an error prone repair mechanism that induces mutations. thus, the study confirmed that airborne mrs are commonly present in indoor and outdoor environment of the residential houses. molecular surveillance, antibiotic stewardship programme, and infection control policies can help to manage increasing mrs burden in developing countries. | methicillin resistant staphylococci (mrs) commonly found in clinical samples or associated environment pose a major health challenge globally. the carriage rate of mrs in human population is high, especially in india but research on airborne distribution of mrs is scanty. the present study aimed to evaluate the prevalence of mrs in indoor and outdoor environment of residential houses. air samples were collected using impactor air sampler. the total counts of viable bacteria, staphylococci, and mrs along with the particles of various sizes were determined from indoor and outdoor environment of 14 residential houses. mrs bacteria were identified as methicillin resistant s. aureus (mrsa) or coagulase negative staphylococci (cns) employing biochemical and pcr assays. the average concentration of mrs inside and outside of the houses was 5.9% and 4.6% of the total bacteria, respectively. the maximum correlation of total indoor and outdoor bacteria with particulate matter was 10 m (r = 0.74) and 5 m (r = 0.84), respectively. statistically, significant positive correlation of staphylococci and mrs was found with particles of 1025 m inside the houses. molecular surveillance, antibiotic stewardship programme, and infection control policies can help to manage increasing mrs burden in developing countries. |
hyperpolarized noble gas magnetic resonance imaging (mri) is an emerging technique that enables novel quantitative analysis of pulmonary physiology (1, 2). although the density of gas introduced into the lungs is low, optical pumping techniques can be used before inhalation to produce extremely high nuclear spin polarization levels that provide a signal size sufficient for mri. by applying this method with the inert, nonradioactive isotope helium-3 (he), mr images of in vivo lung ventilation and oxygen sensitivity the technique has shown great promise in diseases such as cystic fibrosis, asthma, and emphysema (1). as demonstrated with other functional imaging modalities, such as emission tomography, image registration of he - mri with anatomic h - mri and x - ray computed tomography (ct) could provide improved clinical interpretation of the functional data, owing to the superior anatomic localization provided by image fusion. in particular, the combination of he - mri and ct for the management of lung cancer patients has the potential to be a clinically important application of he - mr image registration. for patients with non small - cell lung cancer (nsclc) who are suitable for radiotherapy treatment, functional pulmonary reserve may be severely reduced because radical radiotherapy will cause damage to noncancerous lung tissue in addition to the intended target volume. consequently, radiation pneumonitis is the most common dose - limiting complication of radiotherapy (5). the key to lowering the risk of radiation pneumonitis may be to reduce the dose to healthy regions of lung while maintaining an adequate dose to the tumor. however, this approach requires the healthy (well - ventilated and perfused) lung to be identified. ct images used in conventional radiotherapy treatment planning can not separate these functioning areas from those that have been previously damaged and contribute little to respiration. supplementary images of lung ventilation and perfusion are, therefore, required if radiation fields are to be optimized to reduce the dose to healthy lung. the potential value of incorporating functional information into lung cancer treatment planning has been investigated with single photon emission computed tomography (spect) (69). however, there are several practical limitations to the use of spect for treatment planning, primarily the additional radiation dose and often suboptimal image quality due to difficulties with attenuation correction and image reconstruction. recently, hyperpolarized he - mri has emerged as an alternative lung ventilation imaging modality that eliminates the need for radioisotopes and has the potential to provide functional information superior to that from spect (10). although hyperpolarized he - mri has been shown to detect radiation - induced lung injury in rats (11), no previous work has investigated the potential role of in vivo he - mri for the assessment and treatment planning of lung cancer patients. for nsclc patients who require radiotherapy, he - mr images of ventilation and function could have a considerable impact on patient management. before treatment, many patients have regions of lung with poor ventilation or perfusion that contribute little to effective lung function. by identifying these regions and using the information when planning treatment, he - mr images may enable radiation fields to be targeted to limit the dose to the healthy regions of lung, which could reduce the incidence of treatment complications. in addition, the information provided by he - mr functional images will make it possible to calculate dose volume histograms that are weighted by functional information, which could be a valuable tool for treatment plan analysis (12). for he - mri to be used in radiotherapy however, he - mr image registration has yet to be widely investigated because the lungs represent a nonrigid system with high mobility and thus present a challenging system for image registration. image registration of perfusion mri and he - mri using controlled gas administration has been demonstrated in pigs (13, 14), and a fiducial system for he to h - mri registration has been described (15), but in vivo studies involving the registration of he - mri to ct have not been previously reported. therefore, the first objective of this study was to investigate the feasibility of in vivo he - mr image acquisition and registration to x - ray ct for the management of patients with nsclc. radiotherapy treatment planning of lung cancer with supplementary functional images has been examined for more than a decade (16, 17). using conventional treatment - planning techniques on 104 lung cancer patients, munley. (18) found that 11% of plans were modified according to spect, whereas 48% of plans were considered potentially modifiable owing to the identification of lung regions with relatively low perfusion but unable to be utilized for treatment planning without improved planning tools. however, in recent years there have been significant improvements to treatment planning and delivery with the development of intensity - modulated radiotherapy (imrt). from a study of 41 nsclc patients, (19) concluded that imrt planning can significantly improve target coverage and reduce the volume of normal lung irradiated above low doses with a potential 10% reduction in the risk of radiation pneumonitis. planning with imrt involves the specification of a number of constraints, some of which can conceivably be derived from functional information when available. for the case of he - mri, once the images have been registered to planning ct, the he - mri data can then be incorporated into the treatment - planning process. in related spect studies, the functional data are incorporated into inverse planning with the aim of reducing the dose to healthy regions of lung and improving the sparing of critical structures (68, 16). therefore, the second objective of this study was to compare imrt plans constructed with and without constraints derived from the he - mri data. patients with nsclc underwent hyperpolarized he ventilation mri in addition to conventional x - ray ct for radiotherapy treatment planning. all patients gave written informed consent to participate, and the study was approved by the local research ethics committee (ns02 - 11 - 1507). hyperpolarized he - mr ventilation imaging was performed on a 1.5-t whole body eclipse system (philips medical systems, cleveland, oh), which was fitted with a second radiofrequency (rf) amplifier (2 kw ; analogic corporation, peabody, ma) and a transmit receive circuit tuned to 48.5 mhz for he (2). imaging was performed with a flexible twin saddle quadrature transmit receive rf coil (igc medical advances, milwaukee, wi). three syringes that could be filled remotely with he or water were placed on the chest to act as fiducial markers (15). images were acquired on a flat couch top similar to that used during the acquisition of radiotherapy planning ct. however, accurate reproduction of patient treatment position was not possible owing to the thoracic rf coil, which required the patients to be imaged supine with their arms down. the he images were obtained with a low flip angle, gradient echo acquisition with 112 centric phase encoding views (2), flip angle = 9, 19 coronal slices, 13 mm slice thickness with no gap, field of view = 43 cm, time to echo = 3.4 ms, time to repetition = 6.7 ms, 128 samples, and bandwidth = 16 khz. the he gas (spectra gases, huntingdon, uk) was polarized on site to 30% by optical pumping with rubidium spin exchange apparatus (ge healthcare, princeton, nj). in vivo imaging was then performed during a single 14 s breath - hold of a 300 ml he/700 ml n2 mixture inhaled from a tedlar bag (jensen inert products, coral springs, fl) from a starting point of functional residual capacity. blood oxygen saturation was monitored with an mri - compatible pulse oximeter throughout the procedure. patients were coached in the breathing maneuver and were allowed a trial breath hold with a dummy bag containing room air before imaging. half - fourier single shot fast spin echo h - mr images were also acquired during the same imaging session with the patients in the same position as during the he - mri. on the same day, treatment planning transaxial ct was acquired (512 512 pixels with pixel size 0.9375 mm) with 5 mm slice thickness (philips medical systems pqs ct). the patients were imaged in the conventional treatment position, which is supine with arms raised above the head while breathing freely. the he - mr images were imported into philips medical systems acqsim, which interpolated the 19 coronal he - mr images to match the voxel size and orientation of the transaxial planning ct. acqsim s rigid registration tools were then used to register the he - mri to the planning ct. to assist the manual registration procedure, verification of the image registration methodology was performed with a chest phantom that was scanned with the same he - mri and ct imaging protocols as the patients. using in - house custom matlab software (mathworks, natick, ma), registration accuracy was assessed using an overlap coefficient (), which is calculated as the proportion of the segmented he - mr slice volume (vmri) that intersects the segmented ct lung slice volume (vct) expressed as a percentage of vmri=100vmrivctvmri, where the higher the overlap value the better the registration. segmentation was performed manually by an experienced lung radiotherapy consultant with a suitable windowing of the he - mr images that varied from patient to patient because of differences in the signal - to - noise ratio. the he - mr volume may be different than the ct - defined volume, owing to the possibility of ventilation defects detected in the functional images. this is to be expected because many of the patients have a history of heavy smoking and may have regional obstruction from chronic obstructive pulmonary disease evident in their he - mri (20). hence, the overlap coefficient is a more appropriate measure of he - mr to ct registration accuracy than, for example, the intersection - union ratio, which may have been used if the two volumes were expected to be similar. it is useful to analyze the registration accuracy across the entire lung volume and for individual slices that are the most important when planning treatment. hence, for each patient, the overlap coefficient was first calculated for each image slice, and then the mean and standard deviation was determined for all slices containing (1) ct - defined lung, (2) the planning target volume (ptv), and (3) the gross tumor volume (gtv). the registered he - mri was segmented in acqsim and the contours exported as a dicom structure set to the eclipse planning system (varian medical systems, palo alto, ca) for imrt planning. functional lung tissue was defined as the intersection of the lung ct volume with well - ventilated lung segmented from the registered he images (vmrivct). total lung volume included the ipsilateral and contralateral lung but excluded the gtv, which was contoured by an experienced lung radiotherapy consultant. the ctv - to - ptv margin was 10 mm in the axial plane and 15 mm in the craniocaudal direction. inverse planning for imrt was performed using the dose - volume - optimizer module of the eclipse planning system. plans were produced using five or seven nonopposing, coplanar fields, with the beam angles manually optimized for each patient., plans were produced using two distinct sets of dose volume constraints. in the first set (plan a), constraints were chosen with the aim of minimizing the volume of lung receiving a dose of 20 gy (v20). in the second set (plan b), lung constraints were applied to minimize the v20 of lung tissue identified on the he - mri as functional. additional constraints were selected to produce continuous hyperfractionated accelerated radiotherapy (chart) plans (21) that met the criteria shown in table 1. tighter constraints were applied to the ptv to produce homogeneity in the ptv within the international commission on radiation units and measurements recommended limits of 95% and 107% where possible. a full three - dimensional forward dose calculation with inhomogeneity correction was performed for each plan, and dose volume histograms were produced. for each plan, the following parameters were calculated : (1) flv20 (percentage of functional lung receiving 20 gy), (2) tlv20 (percentage of total lung receiving 20 gy), and (3) mfld (mean dose to functional lung). a measure of the overall quality of the treatment plan, incorporating measures of both target dose and functional lung dose, was given by vptv95/flv20, which is the ratio of the percentage volume of the ptv receiving at least 95% of the prescription dose (vptv95) to the functional lung v20. in addition, a conformity index (ci) was used that accounts for any normal tissue receiving at least 95% of the prescription dose and for any tissue within the ptv that does not receive this dose (22). the maximal value of the index is unity, when the 95% isodose conforms exactly to the ptv, whereas a value of 0 represents a geographical miss. the index is defined as the product of the fraction of ptv receiving at least 95% of the prescription dose and the ratio of the volume of ptv receiving at least 95% to the volume of tissue receiving at least 95% (the treated volume, vt):cl = vptv95vptvvptv95vt. statistical significance of differences between the dose parameters for plan a and plan b were calculated using the wilcoxon signed ranks test. all patients had histologically proven inoperable nsclc and had been selected for radical chart (21). patients had who performance status of 02 and forced expiratory volume in 1 s (fev1) > 1.5 l. during he - mr imaging, no significant decrease in blood oxygen saturation was observed, and no ill effects were reported after inhalation of the he / n2 gas mixture. for the chest phantom, regions of he concentration were well registered to the ct lung regions (= 100%), which demonstrated the validity of the image transfer procedure and the feasibility of he - mri and ct manual rigid registration. for in vivo data, fig. a complete obstruction is evident in the he - mr ventilation image from a patient with a tumor in the right upper lobe that is visible on the h - mri (fig. the he images indicate that the tumor has caused a complete ventilation obstruction of the upper right lung. the registered he - mr image displayed with the external, lung, and gtv contours delineated from the planning ct is shown for 2 patients in fig. 2. in both cases, the ventilation mr images are well registered to the ct lung segments. for patient 5, the image has been windowed to display 2 fiducials that lie on the external ct contour. figure 3 displays the overlap coefficient () for each image slice, and table 3 provides the mean and standard deviation of for each patient. according to all the registered images containing ct - defined lung, for patient 4 (= 97.0 1.9) and patient 5 (= 96.7 2.2) the registration is highly accurate and has low variance. patients 1, 2, and 6 show good accuracy (= 85.5 15.1, = 89.6 5.8, and = 88.2 8.1, respectively), whereas patient 3 exhibits the lowest accuracy and high variability (and = 71.9 14.6). similar values are found when is calculated over the registered images containing the ptv or gtv, except for patient 3, which is 9.8% less accurate over the gtv images compared with overall patient 3 images containing lung (t test, p = 0.01). the markers were not visible for the first 3 patients owing to poor signal - to - noise ratio at the fiducial position due to he gas leakage from the syringe. this problem was rectified for the final 3 patients, which allowed the fiducial markers to be visualized on the he - mr images (fig. the markers assisted the manual alignment of the he - mri with the external ct contour, enabling the fusion of the ventilation mr within the ct lung segments. the total and functional lung volumes for the patients are shown in table 2 along with the ptv. volume parameters achieved by the two sets of optimization constraints are shown in table 4. in all patients, the incorporation of he - mri information in the inverse planning process produced improvements in the volume of lung irradiated. this was reflected in reductions not only in flv20 (median reduction, 3.1% ; range, 0.45.1% ; p = 0.028) but also in tlv20 (median reduction, 1.6% ; range, 0.23.7% ; p = 0.028). the mfld was also reduced for each patient when the functional lung was used in the optimization. the lung dose was improved even in patients, such as patient 5, in whom the optimization using the total lung produced low lung dose statistics. the improvements in lung dose were sometimes at the cost of deterioration of other aspects of the dose distribution. for example, reductions of more than 3.5% in both flv20 and tlv20 and a reduced mfld were seen in patient 3, but the dose to normal tissue was increased when only functional lung was used in the optimization. this is reflected in the lower ci for this patient in plan b. a lower ci was also seen for patient 5, due predominantly to slightly reduced target coverage, although the ptv dose requirements were met by both plans. in all patients, the overall plan quality measure, vptv95/v20, was improved when the functional lung was used in the inverse planning process. the location of the functional lung identified by the he - mri was an important factor in determining the impact of specifically including that tissue in the optimization. in patient 2, relatively modest improvements were seen in plan b (for example, a 0.6% reduction in flv20 and a small increase in ci). although 56% of the patient s total lung volume was functional, the majority of that functional tissue lay inferior to the large ptv and so was not in the path of the coplanar beams. as a result, the scope for influencing the volume of functional lung receiving a given dose was limited. patient 1 also had a large ptv, extending 13 cm craniocaudally, but in this instance the majority of the functional lung tissue was ipsilateral to and close to the ptv. the two sets of constraints produced plans of a generally similar quality ; using the he - mri information in the optimization resulted in similar target coverage, small (< 0.5%) reductions in flv20 and tlv20, and identical vptv95/v20. however, ci was lower when the functional lung information was used, reflecting an increased volume of normal tissue receiving at least 54 gy. this may, in part, be due to the proximity of the functional lung to the ptv optimizing such that the dose to the functional lung tissue close to the high - dose region is minimized provides a greater challenge for the inverse planning software. in this instance it seems to have been achieved only at the expense of an increased dose elsewhere. inverse planning to spare the entire lung consistently produced plans in which the spinal cord dose exceeded tolerance. to maintain an acceptable cord dose, the mean ptv dose was limited to 52.4 gy, so just 80% of the ptv received at least 95% of the 54-gy prescription dose. using only the functional lung in the optimization produced lower spinal cord doses, enabling the desired prescription dose of 54 gy to be achieved. this was not at the expense of the lung dose ; the flv20 for plan b was 3.6% lower than that from plan a, and the mfld was 1.0 gy lower. if the mean ptv dose in plan a was increased to 54 gy such that the cord tolerance was exceeded, these improvements through the use of he - mri information increased to 4.5% and 1.5 gy, respectively. the imrt plans demonstrated the general suitability of inverse planning in the production of clinically acceptable chart treatment plans. in one instance (patient 6), the conventional treatment plan produced at the time of the patient s treatment was limited by the proximity of the ptv to the spinal cord, necessitating a compromise in ptv coverage. both imrt plans produced met the ptv dose constraints while delivering spinal cord maximum doses well within tolerance (22 gy and 32 gy for plans a and b, respectively). in just one case (patient 3), the imrt plans were clinically unacceptable, because of large hot spots in normal tissue that were present irrespective of the beam arrangement. volume constraints were applied, was of limited help in overcoming this problem. for this patient, whose large ptv extended 16.5 cm in the craniocaudal direction, inverse planning using the dose - volume - optimizer may not be a suitable means of producing a treatment plan. however, the inclusion of noncoplanar beams may assist in reducing the high - dose regions and realizing the potential benefits of sparing the functional lung seen in plan b. this preliminary work demonstrates the feasibility of acquiring in vivo hyperpolarized he - mri suitable for image registration to ct for imrt treatment planning. the development of mri for detailed depiction of anatomy in the lungs has been slow when compared with other regions of the body. this is largely owing to the hostile physical environment of the lungs for mri, namely the low proton density and high magnetic field inhomogeneity. thus, h - mri has provided some limited anatomic information on lung parenchyma at the field strengths that are used routinely (1.5 t). more widespread in clinical radiologic practice is the use of mri in assessing pulmonary circulation through perfusion and angiography techniques (23). in recent years, the emergence of hyperpolarized noble gas mri has led to increased interest in the role of mri for pulmonary assessment because hyperpolarized he can provide novel functional information at high spatio - temporal resolution. imaging is safe, noninvasive, and reproducible and does not involve the use of ionizing radiation (1). as an emerging technology, implementation of he however, the problems of cost and access may be addressed by the distribution of polarized gas from a central production facility to a large geographic region (24) and by the advent of new clinical whole body scanners capable of multinuclear imaging. a measure of overlap is used to provide a measure of registration accuracy. with improved image quality it may become possible to also quantify the registration using anatomic landmarks. across all 6 patients there was a variable pattern of registration accuracy measured by the overlap coefficient (fig. however, he - mri and ct were registered with sufficient accuracy to enable functionally guided imrt planning (median overlap, 89% ; range, 7297%). for patients 1 and 3, similarly, a small decrease was observed for patient 4, but otherwise accuracy was consistently high as was the case for patient 5. for patient 6, registration accuracy improved from lower to upper lung. 2 were created using a rigid transformation, which is the most commonly used method of image registration. however, there are a number of potential limitations with this approach when applied to lung. first, the he - mri and ct images were acquired with different breathing schemes. treatment at our institution is planned on ct acquired while the patient is breathing freely. however, he - mri necessitates the use of a breath - hold, and this difference could lead to differences in lung morphology between the two sets of images. furthermore, the coil used for he - mri precludes the simulation of treatment position while acquiring the ventilation images. considering the potential problems in this initial study, the application of rigid registration of he - mri and ct was promising because it was possible to register the edges of ventilation images with reasonable accuracy as quantified by the overlap coefficient. improvements to the methodology, such as the use of ventilation and perfusion he - mri (4), breath - hold ct, treatment position h mri, and nonrigid registration (25, 26) are being explored in a follow - up study that is currently underway. image registration of the ventilation he - mri to ct enabled the functional lung to be taken into account when treatment planning with imrt, and all plans satisfied the spinal cord dose constraint. when minimizing the dose to total lung (plan a), v20 (median, 25% ; range, 1231%) was comparable to other reported values, such as those reported by murshed. (19) (median, 25% ; range, 1343%). in all patients, optimizing with the aim of reducing v20 for functional lung (plan b) reduced v20 not only for functional lung (median reduction, 3.1% ; range, 0.45.1% ; wilcoxon p = 0.028) but also for total lung volume (median reduction, 1.6% ; range, 0.23.7% ; wilcoxon p = 0.028). functional lung dose was also improved in all patients (median reduction, 0.9 gy ; range, 0.12.3 gy ; wilcoxon p = 0.028), and an improvement in plan quality index, vptv95/v20, was achieved across all 6 patients when optimizing to only functional lung (median increase, 0.45 ; range, 06 ; wilcoxon p = 0.043). the differences in conformity index were less consistent : patients 1, 3, and 5 showed an improved degree of dose conformity to the ptv, whereas the other 3 patients had a less well conformed high - dose region. in the patients for whom ci was better when the total lung was included in the optimization, the improved conformity was a result of a lower volume of normal tissue receiving 95% of the prescription dose. this study has demonstrated that hyperpolarized he - mri can be registered to radiotherapy planning ct. using the information provided by the registered he images, statistically significant improvements to functionally guided imrt plans were demonstrated for the v20 for the total and functional lung, in addition to the mean lung dose to the functional lung. | purpose : to demonstrate the feasibility of registering hyperpolarized helium-3 magnetic resonance images (3he - mri) to x - ray computed tomography (ct) for functionally weighted intensity - modulated radiotherapy (imrt) planning.methods and materials : six patients with non small - cell lung cancer underwent 3he ventilation mri, which was fused with radiotherapy planning ct using rigid registration. registration accuracy was assessed using an overlap coefficient, calculated as the proportion of the segmented 3he - mr volume (vmri) that intersects the segmented ct lung volume expressed as a percentage of vmri. for each patient, an imrt plan that minimized the volume of total lung receiving a dose 20 gy (v20) was compared with a plan that minimized the v20 to well - ventilated lung defined by the registered 3he-mri.results : the 3he - mri and ct were registered with sufficient accuracy to enable functionally guided imrt planning (median overlap, 89% ; range, 7297%). in comparison with the total lung imrt plans, imrt constrained with 3he - mri reduced the v20 not only for the well - ventilated lung (median reduction, 3.1% ; range, 0.45.1% ; p = 0.028) but also for the total lung volume (median reduction, 1.6% ; range, 0.23.7% ; p = 0.028).conclusions : statistically significant improvements to imrt plans are possible using functional information provided by 3he - mri that has been registered to radiotherapy planning ct. |
major concerns exist about the quality of water for such uses as human consumption and crop irrigation. thus, the number of samples as well as of analytes (both normal components and pollutants) increases continuously, challenging the capacity of analytical laboratories. in uruguay, the school of chemistry of the universidad de la repblica has been engaged in water analysis due to an agreement with the uruguayan water regulatory body (ursea, unidad reguladora de servicios de energa y agua) with the school acting as a contract laboratory for the analysis of water samples from the public water supply. samples are collected on a regular basis from hundreds of sampling points throughout the country and analysed for almost 60 analytes both organic and inorganic, as well as turbidity, ph, and microbiological parameters. with the exception of a few determinations made on the field, the determinations are carried out in the school 's laboratories by means of apha standard methods. with regard to the inorganic analytes, this means that a large amount of volumetric, colorimetric, and spectrometric determinations are carried out manually, thus presenting a heavy workload for the analysts and resulting in a low throughput. with the goal of solving this problem, although commercial automatic analysers for water samples are available, they are expensive. working in a university environment, it makes sense to engage in the design of the analyser as students, both graduate and nongraduate, can take part in the task. automation of the analysis of water samples has been the subject of a significant number of papers published in the last years. many of these works resort to some kind of flow - based technique [24 ]. however, most of the flow - based analysers proposed in the literature are dedicated systems focusing on the determination of one or two parameters in water samples [58 ], mainly for oceanographic research. recently, the concept of a computer supported system of networked automated water analysers has been introduced, the european awacss system [10, 11 ] being an example of a continent - wide network of analysers. sequential injection analysis (sia) [12, 13 ] was proposed in 1990 as a flow - technique oriented to process analysis but presenting some properties which make it increasingly interesting for the automated routine analysis of water samples. the technique is robust, highly versatile and features low - reagent consumption, resulting in a low volume of chemical residues. thus, it was decided that the analyser to be designed should be based on this technique. the analyser should have multiparametric capability, that is, it should be capable of carrying out not less than three methods without the need of physical modification or reagent changes. it should also be flexible, allowing a fast and easy change of the analytes and methods under software control, and should be capable of interaction with an autosampler. in order to accommodate the analytical methods currently in use, the proposed analyser should resort to photometric detection based on a multiwavelength light - emitting diode (led) light source. this light source has been extensively investigated and found to be suitable for low cost and portable analytical equipment [1520 ]. this work presents the design, construction, and evaluation of an automatic multiparametric analyser based on sia for the analysis of drinking and surface water as well as groundwater samples. the sia flow system (figure 1) was based on a syringe pump (cavro xp-3000, tecan, mnnedorf, switzerland) with a two - position valve and a 2.5 ml barrel, and a 10-position cheminert selector valve with microelectric actuator (valco instruments, houston, tex, usa, model c25 - 3180emh). each of these devices was fed by its own dedicated 24 v dc power supply. both the pump and the syringe were controlled from a notebook computer via the rs232 serial ports of the devices. since the notebook did not have any conventional serial port, commercial plug - and - play usb - serial adapters were used to connect the notebook to the pump and the valve, thus generating virtual com1 and com2 serial ports. connections and coils of the flow system were made with 0.8 mm (internal diameter) fep tubing. a photometric detector was built around a glass flow cell (optical path 10 mm, internal volume 200 l), a photodiode detector (vtb1013, perkinelmer), and a common - anode tricolour high - brightness led (rl5-rgb, superbrightleds, st. the signal from the photodiode was processed by an amplifier implemented with two tl-081 operational amplifiers, and then sent to a 14-bit analogue to digital (a / d) interface (usb-1408fs, measurement computing, norton, mass, usa) controlled via a usb port of the computer. each component of the rgb tricolour led (red, green, blue, nominal wavelengths 630 nm, 525 nm, and 472 nm) was driven by a lm-334 constant - current source, in turn fed by one section of a ln-2803 octal darlington driver ic. the 3 relevant input gates of this driver were controlled from ttl - level signals from the input / output (i / o) ports of the a / d interface. in this way, each section of the led array could be turned on or off at will, allowing selective use of the led with the appropriate wavelength under computer control. a regulated power supply employing lm317 and lm337 voltage regulators provided 12 v for the operational amplifiers and leds. the prototype was built in an industrial plastic box with a hinged lid, measuring 31 30 19 cm (wdh) (figure 3). the front of the syringe pump and the stator of the valve protrude via holes in the case, thus being accessible for fluidic connections and inspection. the detector flow cell is the only component of the flow path situated inside the case. two teflon bulkhead connectors (omnifit, cambridge, uk) allow input and output connections to the cell. a program was written and compiled in visual basic 6.0 (microsoft). a graphic language programming add - on (softwire 3.1, softwire technology) was used together with visual basic to allow an easier programming from a graphic environment. the program controlled the whole operation of the system including the data acquisition, as discussed below. the data (series of absorbance data points) obtained were stored on hard disk as ascii files. these data files were later processed with the peak simple program (sri, torrance, calif, usa) which provided baseline correction, peak - height measurement, and hard copy printout. astm type i water was obtained from a millipore (so paulo, brazil) direct - q5 water purifier. as schematised in figures 1 and 2, the prototype of the analyser basically consists of the following sections : the flow system comprising the syringe pump, the selector valve, and the flow cell ; the light source and its control unit composed by the tricolour led, three adjustable constant - current sources and the uln-2803 driver ; the photo detector and its amplifier ; a power supply for the analogue portion (amplifier, photodetector, and leds) ; the a / d interface and its associated ttl i / o ports ; the notebook computer, with windows xp operating system, and the usb - serial adapters ; the custom - written software. for fluid handling, the cavro pump not only can be easily controlled via a rs232-compliant serial port, but also it has a number of commands available to control speed and acceleration allowing for optimum fluidic handling. it also has its own memory capable of storing complete subroutines that can be called and run when necessary. the 10-position selector valve can accommodate several reagents and auxiliary devices such as minicolumns or mixing coils, allowing for the implementation of several methods simultaneously. it is also controlled via a rs232 serial port by means of simple commands such as go9 the photometric detector was designed around a tricolour led comprising red, green and blue components, and a silicon photodiode as detector. 10-turn potentiometer to a value such that, when water was filling the flow cell, the output signal of the analogue amplifier was about 70% of the saturation voltage. given the high gain of the amplifier, the value of this current was around 1 ma for each led. the use of low currents was chosen to avoid the overheating of the semiconductor and the consequent variations in light output. the program was a critical component as the whole operation of the instrument relied on it. also, the desired flexibility depended heavily on its correct design. visual basic 6.0 was chosen as programming language because of the previous experience of the group with this language. the use of the softwire graphic programming environment (in some way reminiscent of the labview language) was decided because it greatly simplifies programming providing a graphic environment based on activex controls, while simultaneously keeping the possibility of command - based programming of visual basic. the program designed has two modules presented on the screen as formularies : the operating (main) form (figure 4) and the setup form (figure 5). the first one has several command buttons and a scrolling screen simulating a strip - chart recorder, where real - time data is plotted. it also allows calling service routines such as initialisation of the syringe pump and selection of the serial port (com1 or com2) to be connected to the pump and to the valve. it also allows the selection of the a / d board and channel numbers, sampling rate, scale, as well as data paths and file names. other functions currently available (but not essential for the operation) include the possibility of manually turning individual leds on or off and manually setting the photometric scale. after that, each line consists of three parts : a command for the selector valve, a command for the syringe pump, and a third part containing time intervals which may be necessary to wait. once loaded from the setup screen, they are ready to run. since the analyser was to be programmed and operated by trained personnel, no effort was made to use a natural language which would require an interpreter increasing the complexity of the program. in fact, the set of alphanumeric commands necessary to operate the valve and the pump is small and easy to learn by the user. these commands are provided by the manufacturers in the respective manuals and are as simple as go9 to send the valve to position 9, d1500r to dispense a volume equivalent of 1500 steps of the syringe pump, and so forth. the first active line of a method stores the led number (1 to 3) allowing for selection of the led with the appropriate wavelength for the intended method. once the method is loaded and started by means of a command button on the operation form, each line is parsed into its three components. first, a valve command is issued via the corresponding serial com port, sending the selector valve to a given position. then, after waiting a fixed time period of 400 ms (configurable from the setup screen), the pump command is issued via the second serial com port. then, if necessary, the system waits for the specified time interval before executing the next line of the script. the current valve and pump commands, time interval, and line number this screen also supports limited edition capabilities, allowing on the fly modifications of the script to be tested and stored. this feature turned out to be useful during methods development. data acquisition runs as a separate process, which can be started / stopped at any time by command buttons on the operation screen. the program triggers a / d conversions from the a / d interface as required. in order to be able to acquire real - time data, this is done at the beginning of the cycle while pumping water through the flow cell. the program first measures and memorises the dark response with all leds off., the program can then calculate the absorbance each time the a / d interface is triggered. absorbance values are then plotted on the scrolling screen and saved to a raw - data file in ascii format for later processing. after finishing a run, the user can choose to keep the raw - data file with a new name or to delete it. in the first case, the routine creates a new text file containing a header with information to make it compatible with the peak simple chromatography program. this information includes the number of data points and the sampling rate used by the a / d interface, allowing for the establishment of a real - time scale in the peak simple program. at this stage of the project, the use of a commercial chromatography program for data processing and evaluation was preferred in order to save development time. however, it is possible for future versions of the software to provide new routines for data processing such as the establishment of baselines, calculation of peak heights, areas and widths, handling of calibration curves, and so forth. the system price was kept at a minimum, the syringe pump and selector valve being the most expensive items (about us $ 3000 for both) followed by the notebook computer (purchased locally for us $ 600). the cost of the rest of the components, mainly electronics and hardware (including the a / d interface), was under us $ 1000. in order to assess the system 's capabilities, several methods, based on the well - established apha standard methods, chloride was determined by the colorimetric mercuric thiocyanate method (based on apha method 4500-cl g), using the blue led for detection. nitrite was determined using the green led by reaction with n-(1-naphtyl)-ethylenediamine dihydrochloride and sulphanilamide (based on apha method 4500-no2 b). similarly the method of online reduction in a cadmium minicolumn and determination as nitrite (based on apha method 4500-no3 e) was used for nitrate. the latter apha methods, in their batch form, were used as reference methods for validation, while for chloride, the argentometric method apha 4500-cl b was used. table 1 shows the analytical figures of merit of the methods as obtained in the validation stage. for each method, special attention was given to medium - term stability, as there is the possibility of drift from electronic components and temperature changes in the flow cell, especially as components temperature raises during operation. for this purpose, repeated injections were carried out for a period of 60 minutes, at concentrations of 30 mg l (chloride), 0.60 mg l (nitrite), and 10 mg l (nitrate). baseline drift was under 0.010 (absorbance), and sensitivity drift (measured as the relative difference of the average of the height of the first 5 peaks and of the last 5 ones) was 1.9% (chloride), 0.55% (nitrite), and 3.9% (nitrate). only in the latter case the slight baseline drift has no effect as the program used for data processing allows for baseline correction. the number of methods that could be simultaneously run depends on the number of ports in the selector valve. with the 10-position selector valve used and depending on the complexity of the method, it was possible to determine up to 4 analytes sequentially without changing reagents or flow configuration. in order to determine a greater number of simultaneous parameters, it would be necessary to use a selector valve with a higher number of ports. one apparent limitation of the system resides in the use of a tricolour led which limits the number of wavelengths available. however, the system can be used to implement a number of methods involving colorimetric detection, as long as the wavelength required by the method is close to one of the wavelengths emitted by the rgb led. depending on the difference of wavelengths, some loss of sensitivity may be expected if compared to the ideal situation of a perfect wavelength match ; however, this will not necessarily diminish the usefulness of the system for the intended purpose, as demonstrated by the three methods tested in this work. a lab - made autosampler has been designed and is being built at this time. however, a commercial autosampler could conceivably be added to the system with slight modifications in the software allowing for serial communications with the device. further modifications of the system, beside those previously mentioned, would be mainly based on modifications of the software and thus easily implemented. some of these modifications, currently under study, include allowing led operations at any method line, allowing a more complex scheme such as using two leds for background correction by the two - wavelength technique, or periodically resetting the photometric scale, in order to minimise the effects of drift in the zero signal or in the leds emission intensity. the total cost was low enough to make it affordable to a low - budget university laboratory. the analytical figures of merit found for the methods tested were satisfactory for the intended purpose. | an automated multiparametric water analyser was developed and evaluated. the system was based on sequential injection analysis and featured a photometric detection system comprising a tricolour rgb led source and a photodiode. a program compiled in visual basic was used to control the sia flow system, the leds, and the data acquisition and processing. the program loads and executes methods written in ascii and stored as text files. the system was capable of handling up to four methods simultaneously. when used to carry out methods based on the apha standard methods, the figures of merit obtained were considered satisfactory for the purpose. the total cost was under us $ 4600. it was concluded that the analyser is appropriate for routine use and has potential for an increased number of simultaneous methods and for enhanced capabilities if new versions of the software are developed. |
dcs are part of the innate immune system and function as sentinels for pathogens at potential sites of invasion (e.g., the skin or the gastrointestinal tract). proteins are then converted into peptides which are subsequently presented on major histocompatibility complex (mhc) molecules and recognized by t lymphocytes. the dendritic cell drives the immune response depending on the kind of antigen it has taken up. if the host needs defense against an invaded pathogen, dcs present the antigenic peptides to cytotoxic cd8 t cells and proinflammatory cd4 t helper (th1) cells activating the lymphocytes via direct cell - cell contact and proinflammatory cytokines such as interleukin- (il-) 12 or il-6 [3, 4 ]. if autoimmunity or abundant inflammation needs to be dampened dcs interact with regulatory t cells (treg) directly and via secretion of anti - inflammatory cytokines such as il-10 or retinoic acid (ra) [57 ]. what is often hardly considered in experimental settings is the fact that all processes directing the immune response of our organism take place under deprivation of nutrients and oxygen. the interplay of dendritic cells and lymphocytes either takes place in severely inflamed tissue or in secondary lymphoid organs. oxygen distribution in the spleen and lymph nodes is highly variable and hypoxic lymphocytes have been identified in both organs. the adaptation of cells to reduced oxygen tensions is largely coordinated by hypoxia - inducible factors (hifs) which have come into the focus of immunological research during the last ten years. this review focuses on mouse and human dendritic cells and their maturation and activation under hypoxic conditions. by covering publications on in vitro studies a particular emphasis is put on hifs as coordinators of the genetic response to hypoxia. the availability of mice with a dc - specific hif-1 k.- o. now allows a first appreciation of the potential role of hypoxia in dc function in vivo. hypoxia - inducible factors belong to the family of basic helix - loop - helix proteins. they consist of one - (hif-1, hif-2, or hif-3) and one common -subunit (hif-1 or arnt, aryl carbon nuclear translocator) forming the dna - binding transcription factor dimer. whereas the hif-1 protein is not affected by changes in oxygen tension hif- proteins are only detectable under hypoxic conditions. mrna is expressed and transcribed in all nucleated cells whereas the expression of hif-2 is more limited. hif-2 is found especially in endothelial cells but also in immune cells such as macrophages or dendritic cells. the role of hif-3 remains poorly defined so far, particularly in immune cells, and this review will therefore focus on hif-1 and hif-2. hif- proteins are under tight posttranslational control by oxygen. under normoxia, specific prolyl hydroxylase domain containing enzymes (phd 1, 2, and 3) use molecular oxygen to immediately hydroxylate distinct prolyl residues of newly synthesized hif--subunits. hydroxylated hif- (oh - hif-) interacts with the von - hippel - lindau protein (pvhl) e3 ligase complex that polyubiquinates oh - hif- leading to instantaneous proteasomal degradation under normoxia. in contrast, under hypoxia, prolyl hydroxylases lack oxygen as a crucial cosubstrate and are reduced in their enzymatic activity. hif-s can accumulate, translocate into the nucleus, and dimerize with hif-1. the transcription factor complexes hif-1 (hif-1/hif-1) and hif-2 (hif-2/hif-1) recruit cofactors such as p300/cbp (camp - response element binding protein) and bind to hypoxia - responsive elements of target gene dna. in addition to proline hydroxylation, hif-1 and hif-2 can be hydroxylated at an asparagine residue in their c - terminal part. asparagine hydroxylation is controlled by an oxygen - sensitive asparagyl hydroxylase, termed factor - inhibiting hif- (fih) 1. hifs have been shown to regulate more than 100 genes that are involved in glucose metabolism, cell death, cell cycle, angiogenesis, and erythropoiesis. during the last decade it has been recognized that many other factors are able to induce hif- although hif-1 has been studied more thoroughly than hif-2. bacterial lipopolysaccharides (lps) are able to induce the nf-b pathway and hif-1 mrna has been shown to be a target of classical nf-b activation by several groups [1215 ]. recently it was shown that hif-1 is one of the essential modulators of the cytokine response to bacterial lps as it is crucial for the synthesis of il-1. furthermore, hif-1 protein and mrna expression have been shown to be induced in macrophages purified from wounds. the authors could show that elevated protein levels hereby depended on the inflammatory cytokine tnf-. reactive oxygen species (ros) have been shown to induce hif-1 as well, although this effect seemed to be time - dependent as external h2o2 induced hif-1 protein in human osteosarcoma cells at early time points but suppressed it later on. nitric oxide (no) has been shown to induce hif-1 in normoxic macrophages stimulating macrophage migration by modulating the actin cytoskeleton via small gtpases. the role of hif-2 in the function of immune cells has been studied much less. macrophage no production and expression of costimulatory molecules cd86 and mhcii were unaffected by loss of hif-2, but imtiyaz and coworkers found profound changes in cytokine mrna expression and protein release after stimulation with lps and interferon (ifn). murine bone - marrow - derived macrophages lacking hif-2 showed reduced induction of il-1, il12p35, cxcl2, and il-6 mrna levels under inflammatory hypoxia whereas hypoxia alone if at all only moderately affected mrna expression of these genes. moving to in vivo models the authors found that macrophage hif-2 was required for an adequate immune response to cutaneous and peritoneal irritants. furthermore, loss of macrophage hif-2 prevented infiltration of tumor associated macrophages (tams) in models of hepatocellular carcinoma and colitis - associated cancer which inhibited tumor growth. the role of hif-2 in the function of dendritic cells under hypoxia is almost completely unexplored, although filippi. hif-1 activity has been shown to be induced by a number of viral infections as well. hepatitis bx protein (hbx) increases the stability of hif-1 protein via p42/44 mitogen - activated protein kinases (mapk). transactivation of hif-1 is also increased as hbx induces the interaction of the transcription factor with cbp, one of the cofactors hifs recruit for dna binding. kaposi 's sarcoma - associated herpesvirus (kshv) and epstein - barr - virus (ebv) induce hif-1 protein by increasing the proteasomal degradation of proteins involved in the normoxic degradation of hif-1. lana, kshv latency - associated nuclear antigen, increases the degradation of pvhl whereas the phds 1 and 3 are degraded by proteasomes upon ebv infection. furthermore, kshv expresses functional hres in the promoter regions of viral genes which could be activated in vitro by binding of either hif-1 or hif-2. taking these data into consideration, activation of hif-1 (and potentially hif-2) in viral infections seems to bring benefit to the pathogen rather than the host. dcs are central in coordinating immune responses against pathogens : whenever they register pathogens they pick them up, process the proteins of pathogens, and present typical antigenic peptides to cells of the adaptive immunity. these processes do not only activate the cell but also induce dc differentiation and maturation and make them migrate towards secondary lymphoid organs. dc differentiation and maturation are accompanied and at least in part defined by the upregulation of costimulatory molecules such as cd80 and cd86. furthermore, mature dendritic cells express high levels of surface mhcii and cd40 and are able to secrete il-12. this is important as dcs have to function as antigen presenters and producers of il-12 at the same time to induce differentiation and proliferation of th1 cells. until now, several groups have analyzed dc function under hypoxic conditions. for human dcs it has been shown that differentiation of dcs from blood monocytes under hypoxia resulted in a more active phenotype exhibiting a higher ability to stimulate allogeneic t cell responses. furthermore, hypoxic immature dcs have been shown to downregulate bacterial phagocytosis and exhibit an increased migratory capacity. several groups have contributed to the current knowledge on the underlying mechanisms : elia. and ricciardi. have found higher expression of the costimulatory molecules cd80, hla class ii, and cd86 on the cellular surface of hypoxic immature dcs. in contrast, costimulatory molecule expression appeared not to be different between mature hypoxic or normoxic dcs. reported that hypoxic immature like mature dc exhibited higher allo - t cell stimulation than normoxic dcs. this finding was partly supported by elia. who also found this effect for hypoxic immature dcs. exposed immature monocyte - derived dcs to hypoxia but could not detect any differences in the expression of costimulatory markers. in turn, in contrast observed a hypoxia - mediated differentiation of immature human monocyte - derived dcs. they exposed the cells for longer time periods to harsher hypoxic conditions than spirig and coworkers and could detect a higher expression of cd40 after hypoxic treatment. hypoxia also led to higher t cell stimulatory activity of dcs and this effect could be blocked by inhibition of the hif pathway. of note, maturation of dcs under hypoxia leads to a shift in the expression of chemokines and chemokine receptors. while chemokines get downregulated chemokine receptors are upregulated indicating that hypoxia may favor migratory capacity of mature dcs carrying cytokine receptors rather than immunologic functions such as immune cell recruitment [29, 33 ]. could recently show that short - term hypoxia induces the migratory capacity of immature and mature dcs in an in vitro migration assay and that this effect depends on the expression of hif-1. several groups have shown that hypoxia downregulated antigen uptake by immature dcs [28, 30 ] in a manner that seemed to be independent of hif-1. these in part divergent reports may in fact be explained by some still existing uncertainties about the actual degree of cellular hypoxia under different experimental conditions. in addition, the duration of hypoxic exposure will affect the dc response as well as continuous versus intermittent hypoxia. hypoxia did not change costimulatory molecule expression of mature dcs, but several groups have found other hypoxia - dependent changes in mature dc function. chronic hypoxia promoted the onset of a highly proinflammatory gene expression profile in mature dcs generated from human monocytes. hypoxic mature dcs thereby showed induced gene expression of cytokines and chemokines that are known to induce endothelial cell survival, recruitment and adhesion of mononuclear phagocytes, and recruitment and activation of predominantly th1/th17 cells. yang. extended this knowledge and showed that hypoxic mature dcs upregulated the expression of a2b adenosine receptor (a2bar) and thereby predominantly induced th2 activation. in 2011, bosco. found that chronic hypoxia potently induced the cell surface expression of triggering receptor expressed on myeloid cells- (trem-) 1 on mature dcs. the authors illustrated a transient induction depending on the severity of hypoxia and they identified an hre in the promoter region of trem-1. additional work of the same group has shown that hypoxia induces trem-1 expression also in immature dcs. trem-1 cross - linking has been associated with an induced release of inflammatory cytokines such as tnf-, il-6, and chemokines such as cxcl8, ccl4, and ccl5. in addition, trem-1 cross - linking has been shown to induce the release of il12p70, a cytokine inducing th1 immune responses. this would mean that hypoxia does not only favor migration of mature dcs but also modulates their inflammatory repertoire to attract other immune cells and to direct t cell activation. thus, for human dendritic cells it seems appropriate when bosco and varesio claim a examples of how hypoxia affects differentiation and cytokine response of human dendritic cells can be found in table 1. reported that murine bone - marrow - derived dendritic cells upregulate cd80 and cd86 after exposure to hypoxia and bacterial lps in an hif-1-dependent manner. consistent with these findings dcs cultivated under hypoxic conditions were less efficient in antigen uptake as they showed a more mature phenotype. in contrast, hypoxic treatment (24 h) alone was not able to induce expression of costimulatory molecules on the surface of bmdcs. this group used a conditional knockdown of hif-1 in bmdcs (by crossing hif-1 mice with cd11c - cre animals) rather than a sirna approach and they kept bmdcs under normoxic or hypoxic conditions during the whole process of differentiation from bone marrow cells. dcs differentiated for six days under hypoxic conditions showed a marked upregulation of cd80, cd86, and mhcii but none of these molecules showed an hif-1 dependent regulation. the work of khler. instead revealed changes in cytokine expression of hypoxic dcs which were mainly unaffected by loss of hif-1. only secretion of il-22 seemed to be hif-1 dependent when bmdcs were differentiated under hypoxic conditions. in addition, only dcs expressing functional hif-1 showed an increase of surface ccr7 after hypoxic differentiation. further experiments regarding dc stimulation with defined pathogens revealed that bacterial cpgs but not viral poly(i : c) were able to stabilize hif-1 protein. concluded that myd88 is essential to induce inflammatory, hif-1-dependent gene transcription (myd88 is not involved in intracellular tlr3 signaling triggered by poly(i : c)). one potential target gene of hif-1 in dcs cultivated under inflammatory hypoxia (hypoxia + lps or hypoxia + cpgs, resp.) was inducible nitric oxide synthase. cpgs could stimulate hif-1 protein in bmdcs generated from control and conditional hif-1 knockout animals (deletion of the dna binding domain of hif-1 under the control of the lyz2-cre - promoter ; bmdc cultures of these mice show knockout in generated dcs, although circulating dendritic cells do not express lyz2-cre - promoter). furthermore, bmdcs lacking functional hif-1 protein showed severe deficiencies in the release of type i interferons after lps stimulation and could not induce a proper t cell activation in an in vitro cd8 lymphocyte activation assay. last but not least hif-1-induced gene expression has been found to modulate the differentiation of murine plasmacytoid dcs (pdcs). fms - related tyrosine kinase 3-ligand (flt-3l) induced differentiation of pdcs from murine bone marrow cells was dramatically reduced upon hypoxic cultivation and this effect was not found when pdcs were lacking hif-1. inhibitor of dna binding 2 (id2) was identified as suppressor of differentiation. id2 thereby seemed to be exclusively regulated by hif-1 as a loss of hif-2 in the respective cells could not hinder suppression of differentiation. examples on how hypoxia affects the differentiation and cytokine response of murine dendritic cells can be found in table 2. until now, there are only few published studies that address the role of hifs in dendritic cells in inflammatory settings. have analyzed the role of hifs for plasmacytoid dc differentiation and found that hif-1 limited pdc generation in the bone marrow. in its absence, pdc development was encouraged and numbers of pdc increased. when mice with breast tumors in a pymt - mmtv model were crossed with hif-1 lyz2-cre mice lacking functional hif-1 in myeloid cells observed markedly enhanced numbers of pdc within the tumors compared with wt controls. whether this affects tumor progression and disease outcome still has to be analyzed. khler. have assayed the ability of bmdcs differentiated under hypoxic conditions to get recruited to secondary lymphoid organs. for this purpose, they generated differentially labeled bmdcs from hif-1 and hif-1 cd11c - cre mice under normoxic and hypoxic conditions and injected equal amounts of wt and hif-1 k.- o. they could distinguish how many wt or knockout dcs had migrated towards the popliteal lymph nodes and found that the hypoxic increase in dc migration was hif-1 dependent. three other models have been used to elucidate the influence of dendritic cell hif expression on their interaction with t cells. first, vaccination efficacy has been addressed and second two models of inflammation were investigated, namely, leishmania infection and dextran sodium sulfate- (dss-) induced murine colitis [4648 ]. bhandari. studied hif-1 tie2-cre mice showing knockout not only in endothelial but also in hematopoietic cells. they vaccinated hif-1 and hif-1 tie2-cre mice with a synthetic ova peptide that specifically induced a cd8 t cell response. analysis of ifn release by specific t cells in the spleens eight days after vaccination revealed that dendritic cells lacking hif-1 very insufficiently induced t cell activation compared to wt dcs. in addition to this, hif-1 tie2-cre mice showed reduced titers of specific ova antibodies. very recent studies used mouse models with dendritic hif-1 knockout in inflammatory settings in vivo. hammami. analyzed hif-1 and hif-1 cd11c - cre animals in leishmania infection. they found that ablation of hif-1 in dendritic cells resulted in a more efficient immune response. this comprised increased production of il-12, induced expansion of cd8 cells, and higher frequency of short - lived effector cells, a specialized cd8 t cell population that can be found in resolving acute infection. this could be shown to restrict cd8 t cell expansion as hemizygous hif-1 mice exhibited a significantly higher expansion of cd8 cells. fully in line with these findings that hif-1 dampens the inflammatory response, but with a different consequence for the outcome of these mice, flck. reported that hif-1 is essential for dendritic cells to induce regulatory t cells (tregs) in a model of acute dss - colitis. hif-1 mice and hif-1 cd11c - cre animals received equal amounts of dss, but loss of dendritic hif-1 caused more abundant inflammation and worse outcome for the animals. mesenteric lymph nodes of hif-1 cd11c - cre animals showed significantly reduced expression of the anti - inflammatory cytokine il-10 and of transforming growth factor- (tgf-) both are potent inducers of tregs. in addition, the authors found a strongly reduced expression of aldehyde dehydrogenase (aldh) 1a2, which is necessary for dendritic cells to catalyze retinal to retinoic acid (ra). t cells of these lymph nodes in turn showed reduced expression of the retinoic acid receptor (rar) and diminished expression of the gut homing markers ccr9 and 47 integrin. hif-1 deficiency in dcs thus abrogated their ability to induce treg proliferation in secondary lymphoid organs and disrupted treg homing towards the inflamed gut. apart from the interplay between dendritic cells and t cells predominantly occurring in the mesenteric lymph nodes the authors could also show that the loss of hif-1 by dcs affected the crosstalk between dcs and intestinal epithelial cells (iecs) causing iecs to produce more mucins and therefore overcome intestinal barrier damage. another hint that loss of dendritic hif-1 may affect the crosstalk between dcs and iecs was the finding that only hif-1 mice exhibited increased levels of thymic stromal lymphopoietin receptor (tslpr) in dss colitis. a reduced expression of the respective receptor may drive dcs towards a more inflammatory phenotype. figure 1 summarizes the effects of (inflammatory) hypoxia and hif-1 knockout on dendritic cells as it has been discussed above. dendritic cells normally have to function under conditions of inflammatory hypoxia. not only inflammatory stimuli, but also hypoxia profoundly affects their cellular function. the key transcription factors regulating dcs ' adaptation to conditions of (inflammatory) hypoxia are hypoxia - inducible factors (hifs) 1 and 2. hypoxic culture of immature dcs induces expression of costimulatory molecules such as cd80, cd86, and hla class ii / mhcii [28, 29 ]. whether these effects are dependent on hypoxia - inducible factor- (hif-) 1 or not is still under debate [41, 42 ]. in contrast, hif-1 is well recognized to affect migratory capacities of human and murine dendritic cells, most likely by shaping the cellular chemokine / chemokine receptor profile [21, 29 ]. until now, only very few in vivo studies have addressed the role of hifs for dendritic cell function ; however these studies indicate that hif-1 affects (i) differentiation of plasmacytoid dendritic cells in the bone marrow, (ii) migration of dcs towards secondary lymphoid organs, (iii) cd8 t cell activation and release of pathogen - specific antibodies, (iv) expansion of cd8 t cells and short - lived effector cells, and (v) induction of tregs. used the identical dendritic cell - specific hif-1 knockout model (hif-1 cd11c - cre) in different inflammatory settings. very strikingly, both studies reported findings tending in the same direction but with very different outcomes for the treated animals. in both studies, dendritic cells lacking hif-1 were the more potent inducers of an inflammatory response with benefits for the outcome in leishmania infection but abundant inflammation and more severe illness in dss colitis. clearly, more in vivo studies are needed to better understand the fragile balance between necessary induction of immunity to fight a disease and overactivation of immune cells damaging the host. hypoxia - inducible factors may thereby be important transcriptional regulators in the balance of the immune status of dendritic cells. | dendritic cells (dcs) are considered as one of the main regulators of immune responses. they collect antigens, process them, and present typical antigenic structures to lymphocytes, thereby inducing an adaptive immune response. all these processes take place under conditions of oxygen shortage (hypoxia) which is often not considered in experimental settings. this review highlights how deeply hypoxia modulates human as well as mouse immature and mature dendritic cell functions. it tries to link in vitro results to actual in vivo studies and outlines how hypoxia - mediated shaping of dendritic cells affects the activation of (innate) immunity. |
blood donation in israel is voluntary and does not involve any monetary benefit. using records from magen david adom, we registered age, sex, country of birth, and maternal and paternal countries of birth once for each donor, regardless of the number of blood units donated. from 1995 through 1998, donors were screened for antibodies against htlv-1 and htlv-2 by standard elisa (abbott htlv-1/htlv-2 enzyme immunoassay ; abbott laboratories, abbott park, il, usa). since 1998, testing has been performed by chemiluminescent immunoassay with the prism assay (abbott laboratories). the confirmatory assay was western blot htlv blot 2.4 (genelabs diagnostics, singapore science park, singapore). on the basis of virus transmission modes, we developed an algorithm for identifying the ethnic origin of both htlv-1positive and htlv-1negative blood donors (figure 1). we considered infection to be acquired in israel when the donor and both parents were born in israel. we considered infection to be acquired outside israel when the donor or 1 parent was born outside israel. when the donor was born in israel and the mother was born outside israel, country of origin was considered the mother 's country of birth. when the donor and the mother were born in israel, but the father was born outside israel, country of origin was considered the father s country of birth. detailed classification of geographic origin of blood donors (both htlv-1 positive and htlv-1 negative) is given in the technical appendix. data were analyzed by using microsoft access (microsoft, redmond, wa, usa) and epi info (centers for disease control and prevention, atlanta, ga, usa) ; statistical analysis was conducted by using analysis of contingency tables. the odds ratio (or) and 95% confidence interval were calculated. the chaim sheba medical center human subjects research review board approved this study. from january 9, 1995, through december 31, 2003 of these, 73 htlv-1 carriers were identified, for an overall prevalence of 5.8 infections per 100,000 donors. average age at diagnosis was 39.4 11.9 years ; 48 (66%) were men (compared with 72% of all blood donors ; p = 0.3125). all htlv-1positive donors had negative serologic results for htlv-2, human immunodeficiency virus, hepatitis c virus, and hepatitis b surface antigen. donors from middle eastern and eastern european countries were at highest risk for htlv-1 carriage. relative risk for human t - cell lymphotropic virus type 1 carriage in donors from different geographic origins. error bars indicate 95% confidence intervals (ci). per 100,000 population ; p<0.05. the diversity of the population in israel, combined with systematic screening of blood donors, enabled us to examine the global epidemiology of htlv-1 infection. one or both parents of at least 67% of jews in israel were born outside israel. thirteen percent immigrated from asia, 16% from africa, and 36% from europe and the americas (3). we believe that blood donors are representative of healthy persons in the general population of the country because approximately one third of the population are blood donors. the prevalence of htlv-1 infection among donors in israel was 5.8/100,000 but varied depending on donors geographic origins. in blood donors born in israel, the prevalence of htlv-1 infection was 1/100,000, comparable to and even lower than the prevalence of htlv-1 in blood donors from northern europe (technical appendix). we found a similar prevalence of htlv-1 infection in jewish immigrants from known htlv-1 endemic countries to what is known in their native countries. for example, the reported prevalence in blood donors in latin american countries ranged from 30/100,000 to 1,000/100,000 (technical appendix) ; our study found a prevalence of 20/100,000. in donors from the middle east, we found a high prevalence of htlv-1 infection in blood donors from iran (50.4/100,000), turkey (16.0/100,000), and iraq (10.2/100,000). of those countries, only iran can provide information about htlv-1 prevalence, and those data are limited to mashhad (4). our survey was performed after the initial report of htlv-1associated disorders in israelis born in or originating from mashhad (5). this example can highlight the role of immigrants as reflectors of the populations in their countries of origin. we found no systematic information about the epidemiology of htlv-1 in the rest of iran and neighboring countries. however, anecdotal reports suggest that the infection exists in kuwait, turkmenistan, and georgia (68). in persons from eastern europe, we found a high prevalence of htlv-1 carriage in blood donors originating from romania (14.9/100,000), russia (6.3/100,000), and the former yugoslavia (62.9/100,000). most of the literature does not consider eastern europe endemic for htlv-1 infection (2). particularly in romania, htlv-1 prevalence is considered low, and htlv-1 positivity was attributed mainly to immigration from other countries (2). in a careful review of the literature, we found only a few studies involving a small number of blood donors (technical appendix). these studies support our findings and suggest that htlv-1 is endemic in some eastern european countries. several cases of adult t - cell leukemia have been described in patients of romanian origin (914). our findings establish the need for rigorous screening of blood donors in suspected htlv-1endemic areas (middle east and eastern europe) and show the impact of immigrants or travelers as sentinels to the health problem in their country of origin. our data also demonstrate high prevalence of htlv-1 infection among those whose country of origin had been considered nonendemic for htlv-1. viral transmission could have occurred either from the non - jewish local population or from other jewish communities in which htlv-1 is endemic. we are not aware of immigration waves of jewish populations from iran and iraq to eastern europe and vice versa. therefore, it seems reasonable to conclude that our findings reflect local htlv-1 endemicity in the country of origin. | the prevalence of infection with human t - cell lymphotropic virus type 1 (htlv-1) in blood donors from israel is 1 infection/100,000 persons. in donors originating from eastern europe, the middle east, and latin america, prevalences are 7.7, 14.6, and 20.4, respectively. htlv-1 prevalence may be high outside areas where htlv-1 previously was known to be endemic. |
approximately 1 million new and recurrent acute myocardial infarctions (ami) occur annually in the united states.1 whereas st - segment elevation myocardial infarction (stemi) has long been accepted terminology in the literature and clinical setting,2 the term non - stemi (nstemi) became more commonly used in the mid-1990s.3,4 classifying patients with ami into stemi and nstemi has clinical utility since each group is comprised of patients with unique clinical characteristics, different treatment approaches, and in - hospital and short - term survival.58 data from randomized trials have shown that hospitalized patients with nstemi have a lower risk of dying during the first few weeks after an ami, but are at higher risk for adverse cardiovascular outcomes over the long - term than patients with a stemi.9,10 however, data derived from clinical trials may lack generalizability since these study populations generally include younger and healthier individuals than are commonly seen in community - based investigations. some investigations have reported no differences in 1-year mortality rates between stemi and nstemi patients,6 whereas others have observed higher death rates for patients with stemi or for those with nstemi.7,8 these divergent findings may be explained in part by the different sociodemographic and clinical characteristics of ami patients being studied as well as to sampling and methodologic approaches utilized to identify the population of interest. we have previously described the in - hospital and 1-year death rates in patients with nstemi and stemi,11 but more data are needed to delineate the longer - term prognosis of patients with these two major types of ami, particularly from a more generalizable population - based perspective. moreover, few recent studies have examined what factors may be associated with post - discharge mortality in these patients and whether these prognostic factors differ between patients with stemi or nstemi.68 the objectives of this study were to describe relatively contemporary post - hospital discharge all - cause death rates, and factors associated with an increased risk of dying, in patients who were discharged after an ami from all central massachusetts hospitals in the 2000s, further classified according to ami type. data from the worcester heart attack study (whas), an ongoing, population - based investigation describing the clinical epidemiology of ami in residents of central massachusetts, were used in the present investigation.1113 the whas has been collecting data describing the clinical epidemiology of ami in residents of the worcester, ma, usa metropolitan area hospitalized at all eleven central massachusetts medical centers on an approximate alternating yearly basis.1113 for the present study, we included data from only the most recent hospitalized patient cohorts, namely 2001, 2003, 2005, and 2007, in order to describe overall differences, and recent trends, in the epidemiology of stemi and nstemi in this large central new england population. of the 4208 patients hospitalized during the period under study, this study was approved by the committee for the protection of human subjects at the university of massachusetts medical school. the methodology of the whas has been described elsewhere in detail.1113 in brief, the medical records of patients at each central massachusetts medical center who were hospitalized for possible ami were reviewed by trained nurses and physicians and were validated according to predetermined criteria. patients with stemi and nstemi were classified according to standardized diagnostic criteria that we have used in this study on an ongoing basis.11 a diagnosis of stemi was made when new st - segment elevation was present at the j point in two or more contiguous leads. a diagnosis of nstemi was accepted when, in the absence of st - segment elevation, ischemic st - segment or t wave changes were present for at least 24 hours with positive cardiac enzymes and/or a typical clinical presentation. from 2003 on, in the absence of electrocardiogram (ecg) abnormalities, a diagnosis of nstemi was accepted when elevations in various cardiac biomarker assays, including troponin, were accompanied by typical clinical presentation. trained nurse and physician study personnel reviewed all baseline and serial ecgs and quality control activities were routinely conducted with respect to ecg interpretation and abstraction of data from hospital medical records. computer systems at all greater worcester hospitals also over - read all ecgs and were independently reviewed by trained study personnel. we excluded cases of ami that developed in the setting of trauma, as a result of a noncardiac condition, in the perioperative period, as well as cases of ami which occurred in patients who did not reside in the worcester metropolitan area. sociodemographic, medical history, laboratory, and clinical data were abstracted from the hospital medical records of eligible patients.1113 patients were classified as having an initial ami based on the review of information contained in hospital medical records and the absence of a history of prior coronary disease.12,13 information on the development of important inpatient clinical complications was also collected based on the review of hospital medical records. these complications included atrial fibrillation, heart failure, cardiogenic shock, and stroke ; uniform working definitions of each of these complications were defined in a standardized manner.1417 post - discharge mortality data were collected through the review of state and social security death certificates as well as by reviewing medical records for subsequent readmissions. we examined differences in the characteristics of patients discharged from all greater worcester hospitals with stemi versus nstemi by utilizing t - tests for continuous variables and chi - square tests for categorical variables. we examined post - discharge survival through 2009, accounting for the varying length of time patients were followed after hospital discharge. a cox proportional hazards regression approach was utilized to examine the independent association between selected demographic and clinical factors with post - discharge mortality over the period of follow - up. variables were included in this regression model based on a p - value of 0.15 as well as on previously published associations with these factors on long - term prognosis after ami. variables associated with high rates of missing data, which occurred for ejection fraction, serum lipids, and body mass index, were not included in our regression models. post - discharge sociodemographic, clinical, or medical care related variables were not included in our regression models since we did not collect further information about these prognostic factors. hazard ratios (hrs) and accompanying 95% confidence intervals (cis) around various point estimates, including overall differences in long - term survival between patients with stemi and nstemi, were calculated in a standardized manner. using the graphical and numerical methods for model assessment we found that the assumption of proportional hazards was violated by two clinically relevant factors.18 inasmuch, we conducted a sensitivity analysis using an accelerated failure time model to confirm the validity of the cox model in light of the presence of nonproportional hazards among these key study covariates prior medical history of heart failure and systolic blood pressure findings at the time of hospital admission. this analysis confirmed that the violation of the proportional hazards assumption did not affect the reliability of our results. a logistic regression analysis was also carried out for purposes of examining differences in post - discharge prognosis at key long - term points (eg, 3 months, 1 year, 2 years) between patients with stemi versus nstemi, with odds ratios (ors) and accompanying 95% cis calculated. the methodology of the whas has been described elsewhere in detail.1113 in brief, the medical records of patients at each central massachusetts medical center who were hospitalized for possible ami were reviewed by trained nurses and physicians and were validated according to predetermined criteria. patients with stemi and nstemi were classified according to standardized diagnostic criteria that we have used in this study on an ongoing basis.11 a diagnosis of stemi was made when new st - segment elevation was present at the j point in two or more contiguous leads. a diagnosis of nstemi was accepted when, in the absence of st - segment elevation, ischemic st - segment or t wave changes were present for at least 24 hours with positive cardiac enzymes and/or a typical clinical presentation. from 2003 on, in the absence of electrocardiogram (ecg) abnormalities, a diagnosis of nstemi was accepted when elevations in various cardiac biomarker assays, including troponin, were accompanied by typical clinical presentation. trained nurse and physician study personnel reviewed all baseline and serial ecgs and quality control activities were routinely conducted with respect to ecg interpretation and abstraction of data from hospital medical records. computer systems at all greater worcester hospitals also over - read all ecgs and were independently reviewed by trained study personnel. we excluded cases of ami that developed in the setting of trauma, as a result of a noncardiac condition, in the perioperative period, as well as cases of ami which occurred in patients who did not reside in the worcester metropolitan area. sociodemographic, medical history, laboratory, and clinical data were abstracted from the hospital medical records of eligible patients.1113 patients were classified as having an initial ami based on the review of information contained in hospital medical records and the absence of a history of prior coronary disease.12,13 information on the development of important inpatient clinical complications was also collected based on the review of hospital medical records. these complications included atrial fibrillation, heart failure, cardiogenic shock, and stroke ; uniform working definitions of each of these complications were defined in a standardized manner.1417 post - discharge mortality data were collected through the review of state and social security death certificates as well as by reviewing medical records for subsequent readmissions. we examined differences in the characteristics of patients discharged from all greater worcester hospitals with stemi versus nstemi by utilizing t - tests for continuous variables and chi - square tests for categorical variables. we examined post - discharge survival through 2009, accounting for the varying length of time patients were followed after hospital discharge. a cox proportional hazards regression approach was utilized to examine the independent association between selected demographic and clinical factors with post - discharge mortality over the period of follow - up. variables were included in this regression model based on a p - value of 0.15 as well as on previously published associations with these factors on long - term prognosis after ami. variables associated with high rates of missing data, which occurred for ejection fraction, serum lipids, and body mass index, were not included in our regression models. post - discharge sociodemographic, clinical, or medical care related variables were not included in our regression models since we did not collect further information about these prognostic factors. hazard ratios (hrs) and accompanying 95% confidence intervals (cis) around various point estimates, including overall differences in long - term survival between patients with stemi and nstemi, were calculated in a standardized manner. using the graphical and numerical methods for model assessment we found that the assumption of proportional hazards was violated by two clinically relevant factors.18 inasmuch, we conducted a sensitivity analysis using an accelerated failure time model to confirm the validity of the cox model in light of the presence of nonproportional hazards among these key study covariates prior medical history of heart failure and systolic blood pressure findings at the time of hospital admission. this analysis confirmed that the violation of the proportional hazards assumption did not affect the reliability of our results. a logistic regression analysis was also carried out for purposes of examining differences in post - discharge prognosis at key long - term points (eg, 3 months, 1 year, 2 years) between patients with stemi versus nstemi, with odds ratios (ors) and accompanying 95% cis calculated. a total of 3762 individuals with confirmed ami were discharged from all metropolitan worcester hospitals between 2001 and 2007. patients with nstemi were significantly older, included a greater proportion of women, and had more comorbid cardiovascular conditions, but were less likely to have presented with an initial ami compared to patients with stemi (table 1). the hospital course of patients with nstemi was more likely to have been complicated by the development of atrial fibrillation and heart failure (table 1). patients with nstemi had higher systolic blood pressure and serum glucose levels, and lower estimated glomerular filtration rate (gfr) findings, than patients with stemi. expectedly, patients with stemi were more likely to have undergone a percutaneous coronary intervention (pci), whereas coronary artery bypass graft (cabg) surgery was performed in a similar percentage of both patient groups. patients with nstemi were more likely to have been discharged from the hospital on digoxin and calcium channel blockers, while patients with stemi were more likely to have been discharged on aspirin, beta - blockers, lipid - lowering agents, and angiotensin - converting - enzyme (ace)-inhibitors / angiotensin receptor blockers. patients discharged from greater worcester hospitals after nstemi during the years under study experienced higher post - discharge all - cause death rates with 3-month, 1-year, and 2-year death rates of 12.6%, 23.5%, and 33.2%, respectively, compared with 6.1%, 11.5%, and 16.4% for patients with stemi (figure 1). for our initial two study cohorts (2001 and 2003) in which longer - term follow - up was available, the 5-year death rates were 30.2% and 52.4%, respectively, for patients with stemi and nstemi (figure 1). after adjusting for baseline differences in age, sex, length of stay, comorbidities, hospital clinical complications, and physiologic variables, patients with stemi were significantly more likely to have survived at 3 months (or 1.38 ; 95% ci 1.011.87), 1 year (or 1.38 ; 95% ci 1.091.74), and 2 years (or 1.53 ; 95% ci 1.231.89) (all p - values < 0.05) after hospital discharge compared to patients with nstemi. overall, patients with nstemi were significantly more likely to have died during the years under study than patients with stemi (adjusted hr = 1.28 ; 95% ci 1.141.44) (p - value < 0.05). since early coronary revascularization is recommended for patients with stemi, we carried out an additional subgroup analysis in which we examined differences in post discharge death rates in patients with stemi and nstemi who underwent either pci or cabg. the results of this crude unadjusted analysis revealed death rates of 3.1% versus 3.2% at 3 months, 6.0% versus 8.4% at 1 year, and 7.9% versus 12.8% at 2 years in patients with stemi versus nstemi, respectively, in patients who underwent coronary revascularization. after multivariate adjustment for the sociodemographic characteristics, comorbidities, hospital clinical complications, and admission physiologic variables included in table 1, factors significantly associated with an adverse long - term prognosis following hospital discharge for patients with stemi included older age, longer hospital stay, previously diagnosed heart failure, diabetes or stroke, development of atrial fibrillation during hospitalization, elevated systolic blood pressure and blood glucose findings at the time of hospital admission, and lower serum estimated gfr findings (table 2). among patients with nstemi, older individuals, men, those with a longer hospital stay, patients with a history of either stroke, heart failure, or diabetes, and those who developed stroke or heart failure during hospitalization were at greater risk for dying than respective comparison groups. elevated systolic blood pressure and elevated blood glucose levels at the time of hospital presentation as well as lower serum estimated gfr findings were associated with decreased long - term mortality following nstemi (table 2). a total of 3762 individuals with confirmed ami were discharged from all metropolitan worcester hospitals between 2001 and 2007. patients with nstemi were significantly older, included a greater proportion of women, and had more comorbid cardiovascular conditions, but were less likely to have presented with an initial ami compared to patients with stemi (table 1). the hospital course of patients with nstemi was more likely to have been complicated by the development of atrial fibrillation and heart failure (table 1). patients with nstemi had higher systolic blood pressure and serum glucose levels, and lower estimated glomerular filtration rate (gfr) findings, than patients with stemi. expectedly, patients with stemi were more likely to have undergone a percutaneous coronary intervention (pci), whereas coronary artery bypass graft (cabg) surgery was performed in a similar percentage of both patient groups. patients with nstemi were more likely to have been discharged from the hospital on digoxin and calcium channel blockers, while patients with stemi were more likely to have been discharged on aspirin, beta - blockers, lipid - lowering agents, and angiotensin - converting - enzyme (ace)-inhibitors / angiotensin receptor blockers. patients discharged from greater worcester hospitals after nstemi during the years under study experienced higher post - discharge all - cause death rates with 3-month, 1-year, and 2-year death rates of 12.6%, 23.5%, and 33.2%, respectively, compared with 6.1%, 11.5%, and 16.4% for patients with stemi (figure 1). for our initial two study cohorts (2001 and 2003) in which longer - term follow - up was available, the 5-year death rates were 30.2% and 52.4%, respectively, for patients with stemi and nstemi (figure 1). after adjusting for baseline differences in age, sex, length of stay, comorbidities, hospital clinical complications, and physiologic variables, patients with stemi were significantly more likely to have survived at 3 months (or 1.38 ; 95% ci 1.011.87), 1 year (or 1.38 ; 95% ci 1.091.74), and 2 years (or 1.53 ; 95% ci 1.231.89) (all p - values < 0.05) after hospital discharge compared to patients with nstemi. overall, patients with nstemi were significantly more likely to have died during the years under study than patients with stemi (adjusted hr = 1.28 ; 95% ci 1.141.44) (p - value < 0.05). since early coronary revascularization is recommended for patients with stemi, we carried out an additional subgroup analysis in which we examined differences in post discharge death rates in patients with stemi and nstemi who underwent either pci or cabg. the results of this crude unadjusted analysis revealed death rates of 3.1% versus 3.2% at 3 months, 6.0% versus 8.4% at 1 year, and 7.9% versus 12.8% at 2 years in patients with stemi versus nstemi, respectively, in patients who underwent coronary revascularization. after multivariate adjustment for the sociodemographic characteristics, comorbidities, hospital clinical complications, and admission physiologic variables included in table 1, factors significantly associated with an adverse long - term prognosis following hospital discharge for patients with stemi included older age, longer hospital stay, previously diagnosed heart failure, diabetes or stroke, development of atrial fibrillation during hospitalization, elevated systolic blood pressure and blood glucose findings at the time of hospital admission, and lower serum estimated gfr findings (table 2). among patients with nstemi, older individuals, men, those with a longer hospital stay, patients with a history of either stroke, heart failure, or diabetes, and those who developed stroke or heart failure during hospitalization were at greater risk for dying than respective comparison groups. elevated systolic blood pressure and elevated blood glucose levels at the time of hospital presentation as well as lower serum estimated gfr findings were associated with decreased long - term mortality following nstemi (table 2). the results of this community - based study demonstrate that the post - discharge death rates for patients hospitalized with nstemi were higher than for patients discharged after stemi. after multivariate adjustment, increasing age was an important predictor of death for both patient groups after hospital discharge, whereas several factors were differentially associated with mortality depending on ami type. similar to the results of other studies,6,7,19 a previous publication from the whas showed that patients with nstemi were at higher risk for dying during the first year after hospital discharge.11 in the multicenter registry of more than 2100 patients hospitalized with an ami at 56 centers throughout france,6 patients with nstemi experienced higher post - discharge death rates as well as rates of rehospitalization than patients with stemi. among a large cohort of patients undergoing cardiac catheterization between 1999 and 2007 at duke university medical center, patients who developed nstemi experienced a higher risk of dying over the long - term (median follow - up = 4 years) than patients with stemi.7 similarly, when we extended our period of follow - up even further to 5 years after hospital discharge, patients with nstemi continued to fare appreciably worse over the long - term. it has been previously reported that patients with stemi are treated more aggressively with various inpatient therapies that are not based on disease severity.6 we similarly observed differences in the receipt of inpatient procedures and discharge medications according to ami type. while these treatment practices may be accounted for by differential indications and current practice guidelines, it is possible that differences in treatment may have partially accounted for the improved long - term survival we observed in patients with stemi. we did not, however, control for the receipt of either cardiac medications or coronary revascularization procedures in our regression models due to the significant potential for confounding by treatment indication in the context of this nonrandomized observational study. when we did, however, carry out a subgroup analysis in patients who underwent either a pci or cabg surgery during hospitalization, patients with nstemi continued to experience higher all - cause death rates over our follow - up period than patients with stemi. the 1-year all - cause mortality rates for patients discharged from greater worcester hospitals after stemi (12%) and nstemi (24%) were slightly higher for stemi, but considerably higher for nstemi, than what has been reported in prior investigations. a single - center observational study of patients with myocardial infarction undergoing pci reported 1-year death rates of 9.5% and 14.3% for stemi and nstemi, respectively.7 a study of 1486 patients with ami enrolled in the multicenter dynamic registry observed 1-year mortality rates of 7.3% for stemi and 5.5% for nstemi,20 while the 1-year death rates were 9.0% in stemi patients and 11.6% in nstemi patients in the opera registry.6 in the gusto - iib trial, 1-year death rates were 9.6% for patients with stemi and 11.1% for those with nstemi.19 the discrepancies between these results and our findings are likely attributable to the advanced age and comorbid disease burden of patients in our community - based study as compared with those enrolled in other studies with different methodologies and more restrictive inclusion criteria,19 or only patients studied at specialized medical centers.7 our findings may be more generalizable to the overall population of patients experiencing an ami, and are important as they help to resolve the discrepancies in the results between other studies performed over a similar time period in more select patient populations.7,20 the present results lend further support to the notion that patients discharged from the hospital after nstemi constitute a high risk group in whom close surveillance and the use of evidence - based cardiac medications, where appropriate, is warranted. in a multicenter study of patients hospitalized with ami at 56 centers in france in the early 2000s, while advanced age, high heart rate on admission, and low levels of blood pressure at the time of hospital admission were independent, and similar, correlates of 1-year death rates in patients with stemi or nstemi, differences in the predictability of several factors, or their strength of association, were observed in patients according to type of ami.6 results of prior studies examining the association between sex and mortality after ami have been conflicting, especially after controlling for age.21,22 in the present study, men were more likely to have died after hospital discharge for nstemi and stemi. another important prognostic factor was the presence of renal dysfunction, which was associated with long - term mortality in both patient groups. similarly, prior studies have demonstrated higher in - hospital, 30-day, and 3-year mortality rates in patients with ami and renal impairment as compared to those with intact renal function.23,24 renal impairment may increase mortality through multiple mechanisms, including a decreased likelihood of receiving appropriate therapies due to concern over an increased risk of bleeding or worsening renal function, drug toxicity, and endothelial dysfunction.23,24 the long - term mortality for patients with nstemi, in particular, has been related to the extent of their comorbid disease burden.25 in the present study, patients with nstemi were more likely to have several comorbid diseases previously diagnosed, but patients with nstemi and stemi were both found to have decreased survival if they had a history of heart failure. this is not surprising given the dismal long - term prognosis that is associated with this clinical syndrome.26 similarly, we found that a history of prior stroke was associated with decreased survival in both patient groups after hospital discharge. this finding is consistent with prior reports where ami patients with prior stroke were treated less aggressively, and had higher rates of complications and inpatient and early post - discharge mortality, than patients without previously diagnosed stroke.27 interestingly, we found that patients with nstemi who presented with an elevated systolic blood pressure had reduced mortality. a registry study in sweden also found that, among patients admitted to the intensive care unit with ami, an elevated blood pressure was associated with reduced 1-year mortality.28 why a higher systolic blood pressure might be associated with improved outcomes for patients with ami is unknown, but may reflect preserved myocardial function or absence of systemic hypoperfusion. despite similarities in some prognostic factors, there were also differences in the prognostic impact of several physiologic variables and in - hospital clinical complications between patients with stemi and nstemi, with a number of these factors having a particularly adverse impact in those with nstemi. these findings suggest the need for greater surveillance, and intervention where appropriate, for high risk patients with nstemi. the strengths of this study include its community - based perspective, its relatively contemporary population of patients hospitalized with ami, and its large sample size. our study has several limitations, however, that should be considered when interpreting the present results. the greater worcester population is predominantly caucasian and our findings may lack generalizability to other ethnic groups or communities of varying size. we were not able to comment on the influence that hospital and post - discharge treatment practices may have had on long - term prognosis since this was a nonrandomized observational study and we did not collect information about the receipt of different medication treatment regimens or coronary revascularization approaches after a patient s discharge from all central massachusetts hospitals. we were unable to further classify patients with ami into a recently recommended schema29 and to satisfactorily assess, and by extension, analytically control for, other potentially important factors that may have influenced prognosis after ami, such as infarct size, serum lipid or blood pressure levels, or presence of adverse lifestyle practices, such as cigarette smoking. in a multicenter study of patients hospitalized with ami at 56 centers in france in the early 2000s, while advanced age, high heart rate on admission, and low levels of blood pressure at the time of hospital admission were independent, and similar, correlates of 1-year death rates in patients with stemi or nstemi, differences in the predictability of several factors, or their strength of association, were observed in patients according to type of ami.6 results of prior studies examining the association between sex and mortality after ami have been conflicting, especially after controlling for age.21,22 in the present study, men were more likely to have died after hospital discharge for nstemi and stemi. another important prognostic factor was the presence of renal dysfunction, which was associated with long - term mortality in both patient groups. similarly, prior studies have demonstrated higher in - hospital, 30-day, and 3-year mortality rates in patients with ami and renal impairment as compared to those with intact renal function.23,24 renal impairment may increase mortality through multiple mechanisms, including a decreased likelihood of receiving appropriate therapies due to concern over an increased risk of bleeding or worsening renal function, drug toxicity, and endothelial dysfunction.23,24 the long - term mortality for patients with nstemi, in particular, has been related to the extent of their comorbid disease burden.25 in the present study, patients with nstemi were more likely to have several comorbid diseases previously diagnosed, but patients with nstemi and stemi were both found to have decreased survival if they had a history of heart failure. this is not surprising given the dismal long - term prognosis that is associated with this clinical syndrome.26 similarly, we found that a history of prior stroke was associated with decreased survival in both patient groups after hospital discharge. this finding is consistent with prior reports where ami patients with prior stroke were treated less aggressively, and had higher rates of complications and inpatient and early post - discharge mortality, than patients without previously diagnosed stroke.27 interestingly, we found that patients with nstemi who presented with an elevated systolic blood pressure had reduced mortality. a registry study in sweden also found that, among patients admitted to the intensive care unit with ami, an elevated blood pressure was associated with reduced 1-year mortality.28 why a higher systolic blood pressure might be associated with improved outcomes for patients with ami is unknown, but may reflect preserved myocardial function or absence of systemic hypoperfusion. despite similarities in some prognostic factors, there were also differences in the prognostic impact of several physiologic variables and in - hospital clinical complications between patients with stemi and nstemi, with a number of these factors having a particularly adverse impact in those with nstemi. these findings suggest the need for greater surveillance, and intervention where appropriate, for high risk patients with nstemi. the strengths of this study include its community - based perspective, its relatively contemporary population of patients hospitalized with ami, and its large sample size. our study has several limitations, however, that should be considered when interpreting the present results. the greater worcester population is predominantly caucasian and our findings may lack generalizability to other ethnic groups or communities of varying size. we were not able to comment on the influence that hospital and post - discharge treatment practices may have had on long - term prognosis since this was a nonrandomized observational study and we did not collect information about the receipt of different medication treatment regimens or coronary revascularization approaches after a patient s discharge from all central massachusetts hospitals. we were unable to further classify patients with ami into a recently recommended schema29 and to satisfactorily assess, and by extension, analytically control for, other potentially important factors that may have influenced prognosis after ami, such as infarct size, serum lipid or blood pressure levels, or presence of adverse lifestyle practices, such as cigarette smoking. in this relatively contemporary cohort, patients with stemi experienced a better post - discharge prognosis than those with nstemi. the factors associated with increased mortality for each of these patient groups were somewhat distinct, emphasizing the uniqueness of each disease category. additional population - based surveillance efforts are needed to monitor recent trends in the natural history, functional status, and quality of life in patients further classified according to type of ami. studies leading to better understanding of the optimal inpatient and outpatient treatments based on ami type and comorbid disease burden may maximize long - term outcomes for these patients. | backgroundlimited recent data are available describing differences in long - term survival, and factors affecting prognosis, after st - segment elevation myocardial infarction (stemi) and non - st - segment elevation myocardial infarction (nstemi), especially from the more generalizable perspective of a population - based investigation. the objectives of this study were to examine differences in post - discharge prognosis after hospitalization for stemi and nstemi, with a particular focus on factors associated with reduced long - term survival.methodswe reviewed the medical records of residents of the worcester, ma, usa metropolitan area hospitalized at eleven central massachusetts medical centers for acute myocardial infarction (ami) during 2001, 2003, 2005, and 2007.resultsa total of 3762 persons were hospitalized with confirmed ami ; of these, 2539 patients (67.5%) were diagnosed with nstemi. the average age of study patients was 70.3 years and 42.9% were women. patients with nstemi experienced higher post - discharge death rates with 3-month, 1-year, and 2-year death rates of 12.6%, 23.5%, and 33.2%, respectively, compared to 6.1%, 11.5%, and 16.4% for patients with stemi. after multivariable adjustment, patients with nstemi were significantly more likely to have died after hospital discharge (adjusted hazards ratio 1.28 ; 95% confidence interval 1.141.44). several demographic (eg, older age) and clinical (eg, history of stroke) factors were associated with reduced long - term survival in patients with nstemi and stemi.conclusionsthe results of this study in residents of central massachusetts suggest that patients with nstemi are at higher risk for dying after hospital discharge, and several subgroups are at particularly increased risk. |
the trigger finger is in the clinical practice, frequently caused by stenosing tenosynovitis at the a1 pulley characterized by pain, swelling and a triggering sensation with the limitation of finger motion. the first treatment is conservative using anti - inflammatory drugs and steroid injection, but some resistant cases may need surgery. post - traumatic trigger finger is a rare case of resistant trigger finger that can be suspected recording an accurate clinical history and in case of resistant trigger finger after anti - inflammatory drugs therapy and steroid injection.[26 ]. we report a case of post - traumatic trigger finger due to a partial longitudinal tear of the flexor digitorum superficialis. a healthy 22-year - old caucasian male referred to our department suffering from pain and limited range of motion (rom) of the right ring finger. the patient sustained in august 2015 a penetrating injury on the palm of the right hand during his sport activity. he did not refer to any emergency department or general pratictioner, the wound was small and healed naturally. one month later pain started and limitation of rom of the right ring finger gradually worsened. because of his clinical disorders, a trigger finger was diagnosed by another orthopedic who performed a steroid injection. the clinical examination showed a small scar on the palm, located proximally to the fourth metacarpophalangeal joint and the rom of the proximal interphalangeal (pip) joint of the ring finger was limited to 40 degrees of extension and complete (100 degrees) in flexion, both in passive and active motion. the feeling of numbness just under the scar, the first steroid injection that was not effective and the young age of the patient, led us to perform an ultrasound examination of the hand tendons (figure 1), which showed a suspect tear of the flexor digitorum superficialis tendon of the ring finger, proximally to the pulley a1 that appeared thickened. the surgical exploration of the tendon was the treatment of choice, which was performed 2 months and 10 days from the injury. the ultrasounds examination shows an irregular region (arrow) of the flexor digitorum superficialis tendon suggestive of a partial rupture, accompanied by the thickening of the pulley a1 (asterisk). p : proximal phalanx ; m : metacarpus ; mpj : metacarpo - phalangeal joint. the incision was made at the pulley a1 and extended brunner - like proximally in direction of the scar. a partial longitudinal flap - shaped tear was discovered, large approximately half of the entire tendon diameter, of the radial half of the flexor digitorum superficialis tendon that hooked at the entrance of the a1 pulley (figure 2). the flexor digitorum profundus tendon was intact. because of the size of the flap - shaped tear, it was decided not to remove it, but it was sutured to the tendon in anatomic position with simple stitches (nylon 4 - 0) and the pulley a1 release completed the procedure to avoid risk of post - operative re - triggering. the examination during surgery showed the restoration of ring finger rom (0100 degrees) and the disappearance of the trigger, then the exploration of the distal pulleys (a2 and a3) was not necessary. also, a capsulotomy of the pip was not necessary because the limitation of rom was not due to a capsular contracture. the patient was recommended to frequently move the fingers even with the bandage, to prevent the formation of adhesions. there was no need for physiotherapy as the rom has been completely restored almost immediately. after 3 months of follow - up, no evidence of relapse was recorded and the rom was 0100 degrees. the picture shows the longitudinal partial flap - shaped tear of the flexor digitorum superficialis. most of the patients affected by trigger fingers are treated with anti - inflammatory drugs or steroid injection. the success rate of the single - dose of a steroid injection is reported to be 50% and when the conservative treatment fails, open a1 pulley release has a success rate of approximately 100%. in literature some cases of resistant trigger finger caused by rare conditions, such as phalangeal osteochondroma, tendon sheets tumor (fibroma or giant cell tumor) or calcifying tenosynovitis have been reported. more frequent, but still rare in the clinical practice, is the trigger finger due to a partial tear of the flexor digitorum. to our knowledge, there are only few reports in literature.[210 ] previous reports advised the imaging support for the diagnosis of triggering after partial flexor tendon tear. more recently, okano and couceiro in two different reports used mri for the diagnosis. our recent case, together with the other few reported in literature, demonstrates that the diagnosis of trigger finger, although does not present difficulties in the majority of the cases, have to be supported by an accurate clinical history and clinical examination, because in some cases it can be the consequence of rare clinical conditions. in selected cases, mostly if post - traumatic, the ultrasound imaging or the mri can be used to perform a correct diagnosis, and subsequently a correct treatment. the authors declares that there is no conflict of interest regarding the publication of this paper. | abstractwe report a case of post - traumatic trigger finger due to a partial longitudinal tear of the flexor digitorum superficialis. the suspect came from the clinical history and the young age of the patient. it was successfully treated with tendon flap suture and pulley a1 release. |
at the time of the birth of general relativity (gr), experimental confirmation was almost a side issue. einstein did calculate observable effects of general relativity, such as the perihelion advance of mercury, which he knew to be an unsolved problem, and the deflection of light, which was subsequently verified, but compared to the inner consistency and elegance of the theory, he regarded such empirical questions as almost peripheral. but today, experimental gravitation is a major component of the field, characterized by continuing efforts to test the theory s predictions, to search for gravitational imprints of high - energy particle interactions, and to detect gravitational waves from astronomical sources. the modern history of experimental relativity can be divided roughly into four periods : genesis, hibernation, a golden era, and the quest for strong gravity. the genesis (18871919) comprises the period of the two great experiments which were the foundation of relativistic physics the michelson - morley experiment and the etvs experiment and the two immediate confirmations of gr the deflection of light and the perihelion advance of mercury. following this was a period of hibernation (19201960) during which theoretical work temporarily outstripped technology and experimental possibilities, and, as a consequence, the field stagnated and was relegated to the backwaters of physics and astronomy. but beginning around 1960, astronomical discoveries (quasars, pulsars, cosmic background radiation) and new experiments pushed gr to the forefront. experimental gravitation experienced a golden era (19601980) during which a systematic, world - wide effort took place to understand the observable predictions of gr, to compare and contrast them with the predictions of alternative theories of gravity, and to perform new experiments to test them. the period began with an experiment to confirm the gravitational frequency shift of light (1960) and ended with the reported decrease in the orbital period of the hulse - taylor binary pulsar at a rate consistent with the general relativity prediction of gravity wave energy loss (1979). the results all supported gr, and most alternative theories of gravity fell by the wayside (for a popular review, see). since 1980, the field has entered what might be termed a quest for strong gravity. many of the remaining interesting weak - field predictions of the theory are extremely small and diffcult to check, in some cases requiring further technological development to bring them into detectable range. the sense of a systematic assault on the weak - field predictions of gr has been supplanted to some extent by an opportunistic approach in which novel and unexpected (and sometimes inexpensive) tests of gravity have arisen from new theoretical ideas or experimental techniques, often from unlikely sources. examples include the use of laser - cooled atom and ion traps to perform ultra - precise tests of special relativity ; the proposal of a fifth force, which led to a host of new tests of the weak equivalence principle ; and recent ideas of large extra dimensions, which have motived new tests of the inverse square law of gravity at sub - millimeter scales. several major ongoing efforts also continue, principally the stanford gyroscope experiment, known as gravity probe - b. instead, much of the focus has shifted to experiments which can probe the effects of strong gravitational fields. the principal figure of merit that distinguishes strong from weak gravity is the quantity gm / rc, where g is the newtonian gravitational constant, m is the characteristic mass scale of the phenomenon, r is the characteristic distance scale, and c is the speed of light. near the event horizon of a non - rotating black hole, or for the expanding observable universe, 0.5 ; for neutron stars, 0.2. these are the regimes of strong gravity. for the solar system 0). this violation of the massive - body equivalence principle is known as the nordtvedt effect. the effect is absent in gr (=0) but present in scalartensor theory (=1/(2+)+4). the existence of the nordtvedt effect does not violate the results of laboratory etvs experiments, since for laboratory - sized objects, eg / m10, far below the sensitivity of current or future experiments. however, for astronomical bodies, eg / m may be significant (10 for the sun, 10 for jupiter, 4.610 for the earth, 0.210 for the moon). if the nordtvedt effect is present (0) then the earth should fall toward the sun with a slightly different acceleration than the moon. this perturbation in the earth - moon orbit leads to a polarization of the orbit that is directed toward the sun as it moves around the earth - moon system, as seen from earth. this polarization represents a perturbation in the earth - moon distance of the form (38)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta r = 13.1\eta \cos \left ({ { \omega _ 0 } - { \omega _ { \rm{s } } } } \right)t\left [{ \rm{m } } \right ], $ $ \end{document } where 0 and s are the angular frequencies of the orbits of the moon and sun around the earth (see tegp 8.1 for detailed derivations and references ; for improved calculations of the numerical coefficient, see [102, 53 ]). since august 1969, when the first successful acquisition was made of a laser signal reflected from the apollo 11 retroreflector on the moon, the lunar laser - ranging experiment (lure) has made regular measurements of the round - trip travel times of laser pulses between a network of observatories and the lunar retroreflectors, with accuracies that are approaching 50 ps (1 cm). these measurements are fit using the method of least - squares to a theoretical model for the lunar motion that takes into account perturbations due to the sun and the other planets, tidal interactions, and post - newtonian gravitational effects. the predicted round - trip travel times between retroreflector and telescope also take into account the librations of the moon, the orientation of the earth, the location of the observatory, and atmospheric effects on the signal propagation. the nordtvedt parameter along with several other important parameters of the model are then estimated in the least - squares method. several independent analyses of the data found no evidence, within experimental uncertainty, for the nordtvedt effect (for recent results see [56, 154, 96 ]). their results can be summarized by the bound ||>|x| (is the gravitational wavelength/2), the field can be expanded in inverse powers of r=|x| in a multipole expansion, evaluated at the retarded time the leading term in 1/r is the gravitational waveform. for field points in the near zone or induction zone, |x||x|0, during cosmological evolution, the scalar field naturally evolves toward the minimum of the potential, i.e. toward 00, bd, or toward a theory close to, though not precisely gr [47, 48 ]. bounds on the parameters 0 and 0 from solar - system, binary - pulsar and gravitational wave observations (see sec. negative values of 0 correspond to an unstable scalar potential ; in this case, objects such as neutron stars can experience a spontaneous scalarization, whereby the interior values of can take on values very different from the exterior values, through non - linear interactions between strong gravity and the scalar field, dramatically affecting the stars internal structure and the consequent violations of sep. on the other hand, 0 3 \times { 10^{12}}{\rm{km}}{\left ({ \frac{d}{{200{\rm { mpc}}}}\frac{{100{\rm { hz}}}}{f } } \right)^{1/2}}{\left ({ \frac{1}{{f\delta t } } } \right)^{1/2}}. $ $ \end{document } the foregoing discussion assumes that the source emits both gravitational and electromagnetic radiation in detectable amounts, and that the relative time of emission can be established to sufficient accuracy, or can be shown to be sufficiently small. however, there is a situation in which a bound on the graviton mass can be set using gravitational radiation alone. that is the case of the inspiralling compact binary. because the frequency of the gravitational radiation sweeps from low frequency at the initial moment of observation to higher frequency at the final moment, the speed of the gravitons emitted will vary, from lower speeds initially to higher speeds (closer to c) at the end this will cause a distortion of the observed phasing of the waves and result in a shorter than expected overall time ta of passage of a given number of cycles. furthermore, through the technique of matched filtering, the parameters of the compact binary can be measured accurately, (assuming that gr is a good approximation to the orbital evolution, even in the presence of a massive graviton), and thereby the emission time te can be determined accurately. (76) can be measured to an accuracy 1/, where is the signal - to - noise ratio. thus one can estimate the bounds on g achievable for various compact inspiral systems, and for various detectors. for stellar - mass inspiral (neutron stars or black holes) observed by the ligo / virgo class of ground - based interferometers, d200 mpc, f100 hz, and ft1/10. the result is g>10 km. for supermassive binary black holes (10 to 10m) observed by the proposed laser interferometer space antenna (lisa), d3 gpc, f10 hz, and ft1/1000. a full noise analysis using proposed noise curves for the advanced ligo and for lisa weakens these crude bounds by factors between two and 10. these potential bounds can be compared with the solid bound g>2.810 km, derived from solar system dynamics, which limit the presence of a yukawa modification of newtonian gravity of the form (77)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v\left (r \right) = \left ({ gm / r } \right)\exp \left ({ - r/{\lambda _ { \rm{g } } } } \right), $ $ \end{document } and with the model - dependent bound g>610 km from consideration of galactic and cluster dynamics. one of the central difficulties of testing general relativity in the strong - field regime is the possibility of contamination by uncertain or complex physics. in the solar system, weak - field gravitational effects could in most cases be measured cleanly and separately from non - gravitational effects. the remarkable cleanliness of the binary pulsar permitted precise measurements of gravitational phenomena in a strong - field context. still, under suitable conditions, qualitative and even quantitative strong - field tests of general relativity can be carried out. one example is in cosmology. from a few seconds after the big bang until the present, the underlying physics of the universe is well understood, although significant uncertainties remain (amount of dark matter, value of the cosmological constant, the number of light neutrino families, etc.). some alternative theories of gravity that are qualitatively different from gr fail to produce cosmologies that meet even the minimum requirements of agreeing qualitatively with big - bang nucleosynthesis (bbn) or the properties of the cosmic microwave background (tegp 13.2). others, such as brans - dicke theory, are sufficiently close to gr (for large enough bd) that they conform to all cosmological observations, given the underlying uncertainties. the generalized scalar - tensor theories, however, could have small bd at early times, while evolving through the attractor mechanism to large bd today. one way to test such theories is through big - bang nucleosynthesis, since the abundances of the light elements produced when the temperature of the universe was about 1 mev are sensitive to the rate of expansion at that epoch, which in turn depends on the strength of interaction between geometry and the scalar field. because the universe is radiation - dominated at that epoch, uncertainties in the amount of cold dark matter or of the cosmological constant are unimportant. the nuclear reaction rates are reasonably well understood from laboratory experiments and theory, and the number of light neutrino families (3) conforms to evidence from particle accelerators. thus, within modest uncertainties, one can assess the quantitative difference between the bbn predictions of gr and scalar - tensor gravity under strong - field conditions and compare with observations. the most sophisticated recent analysis places bounds on the parameters 0 and 0 of the generalized framework of damour and esposito - farse (see sec. 5.4 and fig. 8) that are weaker than solar - system bounds for 00.3. observations of low - luminosity binary x - ray sources suggest that a form of accretion known as advection - dominated accretion flow (adaf) may be important. in this kind of flow, the accreting gas is too thin to radiate its energy efficiently, but instead transports (advects) it inward toward the central object. if the central object is a neutron star, the matter hits the surface and radiates the energy away ; if it is a black hole, the matter and its advected energy disappear. systems in which the accreting object is believed to be a black hole from estimates of its mass are indeed observed to be underluminous, compared to systems where the object is believe to be a neutron star. this has been regarded as the first astrophysical evidence for the existence of black hole event horizons (for a review, see). while supporting one of the critical strong - field predictions of gr, the observations and models are not likely any time soon to be able to distinguish one gravitational theory from another (except for theories that do not predict black holes at all). another example involving accretion purports to explore the strong - field region just outside massive black holes in active galactic nuclei. here, iron in the inner region of a thin accretion disk is irradiated by x - ray - emitting material above or below the disk, and fluoresces in the k line. the spectral shape of the line depends on relativistic doppler and curved - spacetime effects as the iron orbits the black hole near the innermost stable circular orbit, and could be used to determine whether the hole is a non - rotating schwarzschild black hole, or a rotating kerr black hole. because of uncertainties in the detailed models, the results are inconclusive to date, but the combination of higher - resolution observations and better modelling could lead to striking tests of strong - field predictions of gr. one reason is that gravity is a fundamental interaction of nature, and as such requires the most solid empirical underpinning we can provide. another is that all attempts to quantize gravity and to unify it with the other forces suggest that the standard general relativity of einstein is not likely to be the last word. furthermore, the predictions of general relativity are fixed ; the theory contains no adjustable constants so nothing can be changed. thus every test of the theory is either a potentially deadly test or a possible probe for new physics. although it is remarkable that this theory, born 80 years ago out of almost pure thought, has managed to survive every test, the possibility of finding a discrepancy will continue to drive experiments for years to come. | the status of experimental tests of general relativity and of theoretical frameworks for analysing them are reviewed. einstein s equivalence principle (eep) is well supported by experiments such as the etvs experiment, tests of special relativity, and the gravitational redshift experiment. future tests of eep and of the inverse square law will search for new interactions arising from unification or quantum gravity. tests of general relativity at the post - newtonian level have reached high precision, including the light defl ection the shapiro time delay, the perihelion advance of mercury, and the nordtvedt effect in lunar motion. gravitational wave damping has been detected in an amount that agrees with general relativity to half a percent using the hulse - taylor binary pulsar, and new binary pulsar systems may yield further improvements. when direct observation of gravitational radiation from astrophysical sources begins, new tests of general relativity will be possible. |
hepatocellular carcinoma (hcc) is the sixth most common malignant disease worldwide and the third greatest cause of cancer - related death. the etiology of hcc has been reported to be related to a variety of diseases such as viral hepatitis [2, 3 ], alcoholic hepatitis, nonalcoholic fatty liver disease (nafld) [5, 6 ], and metabolic syndrome including diabetes mellitus [7, 8 ]. the sequences from chronic hepatitis and liver cirrhosis cause de novo hcc (figure 1). hcc is considered to have increased invasiveness with malignant transformation and metastatic potential [10, 11 ]. the clinical therapy for hcc includes various modalities such as liver resection, liver transplantation, transarterial chemoembolization (tace), percutaneous ethanol injection therapy (peit), radiofrequency ablation (rfa), and chemotherapy including molecular - targeted therapy. the reason for the high recurrence rate of hcc could be proliferation of epithelial cells and increased arterial vascularity [19, 20 ]. furthermore, hcc cells themselves express various growth factors such as vascular endothelial growth factor (vegf), platelet - derived growth factor (pdgf), epidermal growth factor (egf), fibroblast growth factor (fgf), and insulin - like growth factor (igf), which induce cell proliferation in an autocrine fashion. the receptors of these growth factors activate intracellular signals such as the raf / mek / erk pathway and the pi3k / akt / mtor pathway, which induce proliferation of both cancer and endothelial cells (figure 2). these growth factors, including their intracellular molecules, are considered to be a specific target for hcc treatment. in clinical trials, sorafenib, which is an inhibitor of the vegf receptor (vegfr) and pdgf receptor (pdgfr), has been proven to have a survival benefit for nonresectable hcc compared a placebo in the best supportive care (bsc) setting [27, 28 ]. phase iii trials are ongoing to determine the survival benefit in patients who receive surgery or ablation. on the other hand, the survival benefit of sorafenib has been limited to a few of months and other pathways need to be blocked to achieve longer survival. side effects of sorafinib therapy are obstacles to continuation of the therapy because normal cells also express vegfr and pdgfr, and sorafenib severely damages both normal and cancer cells. the development of anticancer drugs must focus on a specific target that is restricted to the cancer cells. cancer stem cells have been shown to be a target for cancer - specific therapy recently. the abilities of self - renewal and infinite proliferation are closely related to the nature of hcc development [30, 31 ]. stem cells in the liver are divided into several cell types, including oval cells, small hepatocytes, and progenitor cells (figure 1). liver cancer stem cells and hcc cells could derive from mutation of these stem cells. the origin of the stem cells could be either from mature hepatocytes or from bone marrow cells (figure 1). thus, specific stem cell - based therapy could be another strategy to overcome the high recurrence rate of hcc. we describe the molecular pathogenesis, molecular therapy, and stem cell - targeted therapy for hcc treatment in this review. vegf, pdgf, egf, fgf, and igf, growth factors that facilitate high vascularity and cancer cell proliferation, are expressed not only in cancer cells but also in other surrounding cells. the high expression of the growth factors is also associated with tumor invasion and portal thrombosis [19, 20, 22 ]. among the growth factors, high expression of egf is related to differentiation and invasion by the cancer cells. on the other hand, therefore, growth factors play an important role in proliferation of cancer cells not only in an autocrine fashion but also in a paracrine fashion through surrounding cells. in addition based on the high vascularity of hcc, endothelial cells could be a target for hcc treatment. this approach could be promising because endothelial cells have normal cell physiology with stable genetic regulation, which can be easily manipulated by molecular target therapy. tyrosine kinase type receptors, such as vegfr, pdgfr, egfr, fgfr, and igfr, activate intracellular ras in the raf / mek / erk pathway [1923 ]. subsequently, ap-1 family members such as c - jun and c - fos activate expression of various genes that induce cell proliferation and vasculogenesis (figure 2). the activation of the raf / mek / erk pathway is related to the disease progression of hcc and hbv - related hcc development. furthermore, hcv core protein activates raf and is considered to play a role in the development of hcc. ras and raf play important roles in which intracellular signals activate expression of various genes (figure 2). erk regulates more than one hundred intracellular substrates directly and gene expression indirectly as cell kinase to activate transcription factors and cell cycle regulators [44, 45 ]. activation of erk is closely related to cancer cell proliferation and, thus, inhibition of erk could have an anticancer effect. the phosphatidylinositol-3 kinase (pi3k) pathway plays an important role in the proliferation and survival of cancer cells in various solid tumors, including hcc (figure 2). inactivation of akt has been shown to improve the antitumor effect of sorafenib in an animal model and thus it could have potential use for hcc treatment. the pi3k pathway is regulated by phosphatase and tensin homolog deleted on chromosome 10 (pten) negatively and the expression of pten is suppressed in half of hcc cells clinically (figure 2). in fact, pten expression is suppressed by hbx protein in hbv hepatitis patients, and downregulation of pten is associated with tumor grade progression, tumor stage, and poor overall prognosis. sorafenib is an inhibitor of raf that is activated by vegf and pdgf [5052 ]. it has been tested in phase iii clinical trials, such as the sharp trial and asia - pacific trial (table 1). it improves overall survival in patients with advanced hcc compared to patients administered a placebo in the bsc setting. the clinical phase ii trial of sunitinib for hcc treatment showed severe grade 3 to 4 side effects. brivanib, erotinib, and tsu-68, which are inhibitors of growth factor receptors, have been clinically tested for advanced hcc patients as well. the response rates to single doses of sorafenib [27, 28 ], sunitinib [53, 54 ], brivanib, erlotinib, and tsu-68 were 2.33.3%, 2.72.9%, 5.0%, 9.0%, and 8.6% respectively (table 1). phase ii clinical trials using bevacizumab, a vegfr inhibitor, and cetuximab, an egfr inhibitor, had 13% and 0% rrs, respectively (table 1). although the clinical results of single doses of these molecular - targeted agents were not totally satisfactory, bevacizumab and tsu-68 achieved 2 - 3% complete responses [57, 58 ] (table 1). in addition, a phase ii clinical trial of combination therapy using erlotinib and bevacizumab had a 25% response rate (table 1). therefore, combination of these agents with appropriate management of the side effects could improve survival of patients with advanced hcc in the future. ongoing clinical trials using molecular - targeted agents for hcc are shown in table 2. the storm trial is a phase iii clinical trial using a single dose of sorafenib alone for adjuvant therapy after liver resection and ablation. the silius trial is a phase iii clinical trial using combination therapy with transarterial chemoinfusion (taci) and sorafenib for advanced hcc patients. the space trial and tactics trial are a phase ii clinical trial using tace and sorafenib for advanced hcc patients. the brisk - ps trial is designed for second therapy using brivanib for advanced hcc patients resistant to sorafenib. the brisk - ta employs adjuvant therapy using brivanib after tace, and the brisk - fl trial is a comparative clinical trial using sorafenib alone and brivanib alone. the clinical results of these molecular - targeted therapies have not all been published yet and we will need to interpret the results carefully in the future. molecular markers of cancer stem cells are shared with either normal stem cells or progenitor cells. cd133, cd90, cd44, and epcam [63, 64 ] have been shown to be markers for cancer stem cells in hcc patients (table 3). these biomarkers can be useful to estimate the prognosis of hcc and they could be useful for specific targeted therapy for cancer cells. nsc74859 is a specific inhibitor of signal transducer and activator of transcription 3 (stat3) activation and it decreases cd133-positive cells with suppression of cancer development. in addition, cd133 cells increase in pten deleted mice, which indicates that pten can play an important role to regulate cd133-positive cancer stem cells. these basic studies suggest that cd133 can be a molecular target for hcc treatment. the atp - binding cassette (abc) transporters are a family of membrane transporters such as mdr1, abcg2, and abcc2. a combination of chemotherapy and inhibitors of abc transporters could decrease not only the number of hcc cells but also that of cancer stem cells (table 3). cd90 is expressed in oval cells and progenitor cells and its expression is related to tumor development. cd44 is a receptor of hyaluronate expressed on the cell surface and is often coexpressed with cd90 [62, 74 ]. anti - cd44 treatment induces apoptosis in cd90-positive cells and thus cd44 plays an important role in the survival of cancer cells [75, 76 ]. epcam is another cell marker for progenitor cells and the direct target of the wnt / beta catenin pathway [63, 77 ]. the knockdown of the wnt / beta catenin by small interfering rna (sirna) of wnt / beta catenin decreases the number of epcam - positive cells with suppression of tumor development and induces apoptosis. cd13-positive cells are also potential cancer stem cells (table 3). these cells can be found in the peripheral areas of hcc after tace treatment, which is considered to be related to tumor recurrence. furthermore, inhibitors of cd13 such as 24f can suppress the invasion and angiogenesis of hcc. the molecular pathogenesis of hcc is important to understand the mechanism of tumor development as well as the high - recurrence behavior of hcc. furthermore, each step of the molecular signals could be a target to control tumor progression. further clinical studies using single agents and combination therapies need to be conducted for hcc treatment. the clinical benefits of cell - targeted therapy for cancer stem cells are eagerly awaited. | the prognosis of hepatocellular carcinoma (hcc) is affected by tumoral factors and liver functions ; therefore it is often difficult to select the appropriate therapeutic methods for hcc. recently, two global phase iii trials showed that sorafenib, which is a tyrosine kinase inhibitor, improved the prognosis of patients with advanced hcc. as a new therapeutic strategy for hcc, sorafenib is expected to expand the indication for hcc in the future. however, it alone is insufficient for the molecular - targeted treatment of hcc because the signaling pathway exists not only in cancer cells but also in normal cells. recently, cancer stem cells (cscs) have attracted attention as a novel therapeutic target for hcc. there is now much evidence that stem cell properties such as self - renewal, unlimited proliferation, and differentiation are highly relevant to cancer recurrence and the drug resistance of hcc. in this review, we describe the molecular pathogenesis and the current state and future development of molecular- and csc - therapeutic targeted agents for hcc, citing various reports. |
angiogenesis, the formation of new blood vessels, plays an important role in tumor growth, local invasiveness, and metastatic progression. many preclinical studies and clinical trials confirm the importance of integrin v3 in the process of tumor angiogenesis and metastasis [1, 2 ]. integrin v3 is a cell adhesion molecule and is highly expressed on activated endothelial cells and tumor cells but is not expressed on resting endothelial cells and therefore specific for neoangiogenesis. several extracellular matrix (ecm) proteins like vitronectin, fibrinogen, and fibronectin interact with integrin v3 via the amino acid sequence arg - gly - asp (rgd) [4, 5 ]. vascular endothelial growth factor (vegf - a) also plays an important role in both normal vascular tissue development and tumor neovascularization and is highly expressed in various human tumors [6, 7 ]. in the last few years metal pet isotopes such as ga and both ga and cu are excellent alternatives to f. ga can be obtained from an in - house commercially available ge / ga generator. the advantage of using cyclotron produced cu for pet imaging and radiotherapy is the longer half - life allowing imaging at late time points acquiring additional information. cu can now be produced in high yield and at high specific activity on a small biomedical cyclotron. radiolabeled rgd peptides for positron emission tomography (pet) imaging, targeting integrin v3 in tumors, have been described in several studies with mainly f (t1/2 = 110 min ; emax, + = 634 (97%) kev), ga (t1/2 = 68 min ; emax, + = 1.90 (89%) kev), and cu (t1/2 = 12.7 h ; emax, + = 653 (18%) kev) [917 ]. for ga and cu different chelating systems have been used. some of the latest described tracers have used the chelator 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (nodaga) combined with c(rgd) labeled with ga [9, 12, 15, 18 ] or cu [12, 18 ]. net is one of the tumor types where antiangiogenesis treatment of patients seems promising but patients need to be selected individually. to select the patients, pet scans with a tracer targeting tumor - angiogenesis may be extremely relevant. therefore using a human net xenograft model for preclinical studies to evaluate new pet tracers targeting tumor - angiogenesis the aim of the present study was therefore, as the first, to use and evaluate the pet tracer cu - nodaga - c(rgdyk) using a human net xenograft model. biodistribution, pet / ct scans, correlation between gene expression of angiogenesis markers and tracer uptake, and radiation dosimetry extrapolation for humans were performed. since investigation of tumor - angiogenesis using human xenografts in mice is challenged by the fact that the formation of new vessels in the tumors might be of either human or mice (host) origin, we designed qpcr assays to quantify both mice and human genes of integrin v, integrin 3, and vegf - a. nodaga - c(rgdyk) was purchased from abx gmbh (advanced biochemical compounds, radeberg, germany). cucl2 was produced at risoe technical university of denmark, roskilde, denmark. for high - performance liquid chromatography (hplc) analysis a dionex p580 pump with a pda-100 detector and an in - line scansys radioactivity detector was used. nodaga - c(rgdyk) (2 nmol) dissolved in 450 l ammonium acetate (0.1 m, ph 8.47) was mixed with 50 l of cucl2 (5060 mbq) for 15 min at room temperature. hplc analysis was performed with a jupiter column, 4u proteo 90a, 250 4.6 mm (phenomenex, torrance, ca, usa) and flow 1.5 ml / min. the hplc mobile phase was solvent a, 0.1% trifluoroacetic acid in water, and solvent b, 0.1% trifluoroacetic acid in acetonitrile. gradient : 02 min 15% b, 27 min 1540% b, and 710 min 4015% b. the retention time of cu - nodaga - c(rgdyk) was 5.9 min. the stability of cu - nodaga - c(rgdyk) was investigated in either buffer or plasma from nude mice. for buffer stability, 160 mbq cu - nodaga - c(rgdyk) was incubated for 1, 2, 18, and 24 hours at room temperature in 450 l ammonium acetate buffer. for the stability in plasma, 10 mbq cu - nodaga - c(rgdyk) was incubated for 1, 2, 18, and 24 hours in 500 l plasma. the stability of cu - nodaga - c(rgdyk) in plasma and buffer was analyzed and determined by the previously described hplc conditions. human lung bronchus carcinoid, nci - h727 obtained from atcc (american type culture collection, manassas, va, usa), was used. before taken into experiments, the cell line was tested free of mycoplasma at statens serum institute, copenhagen, denmark. cells were cultured in rpmi (roswell park memorial institute) medium 1640 with glutamax (gibco, life technologies, ny, usa) containing 10% fetal calf serum (biological industries (bi), kibbutz beit - haemek, israel), and 1% penicillin - streptomycin (gibco, life technologies) in 5% co2 at 37c. female nmri (naval medical research institute) nude mice (6 weeks upon arrival) were acquired from taconic europe, lille skensved, denmark. the mice were acclimated one week in the animal facilities before taken into experiments. at the age of 8 weeks (weight : 32.8 4.2 g) the mice had h727 cells (57 10 cells in 100 l medium mixed with 100 l matrigel basement membrane matrix (bd sciences, san jos, ca, usa) inoculated subcutaneously on the left and right flank during anesthesia with 1 : 1 v / v hypnorm (janssen pharmaceutica nv, beerse, belgium)/dormicum (roche, basel, switzerland). tumors were then grown for 3 - 4 weeks (average tumor volume 210 mm). animal care and experimental procedures were performed under the approval of the danish animal welfare council (2006/561 - 1124). tumor bearing mice were anesthetized with 3% sevoflurane (abbott scandinavia ab, solna, sweden) mixed with 35% o2 in n2. mice were tail - vein injected with 2.0 0.7 mbq cu - nodaga - c(rgdyk). biodistribution of cu - nodaga - c(rgdyk) was obtained at 1, 2, and 18 hours after injection (n = 5 mice in each group). mice were sacrificed by neck drawing and tumors, organs (liver, kidneys, lung, spleen, heart, intestine, and muscle) and blood were collected, weighted, and radioactivity measured in a gamma counter (2480 wizard, perkin elmer, ma, usa). tx, usa), stored overnight at 4c, rnalater then removed, and tumors stored at 80c until total rna extraction and further qpcr experiments were performed. for estimation of the human radiation dose using cu - nodaga - c(rgdyk) the mouse biodistribution data were used. assuming similar pharmacokinetics in mice and humans, the activity in organs at 1, 2, and 18 hours post injection (p.i.) was used to calculate single exponential effective half - lives and residence times for each organ. these data were then used for extrapolation to human dosimetry estimates using standard female (58 kg) and male (70 kg) human phantoms and the organ level internal dose assessment / exponential modeling software (olinda / exm ; vanderbilt university, nashville, tn, usa). to ensure a conservative estimate, the effective dose for both adult male and female were then calculated using olinda / exm. a longitudinal dynamic study was performed scanning a mouse at 1, 2, and 18 hours after intravenous injection of 1.6 mbq cu - nodaga - c(rgdyk), allowing dynamic information of the tracer biodistribution in tumors as well as in the other organs of interest. the pet scans were performed using a small animal pet scanner (micropet focus 120, siemens medical solutions, knoxville, tn, usa). the energy window for the emission pet scans was set to 350650 kev and the time resolution was 6 ns. the acquired data for emission scan was stored in list - mode format and postprocessed to obtain 2 bytes 128 144 32 sinograms. finally, the emission sinograms were reconstructed using map algorithms and resulted into 4-byte 256 256 95 image sets with a zoom factor of 1.443 and a voxel size of 0.87 0.87 0.79 mm. the small animal ct scans of the mouse parallel with the pet scans were acquired using a small animal computed tomography (microcat ii, siemens medical solutions). the acquisition time of each ct scan was 6.5 minutes generating 360 projections at 360 arc. the x - ray source settings were 75 kvp, 500 a, and 270 ms for the voltage, the current, and the exposure time, respectively. the ct projections on the 3,000 2,970 ccd crystal were binned by 4 to increase sensitivity and reduce the dataset size. the projections were reconstructed by real - time reconstruction algorithm (the cobra toolbox) using shepp - logan filter into 768 768 512 matrix with a voxel size of 0.092 0.092 0.092 mm. the fusion of the pet and ct images was performed manually using the asipro toolbox (siemens medical solutions). also the measurement of the activity uptake in the tumors and the organs were estimated by manually outlining on ct images. the outlining of the organs on ct images in every slice resulted in 3d region of interest (roi). the results of these rois acquired the concentration of the tracer in the organs expressed in percentage of the injected dose per gram tissue (% id / g). the whole tumors were lysed and homogenized in precellys-24 (bertin technologies, montigny, france) in tubes containing ceramic beads. total rna was isolated using nucleospin rna l kit (macherey - nagel gmbh & co. kg, dren, germany). rna concentration was determined by nanodrop 1000 (thermo fisher scientific, de, usa). rna quality expressed as rna integrity number (rin) was measured on a 2100 bioanalyzer (agilent technologies, ca, usa). 0.3 g total rna was reverse transcribed using affinityscript qpcr cdna synthesis kit (stratagene, ca, usa). human itgav, human itgb3 and human vegfa were tested in a triplex-, mice itgav and mice itgb3 in a duplex, and mice vegfa in a simplex - qpcr assay. the three human housekeeping genes hprt, ubc, and rplp were also measured in a triplex. the brilliant qpcr core reagent kit (stratagene ; cat # 600530) was used for all the multiplex assays. assay optimization resulted in a 50% and 100% increase of taq polymerase and dntp in duplex and triplex assays, respectively. no - rt (no reverse transcription) for all the samples was tested using the housekeeping gene - triplex. the thermal profile for qpcr core reagent was : denaturation at 95c for 10 minutes, followed by 40 cycles with denaturation at 95c for 30 seconds and annealing / elongation at 60c for 1 minute. the thermal profile for brilliant iii reagent was : denaturation at 95c for 3 minutes, followed by 40 cycles with denaturation at 95c for 20 seconds and annealing / elongation at 60c for 20 seconds. the samples were run in triplicates using 1 l of cdna in a total volume of 25 and 20 l, respectively. the optimal housekeeping genes for the h727 tumors were found by testing 12 human reference genes from tataa biocenter (goteborg, se) in 12 different h727 tumors from nmri nude mice. with genorm software the genes and the number of genes giving the most stable endogenous normalization the six genes of interest m - itgav (nm_008402), m - itb3 (nm_016780), m - vegfa (nm_001025250), h - itgav (nm_002210), h - itgb3 (nm_000212), and h - vegfa (nm_001025366), along with the housekeeping genes h - hprt (nm_000194), h - ubc (nm_021009) and h - rplp (nm_001002) were designed using beacon designer (premier biosoft, ca, usa). before designing, all genes were tested for cross homology against the human or the mouse genome and checked for secondary structures. for the optimized conditions of all primers and probes all primers and probes were purchased from sigma - aldrich (st. louis, mo, usa). qbaseplus software based on the comparative method (2) for relative quantification was used including normalization to three housekeeping genes. furthermore an inter - run calibrator for correction between pcr runs within the same gene and an arbitrary overall calibrator for comparison of the gene levels in between mice and human genes were taking into account. as shown in earlier studies the pcr data were found not to be normal distributed when tested by a one - sample kolmogorov - smirnov test. all qpcr data were therefore log10 transformed to obtain normal distribution and the log - transformed data were used for all the subsequent statistical analyses. t - test (two tailed) was used for comparison of mrna levels of each gene human versus mice at the three different sample collecting times. all statistical analyses were performed using spss 17.0 statistical software (spss inc., chicago, illinois, usa). nodaga - c(rgdyk) was labelled with cu in 15 minutes at room temperature with a radiochemical purity of 92.596.6% and a specific activity of 25.125.7 mbq / nmol. the purity of cu - nodaga - c(rgdyk) in buffer was more than 93% at 1, 2, 18, and 24 hours after incubation. the purity was also more than 93% after 24 hours when 4.5 ml of saline was added to 500 l of cu - nodaga - c(rgdyk) solution. in plasma, the purity of cu - nodaga - c(rgdyk) was 88% after 24 hours of incubation. rin (rna integrity numbers) were measured to be between 6.8 and 9.9, verifying fine rna quality with limited degradation. no - rt for each sample was checked in qpcr, finding no ct values, indicating that genomic dna is not present and therefore not disturbing the gene expression values. in h727 tumors (removed 2 hours after tracer injection) integrin v of both human and mice origin was expressed at the same level, whereas gene expression of h - vegf - a was significantly higher than m - vegf - a (p < 0.001). integrin 3 of both human and mice origin was expressed on a very low level (figure 2). regression analyses for cu - nodaga - c(rgdyk) tracer uptake, % id / g (calculated from the biodistribution data) versus quantitative mrna in the h727 tumors showed significant correlation at 2 h p.i. with mice integrin v (r = 0.76, p < 0.05), mice integrin 3 (r = 0.75, p < 0.05), and mice vegf - a (r = 0.81, p < 0.05), also for human integrin v at 2 h p.i. liver, kidneys, lung, spleen, heart, intestine, muscle, and blood were collected from 10 mice at the following time points : 1, 2, and 18 hours post injection. % id / g was calculated from the measured counts by taking counting efficiency of the -counter (f = 0.0943), half - life time for cu, decay from injection to counting time, and tissue weight into account. we found the highest % id / g (mean sem) at 1 h p.i. for the kidneys, intestines, liver and spleen (2.2 0.11, 1.60 0.16, 1.05 0.17, and 0.94 0.11) decreasing already at 2 h p.i. (0.97 0.03, 0.64 0.04, 0.65 0.04, and 0.62 0.04) and decreasing further at 18 h p.i. (0.51 0.02, 0.43 0.04, 0.43 0.02, and 0.33 0.02). tumor - to - blood ratio was 12.7, 37.2, and 20.2, respectively at the 3 collecting times. residence times based on biodistribution data are shown in table 3 and were used for calculating estimates of human radiation - absorbed doses using olinda / exm. table 4 shows the olinda / exm estimates for human adult females and males for each organ and the total effective dose. the highest radiation - absorbed doses were found to be the urinary bladder wall (0.014 and 0.010 mgy / mbq for females and males, resp.). however, this estimate is a worst - case scenario since we used a conservative model with a urinary elimination fraction of 75% with voiding intervals of 5 hours. the whole body effective dose was 0.038 and 0.029 msv / mbq for females and males, respectively. accordingly, an administered dose of 200 mbq cu - nodaga - c(rgdyk) to humans would lead to a radiation burden of less than 8 msv. the tumors were clearly visible after injection of cu - nodaga - c(rgdyk) at all measured time points : 1, 2 and 18 h pi (figure 5). cu - nodaga - c(rgdyk) showed relatively high uptake in h727 tumors ; mean % id / g of right and left tumors were 1.15, 0.99, and 0.76 respectively, at 1, 2, and 18 h pi. tumor - to - muscle (t / m) ratios were 6.9 0.8. both % id / g and t / m ratios were similar to the values found in the biodistribution study using the -counter. tracer activity in organs did not disturb the tumor to background contrast from 2 h pi where most of the nonspecific uptake had been cleared. to the best of our knowledge this is the first study of the pet tracer cu - nodaga - c(rgdyk). the major finding was a close correlation between tumor uptake of cu - nodaga - c(rgdyk) and the target integrin v3 validating its ability to image neo - angiogenesis. the cyclic rgd peptides c(rgdfk) and c(rdgyk) have frequently been used for radiolabeling. c(rgdfk), cyclo [arg - gly - asp - d - phe - lys ] only differs from c(rgdyk), cyclo [arg - gly - asp - d - tyr - lys ], in one amino acid, d - phenylalanine (d - phe) instead of d - tyrosine (d - tyr). d - tyr is more hydrophilic than d - phe and c(rgdyk) has therefore been reported as preferable due to faster renal excretion. in our study we found a faster decrease of radioactivity in kidneys, intestine, and liver from 1 to 18 h (2.2 to 0.5, 1.6 to 0.4, and 1.1 to 0.4%id / g) than that reported for cu - nodaga - c(rgdfk) in the same time intervals (2.2 to 1.6, 2.1 to 1.3, and 1.7 to 1.0%id / g). c(rgdyk), the peptide used in the present investigation, has previously been chelated to 1,4,7,10-tetraazacyclododecane - n, n,n,n-tetraacetic acid (dota) or 1,4,7-triazacyclononane-1,4,7-triacetic acid (nota) and labeled with cu [1113, 25 ] or ga. cu - dota - c(rgdyk) was first described in 2004 by chen and coworkers. the cu - dota complex seemed more unstable than cu - nota or cu - nodaga, because cu was transferred to copper - binding proteins, leading to relatively high blood and liver uptake. nodaga is, compared to the chelators dota and nota [8, 11, 2729 ], particularly useful due to high hydrophilicity leading to rapid renal excretion avoiding radioactivity accumulation in the gastrointestinal tract and in the kidneys. nodaga - c(rgdy / fk) has been labeled with ga [9, 12, 15, 18 ] and nodaga - c(rgdfk) also with cu. one paper compared ga- and cu - nodaga - c(rgdfk) and found the highest tracer uptake using ga - nodaga - c(rgdfk) compared to cu - nodaga - c(rgdfk) (5.2 versus 3.8%id / g, 1 h p.i. in highly angiogenic u87 mg xenograft tumors) but also 3 times higher tumor - to - blood ratio. we found similar t / b ratio (12.7) after 1 hour using cu - nodaga - c(rgdyk) as they did with cu - nodaga - c(rgdfk). the advantage of cu labeling is the possibility of delayed imaging up to 12 or 18 h post injection with expected improved image contrast, and therefore improved visualization of low expressing integrin v3 tumors. indeed, we found that delayed images after 2 and 18 hours showed higher tumor - to - background ratios compared to 1 hour images. in addition cu has a much better image quality than ga due to the shorter positron range. a comparison to f - labeled rgd shows that tumor - to - liver and tumor - to - kidney ratios are similar. for the neuroendocrine h727 xenograft tumors we found moderate levels of tracer uptake (1.2%id / g 1 h p.i. as the highest) explained by the fact that neo angiogenic activity in h727 tumors, reflecting low aggressiveness in general of net tumors, is known to be lower than, for example, in glioblastoma u87 mg xenograft tumors showing high expression of integrins. we investigated the target specificity of the pet tracer by analyzing mrna levels of both integrin v and integrin 3 using quantitative qpcr. our results strongly support that cu - nodaga - c(rgdyk) has high affinity to integrin v3 reflecting neo angiogenesis. a challenge in preclinical studies using human xenografts in mice is, that the formation of new vessels in the tumors might be of both human and mouse origin. the interaction between mouse host stroma and human tumor cells and the role they play in the tumor - angiogenesis and tumor growth are of great importance in antiangiogenesis treatment. therefore in preclinical studies using antiangiogenic drugs for treatment, it is of great importance to take into consideration species specificity. accordingly, the antibody bevacizumab only binds to human vegf - a and not to murine vegf - a, whereas the peptide cilengitide is blocking integrin v3 of both human and mouse origin. in the present study we therefore quantified both the gene expression level of mice and human integrin v, integrin 3, and vegf - a in our human h727 xenograft model. we found that integrin v and integrin 3, of both mouse and human origin, are present at comparable levels. this seems to be true throughout tumor development as we previously (unpublished data) found dual origin from 13 weeks of tumor growth with a confirmed higher level of vegf - a of human origin. species specificity of our pcr designs were verified targeting human or mice gene sequences during blast tests, and as a further control qpcr primers targeting mice genes were tested in cdna from a net patient finding no gene expression. the finding of correlation between tracer uptake of cu - nodaga - c(rgdyk) and mrna levels of mouse integrin v, integrin 3, and vegf - a two hours post injection indicates that cu - nodaga - c(rgdyk) is indeed targeting the tumor - angiogenesis and therefore is a promising radiotracer for pet imaging and quantification of angiogenesis. taken together cu - nodaga - c(rgdyk) seems promising for future use in humans. we therefore performed experiments to estimate dosimetry in humans based on projections from animal data. here we found a favorable estimate of less than 8 msv for a realistic clinical dose of 200 mbq. compared to recent dosimetry estimates for ga - nodaga - c(rgdyk) on a per mbq basis the radiation burden is comparable especially taking into consideration the more conservative urinary elimination model used in our calculations. in both studies we, as the first, have described the use of cu - nodaga - c(rgdyk) as a pet tracer targeting angiogenesis. we found an excellent tumor - to - background ratio due to rapid elimination from nontumor tissue. also an excellent image quality was obtained using cu. in addition, our compound seems stable as indicated by the low liver uptake. using gene expression analyses we found a close correlation with key angiogenesis markers validating the target of the tracer. we therefore suggest that cu - nodaga - c(rgdyk) should be further studied as a promising candidate for human angiogenesis pet. | purpose. the purpose of this paper is to evaluate a new pet tracer 64cu - nodaga - c(rgdyk) for imaging of tumor angiogenesis using gene expression of angiogenesis markers as reference and to estimate radiation dosimetry for humans. procedures. nude mice with human neuroendocrine tumor xenografts (h727) were administered 64cu - nodaga - c(rgdyk) i.v. for study of biodistribution as well as for dynamic pet. gene expression of angiogenesis markers integrin v, integrin 3, and vegf - a were analyzed using qpcr and correlated to the tracer uptake in the tumors (% id / g). from biodistribution data human radiation - absorbed doses were estimated using olinda / exm. results. tumor uptake was 1.2%id / g with strong correlations between gene expression and tracer uptake, for integrin v r = 0.76, integrin 3 r = 0.75 and vegf - a r = 0.81 (all p < 0.05). the whole body effective dose for humans was estimated to be 0.038 and 0.029 msv / mbq for females and males, respectively, with highest absorbed dose in bladder wall. conclusion. 64cu - nodaga - c(rgdyk) is a promising new angiogenesis pet tracer with potential for human use. |
beginning in 1989, oral vaccination using bait that contained the live, attenuated, evelyn - rokitnicki - abelseth (era) (6,7) strain of rabies virus was distributed in ontario to control rabies in red foxes in rural and urban habitats (4,810). the bait matrix consisted of beef tallow, wax, and attractants such as chicken or cod (6). the vaccine was contained in a blister pack, which was embedded in the matrix of the bait (figure 2). evelyn - rokitnicki - abelseth rabies vaccine bait, showing the vaccine container (normally embedded in the matrix of the bait) to the right of the bait. the metropolitan toronto area (centered at latitude 43 42 north, longitude 79 25 west) was defined as a 638-km urban complex that included the cities of toronto, north york, etobicoke, east york, york, and north york. the program in metropolitan toronto was expanded during 19941999 (figure 3) to include the urban corridor from oshawa to hamilton (greater metropolitan toronto, 1,850 km) (figure 3). during 19981999, baiting in rural southwestern ontario extended into the greater metropolitan toronto area (figure 3). during the 1990s, 1,000 foxes (about 1.5/km) lived in close proximity to 3 million people in metropolitan toronto (10,11). in addition, during 19871996, trap - vaccinate - release programs (vaccination by hand - delivered injection) to control rabies in striped skunks and raccoons (procyon lotor) were conducted in a 60-km portion of metropolitan toronto (scarborough) (9). from a public health perspective, rabies control was crucial because since the 1950s, > 63,000 humans had been treated for exposure to potentially rabid animals in ontario (10,12). greater metropolitan toronto area where rabies vaccine bait doses were distributed during 19891999. during 19891999, a total of 332,257 bait doses containing era rabies vaccine were distributed in toronto and the greater metropolitan toronto area (table). bait was distributed primarily by personnel who walked (ground baiting) throughout the ravines and green - belt areas of greater metropolitan toronto ; ecologic studies of red foxes indicated that ravines are used as travel corridors by foxes (11). in 1994, 5,500 bait doses were also distributed aerially along the ravine systems from a turbo beaver aircraft flying at an altitude of 150 m with an approximate airspeed of 140 km / hr. in addition, during 1998, about 16,000 doses were aerially distributed in the greater metropolitan toronto area by twin - otter aircraft. timing of bait placement varied each year but was generally in june and november, from 1989 through 1999. hand - baiting personnel tried to place 1 vaccine - bait dose every 50 m on both sides of waterways in the ravine systems (9). as a modified live virus rabies vaccine was being used in an urban setting, news releases were issued to the media before and during annual baiting operations. the primary objective of the media campaign was to notify the public of the program and ask people not to touch the bait. we documented that 15 persons found bait (but did not touch the vaccine) and 22 dogs had contact with or consumed the bait during hand - baiting operations. clinical signs in dogs after bait ingestion sometimes included diarrhea or vomiting (most likely attributable to the tallow and wax in the bait). three of the dogs had intestinal problems, and 1 had an intestinal obstruction, likely caused by the blister pack. after the 1994 aerial baiting campaign, only 5 persons reported finding bait in their yards. the time needed to hand - distribute 28,000 vaccine - bait doses each year in metropolitan toronto was 145 person - days, which is 193 bait doses / person / day. the annual cost to hand - distribute these 28,000 bait doses was about can $ 25,000 for labor, travel expenses, vehicles, and gas plus $30,000 for the bait (total cost of $1.96/dose). acceptance of vaccine - bait was determined by the presence of tetracycline in tooth sections (6). bait acceptance by foxes sampled in metropolitan toronto during 19891991 was 55%80%, and rabies antibody was detected in 74%100% of the foxes that consumed the bait (9). during this period, 50%68% of the foxes were vaccinated each year (9,10). significantly fewer rabid foxes were reported in metropolitan toronto during the 17 years after fox baiting began (19902006, 5 cases, mean 0.3/yr, standard deviation [sd ] 0.6) than during the 17 years before baiting began (19731989, 96 cases, mean 5.7/yr, sd 7.3) (t=3.01, p<0.005) (figure 1). on the basis of the cyclic nature of outbreaks in metropolitan toronto of rabies in foxes (every 25 years), as well as in skunks, an outbreak should have occurred during the mid-1990s ; but no outbreak occurred. as of september 2006, the last rabid fox in metropolitan toronto, as well as the greater metropolitan toronto area, had been reported in 1996. distribution of vaccine - bait in that urban complex was discontinued in 2000 because metropolitan toronto had been free from reported rabies in foxes for 3 years. metropolitan toronto is connected to rural areas through a series of ravine systems dominated primarily by deciduous trees. these ravines provide a travel corridor through which wildlife, including red foxes, moves into and out of metropolitan toronto (11). the ground and aerial distribution of rabies vaccine bait in metropolitan and greater metropolitan toronto, which resulted in immunization of a substantial portion of the fox population against rabies, eliminated rabies from that urban complex. aerial baiting in rural habitats surrounding metropolitan toronto, as well as greater metropolitan toronto, after 1995 may have contributed to rabies control in metropolitan toronto, as few rabid foxes have been available to disperse rabies into that urban complex. as well, one can not discount the effect that the trap - vaccinate - release programs in scarborough had on the control of rabies in metropolitan toronto. however, the trap - vaccinate - release program targeted raccoons and skunks as opposed to foxes (9). greater metropolitan toronto has been free of reported cases of rabies in red foxes for a decade (19972006) and is a notable success for the ontario ministry of natural resources rabies control programs. the results of this program confirm that distribution of oral rabies vaccine bait is a feasible tactic for controlling rabies in foxes in urban environments. | to control the arctic variant of rabies virus in red foxes, 332,257 bait doses containing live, attenuated evelyn - rokitnicki - abelseth rabies vaccine were distributed in greater metropolitan toronto during 19891999. human and pet contact with bait was minimal, and no adverse reactions to the vaccine were noted. significantly fewer rabid foxes were found during the 17 years after fox baiting (5 cases during 19902006) than in the 17 years before (96 cases during 19731989). the last report of a rabid fox in metropolitan toronto was in 1996 (reporting period through september 2006), which confirms that distributing oral rabies vaccine bait is a feasible tactic for the control of rabies in foxes in urban environments. |
detailed methodology can be found in the supplementary information. in short, luciferase reporter gene assay, immunoprecipitation and immunoblotting analysis, purification of recombinant proteins, in vitro kinase assays, and cell migration assay were performed, as previously described. | summarythe embryonic pyruvate kinase m2 (pkm2) isoform is highly expressed in human cancer. in contrast to the established role of pkm2 in aerobic glycolysis or the warburg effect1,2,3, its nonmetabolic functions remain elusive. here we demonstrate that egfr activation induces translocation of pkm2, but not pkm1, into the nucleus, where k433 of pkm2 binds to c - src - phosphorylated y333 of -catenin. this interaction is required for both proteins to be recruited to the ccnd1 promoter, leading to hdac3 removal from the promoter, histone h3 acetylation, and cyclin d1 expression. pkm2-dependent -catenin transactivation is instrumental in egfr - promoted tumor cell proliferation and brain tumor development. in addition, positive correlations have been identified among c - src activity, -catenin y333 phosphorylation, and pkm2 nuclear accumulation in human glioblastoma specimens. furthermore, levels of -catenin phosphorylation and nuclear pkm2 have been correlated with grades of glioma malignancy and prognosis. these findings reveal that egf induces -catenin transactivation via a mechanism distinct from that induced by wnt / wingless4 and highlight the essential nonmetabolic functions of pkm2 in egfr - promoted -catenin transactivation, cell proliferation, and tumorigenesis. |
acute pancreatitis is an inflammatory condition of the pancreas characterized clinically by abdominal pain and elevated levels of pancreatic enzymes : amylase and lipase in the blood. the incidence of acute pancreatitis in the united states is approximately 17 cases per 100,000 people. although gallstones and alcohol cause > 90% of all cases in adults, medications have been recognized as a potential cause of acute pancreatitis. since the first reported case of acute pancreatitis in the 1950s, hundreds of commonly prescribed medications have been reported to induce acute pancreatitis. even medications are considered a common cause of acute pancreatitis, but the numbers of reported drug - induced acute pancreatitis cases account for only 0.1% to 2% of all cases. benzodiazepines (bzds) are used as sedatives and to treat anxiety, seizure, withdrawal disorder, sleeping disturbance, and agitation. because of their versatility, bzds are widely prescribed, and there are nearly 50 kinds of bzds available worldwide. however, the high incidence of bzd poisoning reflects their universal use and availability. bzd poisoning refers to ingesting the bzd class in quantities greater than those recommended or generally used. the most common symptoms of bzd poisoning include central nervous system depression, impaired balance, and slurred speech. the us food and drug administration has reported that since 2001 to 2012, 81 people (0.33 %) had acute pancreatitis among 24,300 people taking zolpidem with side effects. in addition, lai found these patients actively using zolpidem were at 7-fold increased odds of acute pancreatitis in taiwan. zolpidem is a short - acting non - bzd hypnotic with fewer side effects than bzd, because zolpidem potentiates -aminobutyric acid (gaba) by binding only to subunit. in contrast to zolpidem, bzd has a higher affinity for other subunits of gaba. therefore, we believed bzds overdose could also increase risk of acute pancreatitis. in this study, we used data from the taiwan national health insurance research database (nhird) to investigate whether bzd poisoning increases the risk of acute pancreatitis. the taiwan national health insurance (nhi) program integrated 13 insurance programs into a nationwide, single - payer health insurance program implemented in 1995. in 1998, the coverage rate reached 99% of the 23 million taiwan residents (http://www.nhi.gov.tw/english/index.aspx). the taiwan government appointed the national health research institute to establish and manage the nhird, which contains all historical reimbursement claims data, including a registry for beneficiaries, disease records, and medical services ; the database is updated each year. before releasing the data for research purposes, the national health research institute encrypts all personal identification information and provides an anonymous identification number to protect patient privacy. this study was approved to fulfill the condition for exemption by the institutional review board (irb) of china medical university (cmuh-104-rec2115). disease diagnoses in the nhird are based on the criteria of the international classification of diseases, ninth revision, clinical modification (icd-9-cm). cases of bzd poisoning before 2000 were excluded. the bzd poisoning cohort comprised patients who used bzd - based tranquilizers (icd-9-cm 969.4) during 2000 to 2011 and were aged > 20 years. each bzd poisoning cohort patient was frequency matched to 4 comparison controls according to age and sex (per 5 years) ; the controls had no bzd poisoning. the index date for the comparison controls was set by randomly appointing a month and day with the same index year as that of the matched cases. we excluded the individual with the history of the pancreas cancer (icd-9-cm 157), chronic pancreatitis (icd-9-cm 577.1), and acute pancreatitis (icd-9-cm 577.0) before the index date in bzd poisoning cohort and comparison cohort. follow - up was terminated after 3 months or upon withdrawal from the nhi program, acute pancreatitis occurrence, or december 31, 2011. in addition to demographic factors, we considered the effect of comorbidities on the risk of acute pancreatitis. a history of comorbidities before the index date was collected and included chronic obstructive pulmonary disease (copd, icd-9-cm 491493 and 496), alcohol - related disease (ard, icd-9-cm 291, 303, 305.0, 790.3, and v11.3), cardiovascular disease (cvd, icd-9 410414, 428, 430438, and 440448), gallstone (icd-9-cm 574), chronic kidney disease (icd-9-cm 585586 and 588.8588.9), diabetes mellitus (dm, icd-9-cm 250), hepatitis c virus infection (icd-9-cm v02.62, 070.41, 070.44, 070.51, 070.54), hepatitis b virus infection (hbv, icd-9-cm v02.61, 070.20, 070.22, 070.30 and 070.32), and hypertriglyceridemia (icd-9-cm 272.1). to determine the structure of the study population, we calculated the means and standard deviations for number, percentage, age, age group, sex, and comorbidities. the t test and test was used to determine the distribution difference for continuous variables and category variables, respectively. the incidence density of acute pancreatitis for each group was calculated as number of acute pancreatitis incidences divided by the sum of follow - up time (per 1000 person - months). the cumulative incidence curves were measured using the kaplan meier method, and the difference in the curves was assessed using the log - rank test. the risk of acute pancreatitis between the bzd poisoning and comparison cohorts was presented as hazard ratios (hrs) and 95% confidence intervals (cis) by using single variable and multivariate cox proportional hazards models. in addition, we estimated the acute pancreatitis risk in the bzd poisoning cohort according to different demographic characteristics and comorbidities by conducting a stratified analysis involving the cox model. the data management and statistical analysis was performed using sas 9.4 software (sas institute, cary, nc). the significance level was set at 20 years. each bzd poisoning cohort patient was frequency matched to 4 comparison controls according to age and sex (per 5 years) ; the controls had no bzd poisoning. the index date for the comparison controls was set by randomly appointing a month and day with the same index year as that of the matched cases. we excluded the individual with the history of the pancreas cancer (icd-9-cm 157), chronic pancreatitis (icd-9-cm 577.1), and acute pancreatitis (icd-9-cm 577.0) before the index date in bzd poisoning cohort and comparison cohort. follow - up was terminated after 3 months or upon withdrawal from the nhi program, acute pancreatitis occurrence, or december 31, 2011. in addition to demographic factors, we considered the effect of comorbidities on the risk of acute pancreatitis. a history of comorbidities before the index date was collected and included chronic obstructive pulmonary disease (copd, icd-9-cm 491493 and 496), alcohol - related disease (ard, icd-9-cm 291, 303, 305.0, 790.3, and v11.3), cardiovascular disease (cvd, icd-9 410414, 428, 430438, and 440448), gallstone (icd-9-cm 574), chronic kidney disease (icd-9-cm 585586 and 588.8588.9), diabetes mellitus (dm, icd-9-cm 250), hepatitis c virus infection (icd-9-cm v02.62, 070.41, 070.44, 070.51, 070.54), hepatitis b virus infection (hbv, icd-9-cm v02.61, 070.20, 070.22, 070.30 and 070.32), and hypertriglyceridemia (icd-9-cm 272.1). to determine the structure of the study population, we calculated the means and standard deviations for number, percentage, age, age group, sex, and comorbidities. the t test and test was used to determine the distribution difference for continuous variables and category variables, respectively. the incidence density of acute pancreatitis for each group was calculated as number of acute pancreatitis incidences divided by the sum of follow - up time (per 1000 person - months). the cumulative incidence curves were measured using the kaplan meier method, and the difference in the curves was assessed using the log - rank test. the risk of acute pancreatitis between the bzd poisoning and comparison cohorts was presented as hazard ratios (hrs) and 95% confidence intervals (cis) by using single variable and multivariate cox proportional hazards models. in addition, we estimated the acute pancreatitis risk in the bzd poisoning cohort according to different demographic characteristics and comorbidities by conducting a stratified analysis involving the cox model. the data management and statistical analysis was performed using sas 9.4 software (sas institute, cary, nc). the significance level was set at 1 month (hr = 1.07, p >.05). incidence of acute pancreatitis by age, sex, comorbidity, follow - up time, and cox model measured hazards ratio for patients with bzd poisoning compared to those without bzd poisoning this population - based retrospective cohort study reveals a significant association between bzd poisoning and an increased risk of acute pancreatitis, namely bzd poisoning significantly affects the acute pancreatitis risk. after adjustment for potential confounding factors, patients with bzd poisoning had a 5.33-fold greater acute pancreatitis risk (95% ci = 2.2612.60) compared with patients without bzd poisoning (table 2). the alcohol abuse causes approximately 30% of all acute pancreatitis cases in the united states. alcohol may promote the synthesis by pancreatic acinar cells of the digestive and lysosomal enzymes that is the key to cause acute pancreatitis or the oversensitization of acini to cholecystokinin. in our study, the patients with bzd poisoning and an alcohol - related disorder had a 11.6-fold increased risk of acute pancreatitis compared with the patients without bzd poisoning (table 2).thus, bzd poisoning increased the risk of acute pancreatitis in the patients with alcohol - related disorders. serum triglyceride concentrations exceeding 1000 mg / dl (11 mmol / l) can precipitate attacks of acute pancreatitis ; however, the pathogenesis of inflammation is still unclear. acquired hypertriglyceridemia may be secondary to obesity, dm, hypothyroidism, pregnancy, estrogen or tamoxifen therapy, glucocorticoid excess, nephrotic syndrome, and -blockers. in our study, patients with bzd poisoning and hyperglyceridemia had a significantly higher risk of acute pancreatitis (adjusted hr = 3.57, 95% ci = 0.9613.30) than did patients without bzd poisoning. we also noted that people with dm had a higher risk of acute pancreatitis when they had bzd poisoning (adjusted hr = 2.63, 95% ci = 1.126.19). the gallstone is the most common cause of acute pancreatitis in the world, accounting for 35% to 40% of all cases. but only 3% to 7% of patients with gallstones develop biliary pancreatitis. there were 2 mechanisms of biliary pancreatitis : reflux of bile into the pancreatic duct caused by transient obstruction of the ampulla during passage of gallstones, or obstruction at the ampulla secondary to stone or edema caused by the passage of gallstone. our study revealed that the bzd poisoning did not increase the risk of biliary pancreatitis, possibly because the bzd is unrelated to the obstruction of biliary tract. although its mechanisms remain unclear, some studies have reported that cigarette smoking is an independent risk factor for acute and chronic pancreatitis. tobacco smoking is the most common cause of copd, which is characterized by chronically poor airflow. in our study, although drug - induced acute pancreatitis is rare, some studies show that incidence is increasing. therefore, the diagnosis depends on a high level of suspicion and careful drug history review. the time course of developing acute pancreatitis relative to the drug involved. according to table 3, patients with bzd poisoning exposure had a significantly higher risk of acute pancreatitis within 1 month after the event occurred (adjusted hr = 50.0, 95% ci = 6.39390.8). however, the risk was nonsignificant > 1 month after the event occurred (adjusted hr = 1.07, 95% ci = 0.303.90). we found that female patients with bzd poisoning exposure had a higher risk of acute pancreatitis than did male patients (adjusted hr = 13.2, 95% ci = 3.6048.5). we noted the same trend in another cohort study : in 2015, a cohort study identified several factors associated with acute pancreatitis and chronic pancreatitis that may be specific to older women. heavy smoking (40 + years vs 0 pack - years) was associated with a 2-fold increased odds ratio (or) for chronic pancreatitis. for body mass index (30 kg / m vs < 25 kg / m), the ors were 1.35 (1.071.70) for acute pancreatitis (p for trend =.009) and 0.59 (0.370.94) for chronic pancreatitis (p for trend =.01). the ors for acute and chronic pancreatitis were increased for hormone replacement therapy, heart disease, and hypertension. in taiwan we diagnosed the patient as having bzd poisoning based on careful history taking, clinical symptoms, and urine bzd screen. although we ca nt measure the doses of bzd, we still believed the diagnosis is correct. on the same way, the doctors diagnosed the patient as having acute pancreatitis based on clinical symptoms, blood examination, and image. after we adjusted for the potential confounding factors, the people with bzd poisoning exposure had a 5.33-fold greater acute pancreatitis risk (95% ci, 2.2612.6) than people without bzd 's poisoning exposure. the exact mechanism associated with bzd use and acute pancreatitis can not be fully shown, so we review some of the research in order to explain the possible mechanisms. specific reactions to drugs are adverse effects and its pharmacodynamic mechanisms are not related to drugs directly. these adverse events may occur, which are usually by the abnormal interaction of the drug or its metabolites, or trigger immune - mediated cytotoxicity between the drug and the pancreas. although the accurate mechanism of drug - induced acute pancreatitis is not always known, it should have similar pathogenesis. we found that there are several mechanism assumptions that include duct stenosis, direct pancreatic toxicity, the impact of bile flow, immune - mediated toxicity, metabolic effects, and thrombosis. if acute pancreatitis occurred after drug used for 4 to 8 weeks, it may be caused by hypersensitivity. if acute pancreatitis happened after several months of drug use, it is caused by accumulation of a toxic metabolite. if acute pancreatitis occurred immediately after drug overdose, it usually caused by its intrinsic toxicity. following this hypothesis, we think the mechanism of bzd poisoning induced acute pancreatitis most likely is the intrinsic toxicity or the sphincter of oddi dysfunction. tracing the past studies, we found the diazepam and midazolam had no effect on motility of the sphincter of oddi in human. while there is no direct evidence that bzd has a direct toxicity for pancreas, there are studies that showed that bzds may be related to acute pancreatitis. however, they are also found in other sites, for example, pancreatic cells and immune cells. some studies have implicated the gabaergic system in immune cell functions, inflammatory conditions, and diseases in peripheral tissues. the recent research has investigated the modulation of this autocrine regulatory loop by chronic ethanol and explored the potential prevention of these effects by gaba. we also found the use of zolpidem or zopiclone will increase the incidence of acute pancreatitis. some studies point out the zolpidem is a short - acting agent with half - life of 2.1 to 2.4 hours. although zolpidem and zopiclone is non - bzd class of sleeping pills, but they also are acting on the gaba receptor. therefore, they think bzds may have an acute and direct toxic effect on the pancreas, which further precipitates the pancreatic inflammation. first, we have no information about the amounts and types of bzds that poisoned patients. we also could nt obtain some possible risk factors for acute pancreatitis, such as socioeconomic status, smoking habit, body mass index, and family history. even we ca nt sure whether the patient has alcohol abuse, pancreatic trauma, gene mutation, or coprescribed drugs in detail. however, this cohort study is national research, because nhird covers 99% of the taiwan population. compared to bzd poisoning, the gene mutations, pancreatic trauma, and other drug poisoning are relatively rare. we have the enough database to establish the stronger relationship between bzd poisoning and acute pancreatitis than other resource. second, the evidence derived from a case - control study has lower quality than randomized controlled trials, because a case - control study design adjusted by the relevant confounding factors. although we carefully study design, including adequate control for confounding factors, a key limitation is that bias could still remain if there are unmeasured or unknown confounders. third, the diagnoses in nhi claims are used for administrative billing purposes and do not for scientific purposes. because of the data anonymity, we are unable to contact patients with bzd poisoning directly. however, the data on the acute pancreatitis diagnosis and bzd poisoning were highly reliable. this population - based, retrospective case - control study revealed that the event of bzd poisoning is significantly associated with an increased risk of acute pancreatitis. our findings require confirmation through a large, population - based, unbiased study before any definite conclusions can be drawn. | abstractwe designed a population - based retrospective cohort study to investigate the association between the event of benzodiazepine (bzd) poisoning and the risk of acute pancreatitis.in the present study, 12,893 patients with bzd poisoning during 2000 to 2011 were enrolled and matched with 4 comparison patients according to mean age and sex. we determined the cumulative incidences and adjusted hazard ratios of acute pancreatitis.a significant association was observed between bzd poisoning and acute pancreatitis. after adjustment for potential risk factors, the patients with bzd poisoning had a 5.33-fold increased risk of acute pancreatitis compared with the controls without bzd poisoning (hr = 5.33, 95% ci = 2.2612.60). the results revealed that acute pancreatitis in patients with bzd poisoning occurred in a follow - up time of 1 month (hr = 50.0, p 1 month (hr = 1.07, p >.05).this population - based study revealed the positive correlation between the event of bzd poisoning and an increased risk of acute pancreatitis. the findings warrant further large - scale and in - depth investigation. |
diabetes confers an increased risk for all - site cancer worldwide, except prostate cancer. among the chinese population of hong kong, for example, the prevalence of diabetes was reported to be over 10% in mid 1990s with 30% higher risk for cancer in people with diabetes compared to the general population. with aging and declining mortality from cardiovascular - renal disease, 25% of chinese people with type 2 diabetes now died from cancer, mainly due to liver, pancreatic, colorectal, and breast cancer. despite the health care and socio - economic burden of diabetes and cancer, since insulin has pro - proliferative activity and activation of insulin signaling axis has been implicated in carcinogenesis, some researchers proposed that the use of insulin by diabetic patients might contribute to cancer development. however, this view is controversial, as some epidemiological studies do not show a correlation between use of insulin and all - site cancer while some others have even reported that use of insulin might decrease the risk for cancer. one of the typical features in carcinogenesis is dna mutation which can be caused by dna damage often found in people with diabetes. in this review article, we searched the literature and summarized the evidence in support of a possible linking role of dna damage between diabetes and cancer. dna strand break can be detected using single cell gel electrophoresis or otherwise named comet assay. base oxidation, i.e., the formation of 8-hydroxy-2-deoxyguanosine (8-ohdg), the most studied form of base modification, can be quantified using specific antibody by enzyme - linked immunosorbent assay. these techniques are well - established, and assay kits are easily available and user - friendly. this review focuses on the two forms of dna damage with relevance to diabetes and cancer. dandona. compared the levels of 8-ohdg in mononuclear cells amongst type 1 diabetic patients (n = 12), type 2 diabetic patients (n = 15) and healthy control subjects (n = 10). they found that both type 1 and type 2 diabetic patients had higher levels of 8-ohdg than the nondiabetic subjects. production of reactive oxygen species by mononuclear cells was also significantly greater in diabetic patients than the control subjects. increased serum or urinary levels of 8-ohdg which correlated with poor glycemic control in addition to dna base oxidation, collins, and co - workers used comet assays on white blood cells and reported higher levels of dna strand break in people with type 1 diabetes (n = 10) compared to healthy controls (n = 10). subsequent studies have also confirmed elevated levels of dna strand break in type 2 diabetes, which, similar to 8-ohdg levels, were correlated with poor glycemic control. peripheral blood cells are often used for comet assay to detect dna strand break while urine and serum samples are commonly used for 8-ohdg quantification. a key question is whether the results from the peripheral samples reflect the levels of dna damage in other body tissues. to address this issue, kushwaha. compared the levels of dna strand break in lymphocytes, lung, liver, heart, aorta, kidney, and pancreas from diabetic and control rats. they found that dna strand break was increased in all the tested tissues from diabetic rats, and the level of dna strand break in lymphocytes was positively correlated with that in other tissues, suggesting that lymphocyte dna strand break might be a suitable marker indicating dna damage in internal organs. patients with type 1 or type 2 diabetes have increased plasma levels of glucose and advanced glycation endproducts (ages), which are diagnostic markers for diabetes and glycemic control. type 2 diabetic patients often have increased plasma levels of free fatty acids and insulin, due to obesity - associated insulin resistance, especially during the early stage of the disease. these pathophysiological factors in diabetes have been found to cause dna damage in vitro, which may explain why diabetic patients have increased level of dna damage in vivo. early in 1980s, researchers have reported that a high concentration of glucose (30 mmol / l) could cause dna strand break in cultured human endothelial cells. this observation has been confirmed in a subsequent study using the mouse and human renal cells as experimental models. in addition to dna strand break, high glucose could increase the level of 8-ohdg in endothelial and tubular cells. since dna damage is known to produce various mutations, and that high glucose can promote dna damage, the next question to ask is whether high glucose can cause or promote mutagenesis. indeed, zhang. examined the effect of high glucose on the genomic stability of phosphoribosyltransferase and thymidine kinase loci in human lymphoblastoid cell lines and reported a significant increase in mutations in both loci under high glucose. using mouse embryo as an experimental model, lee. furthermore, since high glucose can cause mutation and dna mutation are believed to play an important role in carcinogenesis, it is logical to speculate that high glucose can promote cancer development. to address this point, berstein and alexandrov injected carcinogen into pregnant rats via an intraperitoneal route and divided them into experimental and control groups. the rats in the experimental group drank 10% glucose in water till delivery, after which the rats and their progeny drank 5% glucose in water for 45 days. the authors found that the progeny of the rats in the experimental group had significantly higher incidence of tumors than those from the control group. in a follow - up study, the authors investigated whether glucose could have an effect on carcinogen - induced mutation in fetal cells, as measured using in vivo / in vitro assay. they found that high glucose not only increased the frequency of mutation but also promoted the proliferation and survival of the fetal cells from the rats in the experimental group. these data suggest that high glucose can promote carcinogenesis, which might be due to its dna damaging and then mutagenic effect. hyperglycemia in diabetes promotes the formation of ages due to nonenzymatic reactions between reducing sugars and free amino groups of proteins. subsequent reactions such as dehydration, oxidation, and condensation result in the irreversible formation of this heterogeneous group of products. stopper. investigated whether ages were genotoxic using pig kidney cells as an experimental model, and found that ages could cause dna strand break. these findings were subsequently confirmed in human liver and colon cells as well as mouse podocytes. in addition to dna strand break, ages can also trigger dna base oxidation and promote the production of 8-ohdg in different types of cells. palmitic acid is the most common saturated free fatty acid, which is often used to represent free fatty acids in experimental studies. beeharry. found that palmitic acid caused dna strand break and apoptosis in insulin - secreting cell line and primary human fibroblasts. under these experimental conditions, the dna damage is the main significant event, and the clinical relevance of the small incremental trend in apoptosis remains uncertain. in a proof of concept study, we quantified 8-ohdg in the culture of hct116 colon cancer cells treated with palmitic acid at concentrations lower than 75 these concentrations were not toxic to the cells but increased the production of 8-ohdg in a dose - dependent manner (lee, unpublished date). in support of these data, obese subjects who had increased risk for cancer also had increased plasma levels of free fatty acids and 8-ohdg, supporting possible causal relationships among free fatty acid, dna damage, and cancer. in subjects with obesity and prediabetes which are associated with insulin resistance, however, in colon cells and human lymphocytes, insulin was found to cause base oxidation by triggering the production of reactive oxygen species. in particular, for colon cancer cells, the lowest tested and lowest active concentration of insulin in vitro was 1 nmol / l for short time treatment (i.e., 2 h) and 0.51 nmol / l for longer exposure. in healthy human subjects, plasma insulin concentrations are in the order of 0.04 nmol / l after fasting, which can increase to 0.2 nmol / l after a meal. thus, the experimental results showed that insulin in pathophysiological concentrations had dna damaging effect. as summarized in table 1, these common pathophysiological features in diabetes, that is, high glucose, high insulin, ages and free fatty acids, can individually cause dna damage, strand break, and base oxidation, although the effect of insulin on base oxidation remains unknown. since these pathophysiological factors frequently co - exist in type 2 diabetes during the clinical course, their potential synergistic effects on causing dna damage is an interesting topic for exploration. dna damaging effects due to the pathophysiological factors in diabetes ages : advanced glycation endproducts. early in 1980s, researchers have reported that a high concentration of glucose (30 mmol / l) could cause dna strand break in cultured human endothelial cells. this observation has been confirmed in a subsequent study using the mouse and human renal cells as experimental models. in addition to dna strand break, high glucose could increase the level of 8-ohdg in endothelial and tubular cells. since dna damage is known to produce various mutations, and that high glucose can promote dna damage, the next question to ask is whether high glucose can cause or promote mutagenesis. indeed, zhang. examined the effect of high glucose on the genomic stability of phosphoribosyltransferase and thymidine kinase loci in human lymphoblastoid cell lines and reported a significant increase in mutations in both loci under high glucose. using mouse embryo as an experimental model, lee. furthermore, since high glucose can cause mutation and dna mutation are believed to play an important role in carcinogenesis, it is logical to speculate that high glucose can promote cancer development. to address this point, berstein and alexandrov injected carcinogen into pregnant rats via an intraperitoneal route and divided them into experimental and control groups. the rats in the experimental group drank 10% glucose in water till delivery, after which the rats and their progeny drank 5% glucose in water for 45 days. the authors found that the progeny of the rats in the experimental group had significantly higher incidence of tumors than those from the control group. in a follow - up study, the authors investigated whether glucose could have an effect on carcinogen - induced mutation in fetal cells, as measured using in vivo / in vitro assay. they found that high glucose not only increased the frequency of mutation but also promoted the proliferation and survival of the fetal cells from the rats in the experimental group. these data suggest that high glucose can promote carcinogenesis, which might be due to its dna damaging and then mutagenic effect. hyperglycemia in diabetes promotes the formation of ages due to nonenzymatic reactions between reducing sugars and free amino groups of proteins. subsequent reactions such as dehydration, oxidation, and condensation result in the irreversible formation of this heterogeneous group of products. stopper. investigated whether ages were genotoxic using pig kidney cells as an experimental model, and found that ages could cause dna strand break. these findings were subsequently confirmed in human liver and colon cells as well as mouse podocytes. in addition to dna strand break, ages can also trigger dna base oxidation and promote the production of 8-ohdg in different types of cells. palmitic acid is the most common saturated free fatty acid, which is often used to represent free fatty acids in experimental studies. beeharry. found that palmitic acid caused dna strand break and apoptosis in insulin - secreting cell line and primary human fibroblasts. under these experimental conditions, the dna damage is the main significant event, and the clinical relevance of the small incremental trend in apoptosis remains uncertain. in a proof of concept study, we quantified 8-ohdg in the culture of hct116 colon cancer cells treated with palmitic acid at concentrations lower than 75 these concentrations were not toxic to the cells but increased the production of 8-ohdg in a dose - dependent manner (lee, unpublished date). in support of these data, obese subjects who had increased risk for cancer also had increased plasma levels of free fatty acids and 8-ohdg, supporting possible causal relationships among free fatty acid, dna damage, and cancer. in subjects with obesity and prediabetes which are associated with insulin resistance, hyperinsulinemia is a frequent phenomenon. whether insulin can cause strand break remains to be examined. however, in colon cells and human lymphocytes, insulin was found to cause base oxidation by triggering the production of reactive oxygen species. in particular, for colon cancer cells, the lowest tested and lowest active concentration of insulin in vitro was 1 nmol / l for short time treatment (i.e., 2 h) and 0.51 nmol / l for longer exposure. in healthy human subjects, plasma insulin concentrations are in the order of 0.04 nmol / l after fasting, which can increase to 0.2 thus, the experimental results showed that insulin in pathophysiological concentrations had dna damaging effect. as summarized in table 1, these common pathophysiological features in diabetes, that is, high glucose, high insulin, ages and free fatty acids, can individually cause dna damage, strand break, and base oxidation, although the effect of insulin on base oxidation remains unknown. since these pathophysiological factors frequently co - exist in type 2 diabetes during the clinical course, dna damaging effects due to the pathophysiological factors in diabetes ages : advanced glycation endproducts. damage of dna by reactive oxygen species can be considered as a direct pathway in diabetes - associated mutation. high serum levels of glucose, ages, free fatty acids, and insulin can all promote the production of reactive oxygen species found to be increased in type 2 diabetes compared to nondiabetic subjects. besides, people with diabetes had low anti - oxidative capacity such as reduced glutathione synthesis which might contribute to their proneness to oxidative damage. despite the high heritability of diabetes, most of the genetic factors discovered by genome - wide association studies only explained small variance of the risk in the majority of people with type 2 diabetes. interestingly, lai. found that genetic variants of peroxisome proliferator - activated receptor- coactivator-1 (ppargc1a) were associated with increased risk of dna damage (urine 8-ohdg) and diabetes. in this light, ppargc1a is known to regulate mitochondrial electron transport, which generates reactive oxygen species while at the same time, activates defending enzymes against reactive oxygen species. imbalance between these two roles of ppargc1a can lead to oxidative stress and possibly dna damage. in hong kong chinese, type 2 diabetes - related genetic variants of hhex, tcf7l2, and cdkal1 have also been reported to be associated with increased all - site cancer although the underlying mechanism remains to be explored. simone. reported the following observations in high glucose experimental models : (1) there were associations amongst akt phosphorylation, tuberin phosphorylation, and 8-oxodg accumulation ; (2) inhibition of akt using pi3 kinase inhibitor reduced high glucose - induced tuberin phosphorylation, and (3) anti - oxidant inhibited reactive oxygen species generation, phosphorylation of akt and tuberin, and 8-oxodg accumulation. these results indicated that the pi3 kinase - akt - tuberin pathway might be important for dna oxidation damage under hyperglycemic condition. in a recent study, habib and liang reported over - activation of akt with decreased protein levels of tuberin and increased 8-ohdg concentration in kidney cancer tissues from diabetic patients, compared with cancer tissues from patients without diabetes. taken together, these data support a possible role of akt / tuberin signaling in the occurrence of dna damage in diabetes. compared the efficacy of removal of damaged dna in peripheral blood lymphocytes between type 2 diabetic patients and healthy individuals, and reported reduced efficacy of repairing dna damage in those with diabetes. dna is sensitive to damage caused by endogenous and exogenous factors, and as such, dna damage occurs frequently in the absence of specific disease. thus, an effective mechanism of dna damage repair is crucial to maintain genomic integrity. since defective dna repair may determine the susceptibility to carcinogenesis, decreased efficiency in dna repair is another potential factor for cancer in diabetes. the akt pathway is an important pathway implicated in cell growth which can be activated by high levels of insulin, glucose, ages and free fatty acids (palmitic acid). here, insulin and high glucose activate akt to phosphorylate tuberin which allows tuberin to interact with binding protein to perform its biological function. although there are reports on associations between dna damage and changes in akt activity, the mechanisms underlying akt activation and dna damage and repair is less clear. as reviewed by xu and coworkers, akt activation led to suppression of atr / chk1 signaling by direct phosphorylation of chk1 or topbp1. it could also inhibit recruitment of double - strand break resection factors (i.e., rpa, brca1, and rad51) to dna damage sites, leading to compromised homologous recombination repair. thus, akt activation may be a potential cause of dna repair inhibition and genomic instability. in a study that used in vitro and in vivo experimental models, akt activation was shown to phosphorylate bim1, which led to increased genomic instability and increased oncogenic potential of bim1. in the case of tuberin, its phosphorylation or inactivation through activation of akt could decrease the gene expression of ogg1, which was also downstream of akt activation by reactive oxygen species, resulting in accumulation of dna damage. while these data support a regulatory role of akt in dna repair, the signal mediators downstream of akt and their interactions remain to be defined. damage of dna by reactive oxygen species can be considered as a direct pathway in diabetes - associated mutation. high serum levels of glucose, ages, free fatty acids, and insulin can all promote the production of reactive oxygen species found to be increased in type 2 diabetes compared to nondiabetic subjects. besides, people with diabetes had low anti - oxidative capacity such as reduced glutathione synthesis which might contribute to their proneness to oxidative damage. despite the high heritability of diabetes, most of the genetic factors discovered by genome - wide association studies only explained small variance of the risk in the majority of people with type 2 diabetes. interestingly, lai. found that genetic variants of peroxisome proliferator - activated receptor- coactivator-1 (ppargc1a) were associated with increased risk of dna damage (urine 8-ohdg) and diabetes. in this light, ppargc1a is known to regulate mitochondrial electron transport, which generates reactive oxygen species while at the same time, activates defending enzymes against reactive oxygen species. imbalance between these two roles of ppargc1a can lead to oxidative stress and possibly dna damage. in hong type 2 diabetes - related genetic variants of hhex, tcf7l2, and cdkal1 have also been reported to be associated with increased all - site cancer although the underlying mechanism remains to be explored. simone. reported the following observations in high glucose experimental models : (1) there were associations amongst akt phosphorylation, tuberin phosphorylation, and 8-oxodg accumulation ; (2) inhibition of akt using pi3 kinase inhibitor reduced high glucose - induced tuberin phosphorylation, and (3) anti - oxidant inhibited reactive oxygen species generation, phosphorylation of akt and tuberin, and 8-oxodg accumulation. these results indicated that the pi3 kinase - akt - tuberin pathway might be important for dna oxidation damage under hyperglycemic condition. in a recent study, habib and liang reported over - activation of akt with decreased protein levels of tuberin and increased 8-ohdg concentration in kidney cancer tissues from diabetic patients, compared with cancer tissues from patients without diabetes. taken together, these data support a possible role of akt / tuberin signaling in the occurrence of dna damage in diabetes. blasiak. compared the efficacy of removal of damaged dna in peripheral blood lymphocytes between type 2 diabetic patients and healthy individuals, and reported reduced efficacy of repairing dna damage in those with diabetes. dna is sensitive to damage caused by endogenous and exogenous factors, and as such, dna damage occurs frequently in the absence of specific disease. thus, an effective mechanism of dna damage repair is crucial to maintain genomic integrity. since defective dna repair may determine the susceptibility to carcinogenesis, decreased efficiency in dna repair is another potential factor for cancer in diabetes. the akt pathway is an important pathway implicated in cell growth which can be activated by high levels of insulin, glucose, ages and free fatty acids (palmitic acid). here, insulin and high glucose activate akt to phosphorylate tuberin which allows tuberin to interact with binding protein to perform its biological function. although there are reports on associations between dna damage and changes in akt activity, the mechanisms underlying akt activation and dna damage and repair is less clear. as reviewed by xu and coworkers, akt activation led to suppression of atr / chk1 signaling by direct phosphorylation of chk1 or topbp1. it could also inhibit recruitment of double - strand break resection factors (i.e., rpa, brca1, and rad51) to dna damage sites, leading to compromised homologous recombination repair. thus, akt activation may be a potential cause of dna repair inhibition and genomic instability. in a study that used in vitro and in vivo experimental models, akt activation was shown to phosphorylate bim1, which led to increased genomic instability and increased oncogenic potential of bim1. in the case of tuberin, its phosphorylation or inactivation through activation of akt could decrease the gene expression of ogg1, which was also downstream of akt activation by reactive oxygen species, resulting in accumulation of dna damage. while these data support a regulatory role of akt in dna repair, the signal mediators downstream of akt and their interactions remain to be defined. genome stability is important for normal cell physiology. however, dna bases are highly vulnerable to being damaged. intrinsically, dna is chemically unstable in an aqueous environment. spontaneous reactions such as hydrolysis and de - amination can occur, resulting in dna damage. other endogenous factors such as metabolic products and exogenous factors such as environmental chemicals can also cause dna damage. dna strand breaks, in particular, double strand breaks, are potentially lethal. in surviving cells, double strand break triggers break - induced replication which is known to produce dna mutations at high frequencies. the break - induced replication and related mechanisms can cause various dna abnormalities which include loss of heterozygosity, telomere maintenance without telomerase, nonreciprocal translocation, copy number variation, and chromosomal rearrangements. take 8-ohdg as an example, it can pair with a to form situ g to t substitution, and if incorporated into dna, it can lead to mis - pairing with da to form a to c substitution [figure 2 ]. moreover, 8-ohdg can change dna conformation, leading to substitution mutations by mis - pairing with da or dt. the presence of 8-ohdg in dna can also have epigenetic consequences such as altering the gene transcription. unfortunately, once mis - pairing occurs in dna, it will not be repaired effectively in cells. although 8-ohdg is the most frequently reported dna damage in diabetes and other diseases, it should be kept in mind that all four dna bases are susceptible to oxidative damage. among these changes, modifications to gc base pairs tend to be more mutagenic while modifications on at base pairs have weaker mutagenic potential. in addition to base oxidation, lipid peroxidation is also harmful, which can cause the formation of reactive aldehydes such as malondialdehyde and 4-hydroxy-2-nonenal. these products are highly reactive to protein and dna and have been shown to be mutagenic. mechanism of mutation caused by 8-hydroxy-2-deoxyguanosine. : oxidized base ; a : mispaired base. about a century ago, boveri speculated that cancer was a problem of cell proliferation due to chromosomal aberrations and/or mutations. indeed, with biotechnological advancement, large number of chromosomal abnormalities and dna mutations had been demonstrated in various types of cancers. this leads to the proposal of somatic mutation theory to explain the mechanisms of cancer development, which considers the accumulation of somatic mutations as the origin of carcinogenesis and cancer progression. in animal models, carcinogenesis is found to involve multiple steps which include at least initiation, promotion, and progression stages. dna mutation is the main molecular feature of the initiation step which is irreversible characterized by micro - lesions such as base pair substitution and frame shift. clinical cancer appears when cancerous cells enter the progression step where the karyotypic alteration of genomic instability and pathological features can be found. if the pathophysiological factors of diabetes, notably, high blood glucose and ages, can contribute to the increased risk for all - site cancer in diabetes, then, we can expect that use of anti - diabetic drugs that reduce blood glucose and ages can also reduce cancer risk in diabetic patients. indeed, this has been supported by epidemiological studies showing reduced cancer risk with metformin which is probably the most commonly used anti - diabetic drug. in addition to its glucose - lowering effect, metformin itself can inhibit oxidative stress and dna damage. using the hong kong diabetes registry, we have also reported the association of reduced cancer risk with all anti - diabetic drugs as well as drugs such as renin - angiotensin system inhibitors and statins which have been shown to have anti - oxidant properties. the available data from the literature support the notion that pathophysiological metabolic factors in diabetes can cause dna damage, making diabetes a state of dna damage. inhibition of antioxidant capacity worsens the oxidative stress state while inhibition of dna damage repair machinery contributes to dna damage accumulation. apart from optimizing glycemic control, future cancer prevention in diabetic patients may target at inhibiting dna damage using alternative drugs like antioxidants. | objective : this review examines the evidence that : diabetes is a state of dna damage ; pathophysiological factors in diabetes can cause dna damage ; dna damage can cause mutations ; and dna mutation is linked to carcinogenesis.data sources : we retrieved information from the pubmed database up to january, 2014, using various search terms and their combinations including dna damage, diabetes, cancer, high glucose, hyperglycemia, free fatty acids, palmitic acid, advanced glycation end products, mutation and carcinogenesis.study selection : we included data from peer - reviewed journals and a textbook printed in english on relationships between dna damage and diabetes as well as pathophysiological factors in diabetes. publications on relationships among dna damage, mutagenesis, and carcinogenesis, were also reviewed. we organized this information into a conceptual framework to explain the possible causal relationship between dna damage and carcinogenesis in diabetes.results:there are a large amount of data supporting the view that dna mutation is a typical feature in carcinogenesis. patients with type 2 diabetes have increased production of reactive oxygen species, reduced levels of antioxidant capacity, and increased levels of dna damage. the pathophysiological factors and metabolic milieu in diabetes can cause dna damage such as dna strand break and base modification (i.e., oxidation). emerging experimental data suggest that signal pathways (i.e., akt / tuberin) link diabetes to dna damage. this collective evidence indicates that diabetes is a pathophysiological state of oxidative stress and dna damage which can lead to various types of mutation to cause aberration in cells and thereby increased cancer risk.conclusions:this review highlights the interrelationships amongst diabetes, dna damage, dna mutation and carcinogenesis, which suggests that dna damage can be a biological link between diabetes and cancer. |
during the course of treatment for autoimmune disease, patients with no history of bleeding sometimes suddenly present with severe ecchymoses or muscle hematoma. in such cases, acquired coagulation factor deficiencies, including acquired hemophilia a (aha), should be considered in the differential diagnosis of the cause of bleeding. as a rare hemorrhagic disorder but the most frequently acquired coagulation factor deficiency, inhibitors, against coagulation factor viii (fviii), which neutralize fviii activity. although aha has previously been reported to have an incidence of 0.2 to 1.0 cases per million population per year, a recent report describes a progressively increasing incidence of 2 cases per million population per year, likely resulting from greater awareness of the disorder. in contrast to the incidence of congenital hemophilia a, a recessive x - linked genetic disorder, the incidence of aha has not been found to differ significantly between men and women. aha has a biphasic age distribution, exhibiting a small peak from age 20 to 30 years and a larger peak at age 60 years and older [4, 5 ]. the majority of patients who present with aha between ages 20 and 30 years are female, as the disease in this age group is associated with pregnancy (i.e., the development of postpartum inhibitors) and autoimmune disorders. while it was previously thought that the majority of patients who present with aha at age 60 years and older are male [4, 6 ], recent studies have revealed no significant difference in the sex ratio of elderly patients. while aha has a high mortality rate, estimated at up to 33%, it has decreased in tandem with the advancement of therapeutic interventions since the 1980s. aha occurs relatively less frequently but develops suddenly and occasionally presents with life - threatening bleeding. furthermore, the management of aha remains difficult and the costs of treatment are often immense. although aha is thus clinically and economically an important disorder, it is often unrecognized or misdiagnosed as other acquired hemorrhagic disorders, such as disseminated intravascular coagulation (dic) and acquired inhibitors against von willebrand factor (acquired von willebrand syndrome) and factor xiii (acquired factor xiii deficiency). in contrast to the fviii - neutralizing inhibitors that develop in congenital hemophilia a after fviii - replacement therapy, which are alloantibodies, the fviii - neutralizing inhibitors that develop in aha are autoantibodies. it is well known that approximately 50% of patients with aha have or have had immune system disorders, such as autoimmune diseases and lymphoproliferative disorders. this fact, as well as knowledge that autoantibodies play a central role in aha pathogenesis, indicates that modulation of the immune system or the autoimmune mechanism that generates autoantibodies is involved in aha. aha patients often present with severe and massive bleeding, which is responsible for their relatively high mortality rate. the most commonly affected organ is the skin, especially at the site of injection or contusion, which often manifests severe ecchymoses. it is notable that hemarthroses most commonly appear in congenital hemophilia a but seldom occur or cause joint damage in aha [11, 12 ]. although relatively uncommon, intra - abdominal or intracerebral hemorrhage in aha patients often leads to life - threatening bleeding. persistent bleeding after surgical procedures, such as intramuscular injection, catheter insertion, and tracheotomy for treatment of underlying or incidentally coexisting diseases, may be the earliest symptom of aha. occasionally, aha is suspected despite the absence of hemorrhagic manifestations by review of the preoperative examination results, especially in patients with low - titer inhibitors. a notable prognostic consideration is that, unlike in congenital hemophilia a, inhibitor titer in aha does not indicate the severity or frequency of bleeding. fviii is a cofactor for activated factor ix (fixa) that forms the xase (tenase) complex in the presence of ca and phospholipids and is essential for the intrinsic coagulation system responsible for blood clotting ; therefore, fviii deficiency causes dysfunction of the intrinsic system and reduces thrombin generation, resulting in a bleeding disorder. fviii is mainly synthesized in the liver as a 2,351 amino acid and 330-kda single - chain precursor glycoprotein with a functional domain structure (a1-a2-b - a3-c1-c2) (figure 1). after proteolytic processing, circulating mature fviii protein is composed of a heterodimer of a heavy (a1-a2) and a light (a3-c1-c2) chain. this chain is noncovalently bound to von willebrand factor (vwf), which protects the fviii from inactivation. vwf has a molecular weight of 226 kda and a multimeric structure consisting of subunits of large molecular weight (> 20,000 kda). the majority of fviii inhibitors observed in aha, which are polyclonal autoantibodies, and in congenital hemophilia a, which are polyclonal alloantibodies, bind to the a2 (454509), a3 (18041819), or c2 domains (21812243) [1417 ]. while anti - c2 antibodies interfere with the binding of fviii to phospholipids and vwf, a2 and a3 inhibitors block the binding of fviii to factor x (fx) and fixa, respectively, and obstruct the formation of the xase complex. previous studies of cd4 t - cell subsets (th1, th2, and th3) specific for fviii revealed that alloantibodies in congenital hemophilia a consist of th1-dependent immunoglobulin (ig) g1 and igg2 and th2-dependent igg4. however, aha autoantibodies are often igg4 autoantibodies and less frequently igg1 and igg2 autoantibodies. further, fviii - neutralizing activity is correlated with the presence of igg4 autoantibodies [3, 18, 19 ]. as igg4 antibodies form nonprecipitating immune complexes and are not complement - fixing autoantibodies, they do not cause the severe organ damage often seen in hemophilia b patients, in whom allergic reactions to fix concentrates are associated with the specific igg1 subclass of alloantibodies against fix. most alloantibodies developed in congenital hemophilia a patients undergoing fviii replacement therapy, which are classified as type i inhibitors of first - order kinetics, inactivate fviii at a rate linearly correlated with their concentration and are able to completely inhibit fviii activity at high concentrations. in contrast to the kinetics of the interaction between fviii and the inhibitors in congenital hemophilia a, the kinetics of the interaction in aha display a nonlinear inhibitory profile. specifically, these type ii inhibitors show a rapid initial inactivation phase followed by a slower equilibrium phase during which some residual fviii activity (fviii : c) is detectable even after incubation at maximum concentrations of inhibitors for a sufficient period (figure 2). however, aha patients with identifiable fviii : c manifest far more severe hemorrhage than congenital hemophiliacs with comparable levels of fviii : c. moreover, addition of excessive fviii concentrates fails to neutralize the inhibitory activity of type ii inhibitors in vitro, making management of aha difficult in the clinical setting and high - dose replacement therapy with fviii concentrates unsuccessful in aha patients with high - titer inhibitors. in a study of the physiological activities of aha inhibitors, identified a subset of inhibitors in congenital hemophilia a that hydrolyze fviii, resulting in fviii inactivation [21, 22 ]. on the basis of their findings, lacroix - desmazes. advocated a unique conception of the inhibitory mechanism in aha that has been supported by further research of the proteolytic activity of igg isolated from patients with aha, demonstrating the presence of autoimmune fviii - hydrolyzing igg. on the basis of the observation that the extent of the fviii hydrolytic activity of acquired inhibitors exhibits a correlation with inhibitory titers of the inhibitors, igg - mediated fviii hydrolysis has been hypothesized to participate in fviii inactivation in aha. this hypothesis is supported by the results of a comparison study of the properties of the proteolytic inhibitors in congenital hemophilia a and aha, which, using proline - phenylalanine - arginine - methylcoumarinamide (pfr - mca), a synthetic substrate for fviii - hydrolyzing autoantibodies, revealed that the rate of fviii hydrolysis differs significantly between hemophilia a and aha patients. while the results of the pfr - mca and bethesda assay revealed a correlation between hydrolytic activity and inhibitor titer in acquired inhibitors, alloantibodies in congenital hemophilia a exhibit little correlation. these findings suggest that populations of proteolytic inhibitors in aha patients differ from those in congenital hemophilia a patients with inhibitors. in addition, some aha autoantibodies can augment fix activity by fix proteolysis in the absence of fviii - proteolytic activity. on the basis of these findings, it has been hypothesized that the fix - potentiating action of autoantibodies may partially compensate for the inhibition of fviii, resulting in restoration of thrombin generation. in approximately 50% of aha patients, especially elderly patients, autoantibody development against factor viii is idiopathic [2, 26, 27 ], indicating that the acquired inhibitors develop via an autoimmune mechanism. the underlying conditions shown in table 1 are observed in the remaining 50% of patients. aha is often associated with autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, thyroid dysfunction, and autoimmune hemolytic anemia. observation of an association between aha and inflammatory bowel disease, pemphigus, and graft versus host disease (gvhd) has been also reported, indicating that aha has an autoimmune origin. in fact, up to 20% of all aha patients present with autoimmune disorders. as fviii inhibitor titers in patients with autoimmune disorders are often high and less frequently resolve spontaneously compared to those associated with pregnancy, the former require aggressive treatment for bleeding management and inhibitor eradication consisting of both hemostatic therapy using bypassing agents and immunosuppressive therapy. although hemorrhagic symptoms commonly present between 1 and 4 months after parturition, they may occur over a year after delivery [29, 30 ]. while the hemorrhagic potential is often low and the inhibitors often spontaneously disappear in almost all patients with low titers of inhibitors, it may be difficult to achieve inhibitor eradication in patients with high titers (5 bu / ml), even with aggressive immunosuppressive therapy. an important consideration is that carrying a fetus might pose the risk of fatal bleeding, as it poses the risk of diaplacental transition of inhibitor igg from pregnant aha patients. when inhibitor eradication in patients with postpartum inhibitors is unsuccessful, other commonly associated conditions, especially autoimmune disorders, should be suspected. underlying malignancy in either solid or nonsolid form presents in approximately 10% of aha patients and commonly develops in elderly patients. an important consideration is that, as the incidence of both solid tumor as well as aha increases with aging, inhibitors might be detected coincidentally in patients with solid tumor. patients with lymphoproliferative diseases complicating aha, which include chronic lymphocytic leukemia, non - hodgkin lymphoma, and multiple myeloma, with altered immune status often have coexisting autoimmune diseases, one of which may be aha. as anticarcinogenic agents induce cell damage and/or modulate immunological reactions through danger signals [31, 32 ], patients with malignancies might be predisposed to autoimmune phenomena and increased risk of developing inhibitors. although it remains unclear whether autoantibody development derives from the tumor itself, the observation that cancer antigens share immunological cross - reactivity with fviii has not been reported to date. suspected medications include antibiotics (penicillin, sulfonamides, and chloramphenicol), anticonvulsants (phenytoin), antihypertensive agents (methyldopa), and bacillus calmette - gurin vaccination. as high titers of inhibitors resulting from drug reactions and allergies disappear after termination of the responsible drug, specific therapy to eradicate fviii autoantibodies may not be provided to patients who experience drug hypersensitivity. it is notable that administration of interferon for hepatitis c virus infection, which, by directly acting on the immune system, results in malfunctioning of the immune response, is associated with aha. anti - fviii autoantibodies are developed in the context of dysfunction of immune system, as discussed above. knowledge of the detailed molecular biological mechanism of inhibitor generation has accumulated gradually over the past decades. variants of the polymorphic cytotoxic t lymphocyte antigen-4 (ctla-4) gene, which is found on the surface of activated and regulatory t - cells, have been associated with autoimmune diseases. the extracellular domain of ctla-4 is similar to the domain of the cd28 that is a component of the costimulatory cd28/b7 receptor / ligand system and competes against cd28 ligands, such as cd80 and cd86, on the surface of dendritic cells. stimulation of the cd28 receptor on t - cells emits a costimulatory signal for t - cell proliferation and activation. in contrast, ctla-4 may inhibit t - cell activation by restricting the ability of b7 to interact with cd28. in regulatory t - cells, tight binding of ctla-4 molecules on regulatory t - cells to costimulatory ligand b7 on antigen - presenting cells strips and destroys b7 molecules. as antigen - presenting cells without b7 ligands can not deliver additional signal 2 (i.e., engage in costimulation), the binding of ctla-4 and costimulatory ligand b7 terminates activation or differentiation of nave t - cells to effector t - cells. thus, ctla-4 acts as a receptor that downregulates the immune system. the results of several studies, including those of a recent study that observed a single nucleotide polymorphism of the ctla-4 gene (+ 49 a / g allele) at a significantly higher frequency in aha patients compared with controls, indicate that ctla-4 variants (ctla-4 single nucleotide polymorphisms) might also be involved in the pathogenesis of aha as well as that other genetic / environmental factors might contribute to the onset of aha. recently, b - cell activating factor belonging to the tumor necrosis factor family (baff), also referred to as blys, has been found to regulate the immune system. known to be involved in the survival and maturation of b - cells, baff binds to tumor necrosis factor - related receptors, such as b - cell - maturation antigen (bcma), transmembrane - activator and calcium - modulator and cyclophilin - ligand interactor (taci), and b - cell activating factor receptor (baff - r). this baff - mediated ligand - receptor interaction forms a complex network that plays a critical role in the induction and regulation of humoral immunity. previous mouse studies demonstrated that constitutive baff overexpression leads to survival of autoreactive b - cells, which in turn induces breakdown of peripheral tolerance. in this setting, autoimmune disorders develop through anomalous b - cell activation with spontaneous production of multiple autoantibodies and polyclonal hypergammaglobulinemia. in humans, elevated baff levels are associated with several b - cell - mediated autoimmune diseases with hypergammaglobulinemia [4042 ]. in a previous study, we found baff levels to be significantly higher in congenital hemophilia a patients with inhibitors compared to healthy controls or hemophilia a patients without inhibitors. these results suggest that elevated baff levels allow anti - fviii antibody - secreting plasma cells to survive and produce inhibitors in congenital hemophilia a patients with inhibitors. despite such research, the typical presentation of baff levels in patients with aha remains to be elucidated. our preliminary measurement of baff in two patients with aha revealed an elevated level of baff in one patient but a normal level in the other, suggesting that baff might be involved in the pathogenesis of aha in at least some aha patients. although further study is warranted before its application in the clinical setting, the targeting of baff as a therapeutic strategy appears promising in the treatment of a subset of aha patients, as well as of hemophilia a patients with refractory inhibitors presenting with elevated baff levels. the first step in diagnosis of aha is tracking signs of bleeding tendency, particularly in the elderly, in the clinical setting and testing for prolongation of activated partial thromboplastin time (aptt) in the laboratory. the next step is review of patient medical history by consideration of the impact of any underlying conditions associated with aha. aptt prolongation reflects decreased levels of coagulation intrinsic factors viii and ix, as well as decreased levels of factors xi and xii, prekallikrein, and high molecular weight kininogen, which are involved in the contact system of coagulation. however, since reduction of proteins involved in the contact system is not associated with bleeding tendencies [44, 45 ], these conditions are ruled out in the differential diagnosis. to diagnose aha, measurement of fviii : c is essential, and consecutive determination of inhibitor titer is a requisite in cases of decreased level of fviii : c. while aptt is prolonged in patients with low levels of fviii : c by anti - fviii neutralizing autoantibodies, pt, fibrinogen and vwf levels, and platelet count are within normal limits and platelet function is normal. since thrombocytopenia, pt and aptt prolongation, and decreased levels of fibrinogen are often observed in dic patients who are erroneously diagnosed with aha, consideration of these findings is helpful in differentiation of dic from aha. several cross - mixing studies have been performed to examine whether aptt prolongation results from a deficiency of intrinsic factor(s) or inhibitor. in one such study, addition of an equal volume of normal control plasma to patient 's plasma was found to correct the aptt value to the normal range in coagulation - factor - deficient patients but not aha patients. as fviii inhibition by autoantibodies is time- and temperature - dependent, the mixture in all such studies should be incubated at 37c for 1 to 2 hours and, if correction of aptt value is unsuccessful, the presence of an inhibitor should be suspected. recently, a cross - mixing test originally developed to differentiate lupus - anticoagulant presence from coagulation - factor deficiency has been established as a more useful laboratory test to determine the cause of aptt prolongation and thus useful in aha diagnosis. the plotting of the results of a cross - mixing test with altering the proportion of normal control plasma to the patient 's plasma yields a convex aptt value curve that faces upward in the presence of inhibitors (including coagulation factor - neutralizing antibodies and lupus anticoagulants) and downward in the presence of a factor deficiency (figure 3). aptt is prolonged in the presence of lupus anticoagulants that interfere with the assembly and activity of the fxa - fva - ca phospholipid complex. lupus anticoagulants are polyclonal immunoglobulins that bind to phospholipids and proteins associated with the cell membrane and show nonspecific inhibitory effects that result in prolongation of both aptt and pt. from the perspective of laboratory testing, since intrinsic coagulation factor activity, including that of fviii, appears to decrease in the presence of lupus anticoagulants, it is often difficult to distinguish aha from lupus anticoagulants even by a mixing test. if the results of a mixing test indicate the presence of an inhibitor, the lupus anticoagulant is therefore evaluated by phospholipid - sensitive functional - coagulation assay, such as the dilute russell 's viper - venom time assay. the coagulant in the venom directly activates fx, indicating that the dilute russell 's viper - venom time assay is dependent on a common pathway, including a pathway with phospholipids, and not influenced by deficiency or inhibition of intrinsic factors. thus, the addition of exogenous phospholipids will correct the prolongation value as measured by clotting assay, confirming the presence of a lupus anticoagulant. one type of clotting test, the platelet neutralization procedure (pnp), takes advantage of the fact that lupus anticoagulants are absorbed onto the phospholipids on the surface of platelets while fviii inhibitors are not absorbed. the addition of washed platelets to the patient 's plasma with lupus anticoagulant will thus decrease aptt prolongation. when the presence of an inhibitor is suspected, the targeted factor should be identified and the extent of inhibitory activity quantified. for the quantification of fviii inhibitors, the bethesda assay is the most commonly used laboratory test worldwide. the classic bethesda method measures the quantity of residual fviii : c of a mixture containing equal amounts of normal control plasma and serially diluted patient plasma after incubation at 37c for 2 h. the level of residual fviii : c in the patient 's plasma with inhibitors increases in tandem with the increasing dilution rate. the inhibitor titer value (1.0 bu / ml) used in the bethesda assay is the reciprocal of the value of the dilution of the patient 's plasma that leads to 50% inhibition. in the nijmegen modification, which permits more accurate measurement of low titers of fviii inhibitor, buffer is added to the bethesda assay to maintain the sample plasma ph within the physiological range for the 2-hour incubation period and thereby stabilize fviii in normal control plasma. although these assays are useful for determination of titers of alloantibodies against fviii in congenital hemophilia a patients with type i kinetics, exact determination of autoantibody titer in aha is difficult in patients with type ii kinetics, in whom the acquired inhibitor - fviii complex may show some residual fviii : c, even in the presence of high concentrations of inhibitors. therefore, measurement of levels of fviii - binding antibodies is necessary for performing meaningful clinical assessment of the inhibitors present in aha. prior to development of enzyme - linked immunosorbent assay (elisa), measurement of fviii - binding antibodies was traditionally performed using the agarose gel method [52, 53 ]. previous studies have demonstrated that noninhibitory antibodies can be detected by elisa in hemophilia patients in whom no inhibitors were detected using the bethesda method [5457 ]. in accordance with previous research into the use of the immunoprecipitation method for measurement of noninhibitory antibodies [58, 59 ], our investigation of the efficacy of immune - tolerance induction therapy in hemophilia a with refractory inhibitors using the immunoprecipitation method revealed that the method yields results of sufficient sensitivity. however, use of all of these methods has certain drawbacks, such as the need to use radioactive materials and perform complex, time - consuming procedures. fortunately, the ability of fluorescent microbeads method to overcome these drawbacks has been demonstrated in several studies, including one study in which we demonstrated its usefulness for assessing aha patients as well as hemophilia a patients with inhibitors (figure 4). as measurement using the fluorescent microbeads method is almost completely unaffected by the presence or absence of residual fviii : c, use of the method allows for detection of antibodies without the undue influence of the presence of lupus anticoagulants or heparin. favorable outcome in aha depends on selection of an appropriate therapeutic approach based on early, correct diagnosis. the therapeutic strategy should aim for the achievement of 2 targets : control of bleeding and eradication of inhibitors. bleeding episodes in aha are often severe and life threatening and presents with severe anemia. as massive subcutaneous or intramuscular hemorrhage may continuously worsen if left untreated, provision of immediate hemostatic therapy and monitoring of its efficacy by observation of improvement in anemia and clinical manifestations is required. the first - line treatment for severe bleeding episodes, especially in patients with high titers of inhibitors, is administration of bypassing agents [63, 64 ]. activated prothrombin complex concentrates (apcc) containing factors ii (prothrombin), vii, ix, and x or recombinant activated factor vii are commonly administered and have shown to be beneficial in treating patients with aha as well as congenital hemophilia a patients with inhibitors [6366 ]. use of the immunoadsorption technique for removal of high - titer inhibitors has also proven beneficial in aha patients with acute, life - threatening bleeding. another hemostatic treatment, the provision of inhibitor - neutralizing therapy with administration of fviii concentrates at a level sufficient for neutralizing inhibitors, may also be beneficial for these patients. however, it is difficult to determine the quantity of fviii required and calculate the half - life of the infused fviii owing to the presence of type ii inhibitors in aha. in contrast, the requisite quantity of fviii for neutralizing type i inhibitor in congenital hemophilia a can be determined theoretically. therefore, frequent monitoring of hemostatic functioning accompanied by measurement of fviii : c and/or aptt should be performed while providing neutralizing therapy to aha patients. administration of desmopressin, which stimulates the release of fviii and vwf from endothelial cells and can provide a transient rise in fviii : c levels to therapeutic levels, may also be effective in aha patients with low titers of inhibitors or an fviii : c level > 5%. while desmopressin has the advantages of being of low cost and safety, it does not entirely increase fviii : c level to a therapeutic level and becomes less efficacious with repetitive administration. as neither therapy is adequate for aha patients with high titers of inhibitors or severe bleeding symptoms, a with inhibitors, suppression and eradication of inhibitors are essential for normalization of hemostatic function and elimination of the risk of hemorrhage in aha. for this, provision of immunosuppressive therapy is critical. in some cases of postpartum and drug - induced acquired hemophilia that resolves spontaneously however, even if bleeding symptoms are mild, the risk of severe and fatal hemorrhage persists unless inhibitors are eradicated. therefore, immediate initiation of immunosuppressive therapy after confirmation of aha diagnosis is recommended [7073 ]. several studies have established the effectiveness of immune - tolerance - induction therapy based on repetitive high - dose fviii infusion for the eradication of inhibitors developed in congenital hemophilia a. immune - tolerance - induction methods that have been reported to be effective for treating aha include not only administration of high - dose fviii but also immunoadsorption and immune suppression therapy, the latter of which is likely essential for therapeutic success. agents used in immunosuppressive therapy for suppression of inhibitors include immunosuppressive agents such as prednisone, azathioprine, and cyclosporine and antineoplastic agents such as cyclophosphamide (cpa), mercaptopurine, and vincristine. among these, administration of prednisone alone or in combination with cpa combined prednisone - cpa administration has been reported to yield favorable outcomes [29, 64, 76 ], indicating that combined use of immunosuppressive or antineoplastic agents and prednisolone may yield beneficial effects. high - dose intravenous immunoglobulin therapy can be provided as an adjunctive therapy but should not be used as an initial treatment. physical removal of inhibitors by plasma exchange therapy or protein a adsorption column is effective for transient removal of inhibitors in patients with acute, severe bleeding. recently, several case studies of successful treatment with chimeric monoclonal antibodies targeted against the pan - b - cell marker cd20 (rituximab) in patients refractory to initial immunosuppressive therapy have been reported. in cases where increased baff levels activate b - cells, use of a strategy to suppress b - cell activation appears rational. there is no evidence that one immunosuppressive therapy is clinically superior to all others in treating aha or that a certain therapy should be chosen depending on inhibitor titer or the hemorrhagic status. therefore, first - line treatment is determined by evaluation of disease condition and consideration of possible adverse effects. although acute hemorrhage in aha is potentially lethal, infectious diseases, such as pneumonia and sepsis, are responsible for approximately 50% of mortality associated with aha. therefore, sufficient attention to prevention and early detection of infectious disease is warranted when aggressive and prolonged immune suppression therapy is provided. aha is characterized by the presence of an autoimmune mechanism that alone or accompanied by autoimmune disease, aging, pregnancy, or drug exposure causes breakdown of immune tolerance to fviii associated with cd4 t - cells and results in development of autoantibodies against fviii. in addition to treatment for acute bleeding, which is often required for aha patients, immune suppression is essential for eradication of the inhibitors that play a central role in aha pathogenesis. while provision of immunosuppression therapies, such as combined prednisone - cpa therapy, is currently the first - line treatment, administration of anti - cd20 monoclonal antibody (rituximab) appears to be a promising alternative treatment for aha. consideration of the findings regarding the association between the autoimmune mechanism responsible for aha development and the innate immune system presented here and further elucidation of this association in future research will provide for better understanding of aha pathophysiology and the development of novel therapies for eradication of inhibitors. | acquired hemophilia a (aha) is a rare hemorrhagic disease in which autoantibodies against coagulation factor viii- (fviii-) neutralizing antibodies (inhibitors) impair the intrinsic coagulation system. as the inhibitors developed in aha are autoantibodies, the disease may have an autoimmune cause and is often associated with autoimmune disease. although acute hemorrhage associated with aha may be fatal and is costly to treat, aha is often unrecognized or misdiagnosed. aha should thus be considered in the differential diagnosis particularly in postpartum women and the elderly with bleeding tendency or prolonged activated partial thromboplastin time. cross - mixing tests and measurement of fviii - binding antibodies are useful to confirm aha diagnosis. for treatment of acute hemorrhage, hemostatic therapy with bypassing agents should be provided. unlike in congenital hemophilia a with inhibitors, in which immune tolerance induction therapy using repetitive infusions of high - dose fviii concentrates is effective for inhibitor eradication, immune tolerance induction therapy has shown poor efficacy in treating aha. immunosuppressive treatment should thus be initiated to eradicate inhibitors as soon as the diagnosis of aha is confirmed. |
sphingomonas is a group of gram - negative, rod - shaped, non - spore - forming, chemoheterotrophic, strictly aerobic bacterium that produces yellow or off - white pigmented colonies. the distinctiveness of sphingomonas lies in its possession of ubiquinone 10 as its major respiratory quinone, presence of glycosphingolipids (gdls) in their cell envelopes, and its metabolic versatility.1 the genome of sphingomonas is approximately 3,948 kb and contains 70 structural rnas. it encodes for approximately 3,914 proteins.2 sphingomonas utilize glucose as its primary source of carbon. however, it can also utilize a wide variety of other sugars such as arabinose, fucose, glactose, lactose, mannose, melibiose, sucrose, trehalose, and xylose. moreover, sphingomonas can also degrade polysaccharides, and many of the strains were also able to utilize one or more of the contaminants as their source of carbon.2 the capability of sphingomonas to utilize a wide range of organic compounds, and to grow and survive under low - nutrient conditions has resulted in its widespread distribution in various environments, including drinking water, soil, air, sinks, shower curtains, and corroding copper pipes. in addition, sphingomonas paucimobilis has also been found in several clinical specimens, which include hospital water supplies ; temperature probe respirators ; stocked distilled water ; blood ; removes ; hospital dialysis equipment ; patients with meningitis, septicemia, bacteremia, and peritonitis ; and wound infections.3 reported cases of nosocomial infections caused by s. paucimobilis are rarely serious and could be effectively treated with antibiotics. on the contrary, some other reports have concluded that s. paucimobilis nosocomial infections have the ability to severely threaten immune - compromised or ill patients causing health problems with consistent exposure to the source of infection such as shower curtains.4 for example, in 2007, reports concluded that s. paucimobilis was the cause of bacteremia outbreak in the hemato / oncology units in glhane military hospital in ankara, turkey. the reports also showed that the clinical isolates were traced back neither to the health care workers nor to the environmental isolates.5 moreover, it was strongly documented that s. paucimobilis created significant problems in various clinical settings, being the most widespread cause of nosocomial infections including bacteremia / septicemia caused by contaminated solutions such as distilled water, hemodialysis fluid, and sterile drug solutions. cases of pseudo - bacteremia have been recorded in association with s. paucimobilis, as have many cases of unusual infections both invasive and severe, eg, septic arthritis and osteomyelitis.6 moreover, s. paucimobilis caused bloodstream infection in a patient with down syndrome. it was thereby concluded that s. paucimobilis should be recognized as a nosocomial infectious agent in patients with down syndrome and immunosuppressive disorders.7 in addition, it was also reported that s. paucimobilis has the ability to cause infections in both previously healthy and immune - compromised children8 and can act as a causal agent of osteomyelitis in an immune - competent patient.9 frequent s. paucimobilis infections were observed among our diabetic foot ulcer patients (23% of observed gram - negative infections). s. paucimobilis general infection rate was 9.5%, falling just behind staphylococcus aureus (unpublished data). there is still an ongoing debate about the clinical virulence of s. paucimobilis with a possible conclusion that its clinical importance can not be neglected. henceforth, this study employs comparative genomics and bioinformatics techniques in order to investigate the pathogenic potentials of sphingomonas spp. partial 16s rdna sequences of selected pathogenic bacteria, namely, s. paucimobilis (d16144.1), bacillus anthracis (gq280034.1), bartonella bacilliformis (af442955.1), brucella sp. (ab379686.1), campylobacter jejuni (ay621112.1), clostridium difficile (hm245939.1), corynebacterium sp. (ay034821.1), legionella pneumophila (nr_041742.1), neisseria meningitidis (af059671.1), pseudomonas sp. (jn626189.1), vibrio cholerae (z21856.1), yersinia pestis (aj232235.1), were all acquired from the genbank. these sequences were then aligned using the bioedit built - in clustal w program (gap opening penalty = 10, gap extension penalty = 5, delay divergent sequences = 40%). the resulting alignments were compared, and the final alignments were improved manually and prepared in fasta and mega formats using format converter tool v2.2.5 available online at http://www.hiv.lanl.gov/content/sequence/format_conversion/form.html. in order to establish the phylogenetic relationships among taxa, tree was constructed using the maximum likelihood (ml) method based on the tamura nei model.10 the percentage of trees in which the associated taxa clustered together is shown next to the branches. initial tree(s) for the heuristic search was(were) obtained automatically by applying neighbor - joining and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach, and then the topology was selected with superior log - likelihood value. the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. evolutionary analyses were conducted in mega6.11 virulence genes sequences and functions, corresponding to different major bacterial virulence factors of selected pathogens, were collected from genbank and validated in virulence factors of pathogenic bacteria database at http://www.mgc.ac.cn / vfs/. supplementary table 1 shows the tested major pathogenic virulence factors. selected gene sequences were tested against available sphingomonas gene information using sphingomonas nucleotide blast tool available at http://blast.ncbi.nlm.nih.gov/blast.cgi?page_type=blastsearch&prog_def=blastn&blast_prog_def=megablast&blast_spec=microbialgenomes_13687&db_group=allmg. the search sphingomonas complete genomes, and selected organism was sphingomonas (tax i d : 13687). the program selected for the search was blastn, optimizes for fairly similar sequences because of evolutionary divergence of the tested and query taxa. partial 16s rdna sequences of selected pathogenic bacteria, namely, s. paucimobilis (d16144.1), bacillus anthracis (gq280034.1), bartonella bacilliformis (af442955.1), brucella sp. (ab379686.1), campylobacter jejuni (ay621112.1), clostridium difficile (hm245939.1), corynebacterium sp. (ay034821.1), legionella pneumophila (nr_041742.1), neisseria meningitidis (af059671.1), pseudomonas sp. (jn626189.1), vibrio cholerae (z21856.1), yersinia pestis (aj232235.1), were all acquired from the genbank. these sequences were then aligned using the bioedit built - in clustal w program (gap opening penalty = 10, gap extension penalty = 5, delay divergent sequences = 40%). the resulting alignments were compared, and the final alignments were improved manually and prepared in fasta and mega formats using format converter tool v2.2.5 available online at http://www.hiv.lanl.gov/content/sequence/format_conversion/form.html. in order to establish the phylogenetic relationships among taxa, tree was constructed using the maximum likelihood (ml) method based on the tamura nei model.10 the percentage of trees in which the associated taxa clustered together is shown next to the branches. initial tree(s) for the heuristic search was(were) obtained automatically by applying neighbor - joining and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach, and then the topology was selected with superior log - likelihood value. the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. virulence genes sequences and functions, corresponding to different major bacterial virulence factors of selected pathogens, were collected from genbank and validated in virulence factors of pathogenic bacteria database at http://www.mgc.ac.cn / vfs/. supplementary table 1 shows the tested major pathogenic virulence factors. selected gene sequences were tested against available sphingomonas gene information using sphingomonas nucleotide blast tool available at http://blast.ncbi.nlm.nih.gov/blast.cgi?page_type=blastsearch&prog_def=blastn&blast_prog_def=megablast&blast_spec=microbialgenomes_13687&db_group=allmg. the search sphingomonas complete genomes, and selected organism was sphingomonas (tax i d : 13687). the program selected for the search was blastn, optimizes for fairly similar sequences because of evolutionary divergence of the tested and query taxa. in this study, the presence of the major known bacterial virulence factors in sphingomonas spp. was examined. in order to decide on the accurate common pathogenic bacterial species for comparison with sphingomonas spp., a phylogenetic tree using the ml method was constructed using partial 16s rdna sequences of selected pathogenic bacteria as mentioned above (fig. the phylogenetic tree showed that the selected bacterial species are divided into two major clades (groups), namely, gram - positive bacteria and gram - negative bacteria. the gram - positive bacterial group contained clostridium spp., corynebacterium sp., and bacillus spp., whereas the gram - negative bacterial group contained the remaining bacterial species. these results also agree with the known taxonomic arrangement of the tested bacterial species with the exception of bartonella spp. more importantly, the 16s rdna phylogenetic tree showed brucella sp. to be the closest bacterial taxon to sphingomonas with a bootstrap value of 87 followed by helicobacter spp., pseudomonas sp., and then legionella sp. based on the suggested phylogenetic relationships, the following bacterial species which include brucella sp. table 1 presents the five selected bacterial genera with its corresponding species, the selected hosts, and the diseases caused by these pathogens. the selected pathogens were mainly human pathogens having also the ability to infect mammals, protozoa (legionella sp.), and plants (pseudomonas syringae). all the virulent factors acquired by these pathogens (table 2) were tested for their presence in sphingomonas genomic information. the major categories of bacterial virulence factors such as adherence, endotoxin, mobility, secretion systems, quorum sensing, and many others are shown in (table 2). table 3 and figure 2 present the shared virulence factors among sphingomonas and the selected five bacterial pathogens. shares the genes responsible for intracellular survival ability (cgs, manc, and pgm) with brucella sp. with e - values ranging from 0 to 3.00e 09.1214 in addition, sphingomonas spp. shares the genes encoding for type iv secretion system such as bmeii0026 with brucella sp. with e - value of 6.00e 04 and identity similarity of 90%. on the contrary, sphingomonas spp. shared no virulence factors with helicobacter spp. or campylobacter sp., despite their close phylogenic relationship when compared to pseudomonas sp. and legionella sp. the sodb encodes for superoxide dismutase, which is a cytoplasmic iron superoxide dismutase important for intracellular survival and transmission.15 regardless of the phylogenetic divergence between sphingomonas spp. and, it was observed from our results that they shared several major virulence factors such as adherence, antiphagocytosis, iron uptake, proteases, quorum sensing, and others. with regard to adherence, they shared 20 genes, including flgk with e - value of 1.00e 04 and identity similarity of 94%, and flgj with e - value of 6.00e 05 and identity similarity of 89%. flagella plays an important role as a virulence factor such as in swimming motility toward the infection site and a role in biofilm formation and other pathogenic adaptations.1620 type iv pili play an important role in adherence by assisting the pathogens to attach with their host cells and causing a twitching motility that allows the bacteria to move along the cell surface, and in biofilm formation.17,2125 both sphingomonas spp. and pseudomonas sp. shared many genes implicated in type iv pili biogenesis and mechanical function of pili, such as pilf with e - value of 4.00e 05 and identity similarity of 97%, and pilb with e - value of 3.00e 04 and identity similarity of 93%. they shared 10 alginate genes, including algj with e - value of 7.00e 04 and identity similarity of 94%, and algz with e - value of 5.00e 05 and identity similarity of 91%. the production of alginate permits pathogenic bacteria to form biofilm and contributes to the persistence of bacteria in the lung by acting as an adhesin, which prevents the bacteria from being expelled from the infection site, and the alginate slime layer makes it more difficult for phagocytes to ingest and kill the bacteria.2630 another important bacterial virulence factor shared between sphingomonas spp. and pseudomonas sp. is quorum sensing.. showed acquiring of both rhll and rhlr with e - values of 3.00e 4 and 5.00e 9, respectively., quorum sensing consists of two separate but interrelated systems, namely, las and rhl, which are found to regulate the production of multiple virulence factors and are also crucial for proper biofilm formation.3133 it is also worth mentioning that both sphingomonas spp. and pseudomonas sp. share seven genes encoding for xcp secretion system (type ii secretion system), including xcpx and xcpr with e - values of 1.00e 5 and 3.00e 174, respectively. the xcp secretion system is found to be responsible for secretion of toxins and enzymes into the extracellular fluid.34,35 it was also observed that both sphingomonas spp. and pseudomonas sp. shared many genes involved in iron uptake using both pyochelin (10 genes) and pyoverdine (4 genes). the pyochelin is effective at promoting iron uptake in pseudomonas aeruginosa, catalyzes the formation of tissue - damaging free radicals, and also binds other transition metals (eg, mo(iv), co(ii)) with appreciable affinity, and is also implicated in the delivery of both co(ii) and mo(iv) to p. aeruginosa cells.36,37 the pyoverdine is effective at acquiring iron from transferrin and lactoferrin. moreover, pyoverdine is cytotoxic because of its ability to stimulate the production of reactive oxygen species.38,39 interestingly, sphingomonas spp. and pseudomonas sp. shared only one gene responsible for toxin production, namely, plch that is responsible for degrading the phospholipids surfactant, which functions to reduce the surface tension so that the alveoli do not collapse completely when air leaves them during breathing. it is noteworthy that sphingomyelin (plch is a multifunctional enzyme) has been isolated from pseudomonas aeruginosa in 2003.40,41 our comparative analysis was further extended to search for other bacterial toxins that sphingomonas spp. table 4 shows other toxins that were found to be shared between sphingomonas spp. and b. pertussis is a strict human pathogen causing whooping cough, a highly contagious respiratory disease marked by severe, spasmodic coughing episodes.42 it was also observed that sphingomonas spp. contains genes for invasive adenylate cyclase / hemolysin, cyclolysin secretion protein, which is a bifunctional toxin harboring both adenylate cyclase and hemolytic activities, and functions primarily as an anti - inflammatory factor.4345 moreover, sphingomonas spp. contains genes responsible for pertussis toxin and its secretion system, which assists in the attachment of b. pertussis to ciliated respiratory cells, important immunogen and activate cyclic adenosine phosphate (camp), histamine sensitizing factor (hsf), lymphocytosis promoting factor (lpf), islet - activating protein (iap) ; interferes with leucocyte function ; and is hemolytic.46,47 contains several major virulence factors, mainly resembling those of pseudomonas sp. other virulence factors from legionella sp. these findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor. we selected sphingomonas spp. to test its potentiality for being potential virulent pathogen.we tested the phylogenetic relationship of sphingomonas spp. against known virulent pathogens.we screened the presence of various virulent factors from different virulent pathogens in sphingomonas spp.sphingomonas spp. contains several major virulence factors, mainly resembling those of pseudomonas sp. we selected sphingomonas spp. to test its potentiality for being potential virulent pathogen. we screened the presence of various virulent factors from different virulent pathogens in sphingomonas spp. | there is an ongoing debate about the clinical significance of sphingomonas paucimobilis as a virulent bacterial pathogen. in the present study, we investigated the presence of different virulence factors and genes in sphingomonas bacteria. we utilized phylogenetic, comparative genomics and bioinformatics analysis to investigate the potentiality of sphingomonas bacteria as virulent pathogenic bacteria. the 16s ribosomal rna gene (16s rdna) phylogenetic tree showed that the closest bacterial taxon to sphingomonas is brucella with a bootstrap value of 87 followed by helicobacter, campylobacter, pseudomonas, and then legionella. sphingomonas shared no virulence factors with helicobacter or campylobacter, despite their close phylogenic relationship. in spite of the phylogenetic divergence between sphingomonas and pseudomonas, they shared many major virulence factors, such as adherence, antiphagocytosis, iron uptake, proteases, and quorum sensing. in conclusion, sphingomonas spp. contains several major virulence factors resembling pseudomonas sp., legionella sp., brucella sp., and bordetella sp. virulence factors. similarity of virulence factors did not match phylogenetic relationships. these findings suggest horizontal gene transfer of virulence factors rather than sharing a common pathogenic ancestor. sphingomonas spp. is potential virulent bacterial pathogen. |
let dj, j=1,,m, mn, be bounded and open subsets of rn, n=2,3, such that rndj are connected and assume that they are disjoint.,m, of dj is of class c2 and the unit normal vector is directed into the exterior of dj. finally, we set dj=1mdj. we denote by the density function such that =1 in rnd, continuous inside d and has a discontinuity across d. we also denote by and the lam coefficients and we assume that those coefficients are constant in rn and satisfy the conditions >0 and 2+3>0. we are concerned with the scattering problem of elastic waves by the obstacle d at a fixed frequency. precisely, if ui, which is a vector field satisfying ui+(+)divui+2ui=0 in rn, is the incident field, then the total field utui+u, with uc2(rnd) as the scattered field, is the solution to the following inhomogeneous problem associated with the lam system (1){ut+(+)divut+2ut=0,in rnlim|x||x|n12(up|x|ikpup)=0,andlim|x||x|n12(us|x|iksus)=0, where the last two limits are uniform in all directions xx|x|sn1 the unit sphere in rn. here, we denoted upkp2divu to be the longitudinal (or the pressure) part of the field u and usks2curl curl u to be the transversal (or the shear) part of the field u corresponding to the helmholtz decomposition u = up+us. the constants kp2+ and ks are known as the longitudinal and the transversal wavenumbers, respectively. it is well known that the scattering problem (1) is well posed ; see for instance [13 ]. the scattered field u satisfies the following asymptotic expansion at infinity (2)u(x)eip|x||x|n12up(x)+eis|x||x|n12us(x)+o(1|x|n+12),|x| uniformly in all directions xsn1 ; see for instance. the fields up(x) and us(x) defined on sn1 are called the longitudinal and transversal parts of the far field pattern, respectively. the longitudinal part up(x) is normal to sn1 while the transversal part us(x) is tangential to sn1. due to this property note that it is not necessarily true for near field measurements. in this case, see for an approximate separation of these two components. now, we specify the type of incident waves used in this work. as usual in the scattering problems, we use plane waves as incident waves. for the lam system, they have the analytic forms (3)ui, p(x,)eipxandui, s(x,)eisx, where is any vector in sn1 orthogonal to. remark that ui, p(,) is normal to sn1 and ui, s(,) is tangential to sn1. hence, we can define the matrix (4)(ui, p, ui, s)f(ui, p, ui, s)[up,p(,)up,s(,)us,p(,)us,s(,), ] where 1.(up,p(,),us,p(,)) is the far field pattern associated with the pressure incident field ui, p(,).2.(up,s(,),us,s(,)) is the far field pattern associated with the shear incident field ui, s(,). (up,p(,),us,p(,)) is the far field pattern associated with the pressure incident field ui, p(,). (up,s(,),us,s(,)) is the far field pattern associated with the shear incident field ui, s(,). in this paper, we are interested in the following inverse scattering problem. from the knowledge of the matrix (4) for all directions x and in sn1, determine d. several works have been published regarding this inverse problem, see for instance [68 ] using the full matrix (4) for all directions x and in sn1. for near field measurements, we also mention the works [1417 ] regarding small obstacles and for imaging extended obstacles. we consider now the cavity problem (5){u+(+)divu+2u=0,in rnd(u)=(ui),on dlim|x||x|n12(up|x|ikpup)=0,andlim|x||x|n12(us|x|iksus)=0, where (u)(2+div+curl)u. this problem is well posed, see, and we have a similar asymptotic behavior as (2). with this at hand, we state the similar inverse problem as for the inhomogeneous medium case. it says that every column of the matrix (4) for all directions x and in sn1, determines d. sampling type methods for solving this obstacle inverse scattering problem have been developed by several authors, see using the full matrix (4) for all directions x and in sn1. we remark that in the above works, not only the information over all directions of incidence and observation, but also both pressure and shear parts of the far field pattern are needed. in, we proved that it is possible to reduce the amount of data for detecting d as follows. theorem 1.1every component of the matrix (4) known for all directions x and in sn1, determines d for the model (5). every component of the matrix (4) known for all directions x and in sn1, determines d for the model (5). remark that in theorem 1.1, we need only the longitudinal part (or only the transverse part) of the far field pattern if we use longitudinal incident waves or transversal incident waves. the result in theorem 1.1 is also valid for the inhomogeneous medium model (1). the proof is based on the asymptotic expansion of the singular solutions of the models in (1) and (5), see for the impenetrable case and for the penetrable case related to the scalar equations. first, we would like to propose numerical methods corresponding to the uniqueness result in theorem 1.1. second, we would like to see whether the choice of the type of incident field is relevant or not. for this, we restrict ourselves to the case of point - like obstacles for which more explicit calculations can be done. note that none of the known methods (iterative, sampling, probe, etc.) have been applied for detection by elastic waves using the reduced amount of data mentioned in theorem 1.1. to our knowledge, the only result considering the use of one type of elastic scattered waves for the detection is the one in who used p incident waves and the p part of the scattered waves to detect point - like obstacles. however, no mathematical justification, as in theorem 1.1 or in theorem 3.1, was given there. using a music type algorithm, we show that indeed one type of waves is enough for the reconstruction. in addition, using s incident waves we obtain better resolution than when using p incident waves in the presence of noise. this can be explained by the fact that the s incident waves have shorter wavelengths than the p incident waves. we note that, since we make use of a weak scattering model to simulate the measured data, it is not physically meaningful to apply this model to the case of close scatterers. therefore, the notion of resolution in this paper should be understood as the minimum distance between two point - like scatterers that can be resolved by the algorithm in the presence of measurement noise. however, we should remark that this weak scattering assumption is merely for the simplicity of the forward modeling. in a future work, we will investigate the resolution of the music type algorithms using a more physically meaningful model which can be used also for close scatterers. the rest of the paper is organized as follows. in section 2, we describe briefly the scattering of point - like obstacles including weak (born) approximation. section 3 is devoted to the music algorithms for scalar and elastic waves. finally, section 4 shows numerical examples of the music algorithms and to confirm our discussions on the resolution limits. suppose that they are illuminated by an incident plane elastic wave ui(x,),xrn,sn1. as described in the introduction, here ui(x,)=ui, p(x,) or ui(x,)=ui, s(x,). as it is shown in [24, section 8.4 ], the total scalar field ut corresponding to the scalar model (acoustic model for instance) is written as follows(6)ut(x)=ui(x)+m=1mmut(ym)(x, ym), where ui is the incident scalar field and is the fundamental solution of the associated helmholtz model. schwinger equation by replacing the source, given by the density in each dm, m=1,,m, by m(ym). here, is the dirac measure. following this approach, using the lippmann schwinger equation corresponding to problem (1), under the assumption that the lam coefficients and are constant in rn, the total vector field corresponding to the lam system can be described as follows(7)ut(x)=ui(x)+m=1mmg(x, ym)ut(ym), where ui is the incident vector field and g is the fundamental tensor associated with the lam system. the constant mc,m0, represents the scattering strength of the m - th scatterer dm. the main difficulty in using the model (7) to generate the far field is the calculation of u(yj). this is due to the singularities of g on the points yj, see for more details. to avoid this however, we should note that the music type algorithms are applicable for the nonlinear model (7) since the proofs of theorem 4.1 of and theorem 3.1 are also valid for this case. for results using the scalar model (6), a current work is being carried out for the elastic model (7) and we will discuss this in a future work. assume that there is no multiple scattering between the scatterers (born approximation), then the scattered wave can be written in the form(8)u(x,)=m=1mmg(x, ym)ui(ym,), by replacing in the right hand side of (7)ut by ui. the asymptotic behavior of the green tensor at infinity is given as follows(9)g(x, ym)=apxxeikp|x||x|n12eikpxym+as(ixx)eiks|x||x|n12eiksxym+o(|x|n+12), with x=x|x| and i being the identity matrix in rn, ap = kp242 and as = ks242, see for instance. it follows from (8) and (9) that the p and s parts of the far field pattern associated with the p incident wave ui, p are given by(10)up,p(x,)=apm=1mm(xx)eikpym(x),(11)us,p(x,)=asm=1mm(ixx)eikpymeiksymx. similarly, the p and s parts of the far field pattern associated with the s incident wave ui, s can be written as(12)us,p(x,)=apm=1mm(xx)eiksymeikpymx,(13)us,s(x,)=asm=1mm(ixx)eiksym(x). here we have used the subscripts p and s to represent the p and s parts of the far field pattern and the superscripts p and s to represent the p and s incident waves, respectively. the main difficulty in using the model (7) to generate the far field is the calculation of u(yj). this is due to the singularities of g on the points yj, see for more details. to avoid this however, we should note that the music type algorithms are applicable for the nonlinear model (7) since the proofs of theorem 4.1 of and theorem 3.1 are also valid for this case. for results using the scalar model (6), a current work is being carried out for the elastic model (7) and we will discuss this in a future work. assume that there is no multiple scattering between the scatterers (born approximation), then the scattered wave can be written in the form(8)u(x,)=m=1mmg(x, ym)ui(ym,), by replacing in the right hand side of (7)ut by ui. the asymptotic behavior of the green tensor at infinity is given as follows(9)g(x, ym)=apxxeikp|x||x|n12eikpxym+as(ixx)eiks|x||x|n12eiksxym+o(|x|n+12), with x=x|x| and i being the identity matrix in rn, ap = kp242 and as = ks242, see for instance. it follows from (8) and (9) that the p and s parts of the far field pattern associated with the p incident wave ui, p are given by(10)up,p(x,)=apm=1mm(xx)eikpym(x),(11)us,p(x,)=asm=1mm(ixx)eikpymeiksymx. similarly, the p and s parts of the far field pattern associated with the s incident wave ui, s can be written as(12)us,p(x,)=apm=1mm(xx)eiksymeikpymx,(13)us,s(x,)=asm=1mm(ixx)eiksym(x). here we have used the subscripts p and s to represent the p and s parts of the far field pattern and the superscripts p and s to represent the p and s incident waves, respectively. the first music algorithm for determining the locations of point - like scatterers was first developed in in 2000 using near field measurements of electromagnetic waves. so far, several works have studied this type of algorithms for both near field and far field measurements and for different types of waves. for the elasticity, ammari. used the music algorithm with full green s matrix as the measurements to reconstruct the locations of small inclusions and simonetti showed some numerical results using a music algorithm for only one part (s or p) of the scattered waves. in this paper, we also use the music type algorithms for reconstructing the locations of the scatterers but using only one part of the far field patterns and one type of incident plane waves as described in the previous section. the idea is to convert the vector - type far field pattern to scalar one and make use of the music algorithm for scalar waves with some modifications. we first briefly recall the classical music algorithm for scalar waves with far field measurements in the next subsection. consider the scattering of acoustic wave by point - like scatterers associated with incident plane wave ui(x,)=eikx, where k is the wavenumber and sn1 is the direction of incidence. then under the assumption of weak scattering, it follows from (6) that the far field pattern can be given in (14)u(x,)=m=1mmeikym(x),xsn1.,m, of the scatterers from the measured far field pattern u(x,) for a finite set of incidence and scattered directions, i.e., x,{j, j=1,,n}sn1. here we assume that the number of scatterers is not larger than the number of incidence (and observation) directions, i.e., nm. given the measured far field pattern, we define the multistatic response matrix fcnn by (15)fj, l = u(j,l)=m=1mmeikym(lj). in order to determine the locations ym for each point z, we define the vector zcn by (16)z=(eikz1,eikz2,,eikzn)t. the use of the music algorithm is based on the property that z is in the range r(f) of f iff z is at one of the locations of the scatterers. that is, z{y1,y2,,ym } iff pz=0, where p is the projection onto the null space n(f)=r(f) of the adjoint matrix f of f[25, chapter 4 ]. in applying the music algorithm for elastic waves, we have noticed that care must be taken in designing measurement setups as well as some modifications are needed in forming the multistatic response matrix. for example, if we use the p part of the far field patterns of the p incident plane waves, i.e., up,p, it is clear that the measured data vanishes in the directions orthogonal to the incidence direction. that is, the measured data in these directions are useless. more generally, the information contained in the far field patterns is proportional to |x| the cosine of the angle between the incidence and observation directions. therefore, to obtain usable data, the measurement system should be set up in such a way that |x|>0. with this system setup, given the p part of the far field patterns, we can calculate the scalar far field pattern (17)u(x,)=up,p(x,)ap(x)2=m=1mmeikpym(x). in this case, we can use the same algorithm as in the scalar case to find the locations of the scatterers, with z in (16) being replaced by the test vector (18)zp=(eikpz1,eikpz2,,eikpzn)t which corresponds to the longitudinal far field of the p part of a point source located at z. the case of s incident waves and s part of the far field patterns is treated in the same way by using the test vector (19)zs=(eiksz1,eiksz2,,eikszn)t, which represents the transversal far field of the s part of the point source. now consider the mixed cases, i.e., s incident waves and p part of the far field patterns or p incident waves and s part of the far field patterns. by similar arguments as above, we note that the observation directions should not be parallel or anti - parallel to the incidence directions. in these cases, modifications are needed in applying the music algorithm since we have the presence of both s and p wavenumbers ks and kp. similar to the above case, we assume that |x|>0. under this assumption, we can calculate from the p far field pattern (12) the following modified multistatic response matrix fcnn by (20)fj, l=m=1mmeiksymleikpymxj with l, l=1,,n, the observation directions. note that this modified multistatic response matrix is different from the scalar one due to the presence of two different s wavenumber ks and p wavenumber kp. following the same arguments as in, we factorize the matrix f as (21)f=hpths, where hp and hs are matrices in cmn defined by (22)hmjp=|m|eikpymxj, hmjs=|m|eiksymj, m=1,,m;j=1,,n. now for each sampling point zrn, we also make use of the test vector zs defined by (19). as in the scalar case, the key properties of the music algorithm are (i) the vector zs belongs to r(hs) iff z{y1,,ym } and (ii) the range r(ff) of ff coincides the range r(hs) of hs. they are proved in the following theorem. theorem 3.1suppose that the sets { n, nn}sn1 and { xn, nn}sn1 are dense on sn1 in the sense that any analytic function on sn1that vanishes on one of these sets vanishes on the whole sn1. let k be a compact set of rn containing { ym, m=1,,m }. then there exists a number n0n such that for all nn0, the following properties are satisfied(i)z{y1,y2,,ym } iff zsr(hs) for zk.(ii)r(ff)r(hs).therefore, z{y1,y2,,ym } iff zsr(ff) or equivalently, pzs=0, where p is the projection onto the null space of the self - adjoint matrix ff. suppose that the sets { n, nn}sn1 and { xn, nn}sn1 are dense on sn1 in the sense that any analytic function on sn1that vanishes on one of these sets vanishes on the whole sn1. let k be a compact set of rn containing { ym, m=1,,m }. then there exists a number n0n such that for all nn0, the following properties are satisfied(i)z{y1,y2,,ym } iff zsr(hs) for zk.(ii)r(ff)r(hs).therefore, z{y1,y2,,ym } iff zsr(ff) or equivalently, pzs=0, where p is the projection onto the null space of the self - adjoint matrix ff. z{y1,y2,,ym } iff zsr(hs) for zk. the only difference is that in this case we make use of two different sets of incidence and observation directions. using the same arguments as of the mentioned theorem, we can prove first that there exists a number n1n such that the vectors y1s,,yms,zs are linearly independent for nn1 and zk{y1,,ym } and the point (i) of the theorem exactly follows from the proof of theorem 4.1 of.now consider the point (ii). equivalently, hs is surjective from cn to cm.concerning the matrix hp, we define the following vector (24)zp=(eikpzx1,eikpzx2,,eikpzxn)t. following the same arguments of the point (i), we also can prove that there exists a number n2n such that the vectors y1p,,ymp are linearly independent for nn1. now for nn0=max{n1,n2 }, there exists ycn such that x = thsy as hs is surjective. that is y = hsthsyr(ff). the only difference is that in this case we make use of two different sets of incidence and observation directions. using the same arguments as of the mentioned theorem, we can prove first that there exists a number n1n such that the vectors y1s,,yms,zs are linearly independent for nn1 and zk{y1,,ym } and the point (i) of the theorem exactly follows from the proof of theorem 4.1 of. concerning the matrix hp, we define the following vector (24)zp=(eikpzx1,eikpzx2,,eikpzxn)t. following the same arguments of the point (i), we also can prove that there exists a number n2n such that the vectors y1p,,ymp are linearly independent for nn1. now for nn0=max{n1,n2 }, there exists ycn such that x = thsy as hs is surjective. that is y = hsthsyr(ff). remark 3.1instead of the test vector zs, we can also use the vector zp for the music algorithm. however, in this case, the matrix ff must be replaced by ff. instead of the test vector zs, we can also use the vector zp for the music algorithm. consider the scattering of acoustic wave by point - like scatterers associated with incident plane wave ui(x,)=eikx, where k is the wavenumber and sn1 is the direction of incidence. then under the assumption of weak scattering, it follows from (6) that the far field pattern can be given in (14)u(x,)=m=1mmeikym(x),xsn1.,m, of the scatterers from the measured far field pattern u(x,) for a finite set of incidence and scattered directions, i.e., x,{j, j=1,,n}sn1. here we assume that the number of scatterers is not larger than the number of incidence (and observation) directions, i.e., nm. given the measured far field pattern, we define the multistatic response matrix fcnn by (15)fj, l = u(j,l)=m=1mmeikym(lj). in order to determine the locations ym for each point z, we define the vector zcn by (16)z=(eikz1,eikz2,,eikzn)t. the use of the music algorithm is based on the property that z is in the range r(f) of f iff z is at one of the locations of the scatterers. that is, z{y1,y2,,ym } iff pz=0, where p is the projection onto the null space n(f)=r(f) of the adjoint matrix f of f[25, chapter 4 ]. in applying the music algorithm for elastic waves, we have noticed that care must be taken in designing measurement setups as well as some modifications are needed in forming the multistatic response matrix. for example, if we use the p part of the far field patterns of the p incident plane waves, i.e., up,p, it is clear that the measured data vanishes in the directions orthogonal to the incidence direction. that is, the measured data in these directions are useless. more generally, the information contained in the far field patterns is proportional to |x| the cosine of the angle between the incidence and observation directions. therefore, to obtain usable data, the measurement system should be set up in such a way that |x|>0. with this system setup, given the p part of the far field patterns, we can calculate the scalar far field pattern (17)u(x,)=up,p(x,)ap(x)2=m=1mmeikpym(x). in this case, we can use the same algorithm as in the scalar case to find the locations of the scatterers, with z in (16) being replaced by the test vector (18)zp=(eikpz1,eikpz2,,eikpzn)t which corresponds to the longitudinal far field of the p part of a point source located at z. the case of s incident waves and s part of the far field patterns is treated in the same way by using the test vector (19)zs=(eiksz1,eiksz2,,eikszn)t, which represents the transversal far field of the s part of the point source. now consider the mixed cases, i.e., s incident waves and p part of the far field patterns or p incident waves and s part of the far field patterns. by similar arguments as above, we note that the observation directions should not be parallel or anti - parallel to the incidence directions. in these cases, modifications are needed in applying the music algorithm since we have the presence of both s and p wavenumbers ks and kp. similar to the above case, we assume that |x|>0. under this assumption, we can calculate from the p far field pattern (12) the following modified multistatic response matrix fcnn by (20)fj, l=m=1mmeiksymleikpymxj with l, l=1, note that this modified multistatic response matrix is different from the scalar one due to the presence of two different s wavenumber ks and p wavenumber kp. following the same arguments as in, we factorize the matrix f as (21)f=hpths, where hp and hs are matrices in cmn defined by (22)hmjp=|m|eikpymxj, hmjs=|m|eiksymj, m=1,,m;j=1,,n. now for each sampling point zrn, we also make use of the test vector zs defined by (19). as in the scalar case, the key properties of the music algorithm are (i) the vector zs belongs to r(hs) iff z{y1,,ym } and (ii) the range r(ff) of ff coincides the range r(hs) of hs. they are proved in the following theorem. theorem 3.1suppose that the sets { n, nn}sn1 and { xn, nn}sn1 are dense on sn1 in the sense that any analytic function on sn1that vanishes on one of these sets vanishes on the whole sn1. let k be a compact set of rn containing { ym, m=1,,m }. then there exists a number n0n such that for all nn0, the following properties are satisfied(i)z{y1,y2,,ym } iff zsr(hs) for zk.(ii)r(ff)r(hs).therefore, z{y1,y2,,ym } iff zsr(ff) or equivalently, pzs=0, where p is the projection onto the null space of the self - adjoint matrix ff. suppose that the sets { n, nn}sn1 and { xn, nn}sn1 are dense on sn1 in the sense that any analytic function on sn1that vanishes on one of these sets vanishes on the whole sn1. let k be a compact set of rn containing { ym, m=1,,m }. then there exists a number n0n such that for all nn0, the following properties are satisfied(i)z{y1,y2,,ym } iff zsr(hs) for zk.(ii)r(ff)r(hs).therefore, z{y1,y2,,ym } iff zsr(ff) or equivalently, pzs=0, where p is the projection onto the null space of the self - adjoint matrix ff. z{y1,y2,,ym } iff zsr(hs) for zk. the only difference is that in this case we make use of two different sets of incidence and observation directions. using the same arguments as of the mentioned theorem, we can prove first that there exists a number n1n such that the vectors y1s,,yms,zs are linearly independent for nn1 and zk{y1,,ym } and the point (i) of the theorem exactly follows from the proof of theorem 4.1 of.now consider the point (ii). equivalently, hs is surjective from cn to cm.concerning the matrix hp, we define the following vector (24)zp=(eikpzx1,eikpzx2,,eikpzxn)t. following the same arguments of the point (i), we also can prove that there exists a number n2n such that the vectors y1p,,ymp are linearly independent for nn1. now for nn0=max{n1,n2 }, there exists ycn such that x = thsy as hs is surjective. the only difference is that in this case we make use of two different sets of incidence and observation directions. using the same arguments as of the mentioned theorem, we can prove first that there exists a number n1n such that the vectors y1s,,yms,zs are linearly independent for nn1 and zk{y1,,ym } and the point (i) of the theorem exactly follows from the proof of theorem 4.1 of. concerning the matrix hp, we define the following vector (24)zp=(eikpzx1,eikpzx2,,eikpzxn)t. following the same arguments of the point (i), we also can prove that there exists a number n2n such that the vectors y1p,,ymp are linearly independent for nn1. now for nn0=max{n1,n2 }, there exists ycn such that x = thsy as hs is surjective. that is y = hsthsyr(ff). remark 3.1instead of the test vector zs, we can also use the vector zp for the music algorithm. however, in this case, the matrix ff must be replaced by ff. instead of the test vector zs, we can also use the vector zp for the music algorithm. in this section, we illustrate the performance of the music algorithm for elasticity using one type of wave and compare the results for the case of s and p incident plane waves. it is expected that, since they have shorter wavelength, the s incident waves should provide sharper results compared to the p incident waves. this is confirmed in the following numerical examples. for the convenience in visualizing the results, we should mention that the algorithm in two and three dimensional spaces are the same. but we are aware that the three dimensional case is a bit more complicated since there are two linearly independent directions for the transverse waves. as we have mentioned in the previous section, care must be taken in setting up the measurements to avoid small values of the measured far field patterns. for this purpose, we propose the following setups in two situations. for p incident waves and p part of far field patterns (pp case), or s incident waves and s part of far field patterns (ss case) denote by nd the number of incidence directions (angles) used in a quarter of the unit circle. in the first and the third quarter, we use the following incidence angles (see fig.,nd, and in the second and the fourth quarter, we make use of the incidence angles nd+j=2+4nd+(j1)2nd,3nd+j=32+4nd+(j1)2nd, j=1,,nd., we have |x|sin(4nd) for all x,{j, j=1,,4nd}. to avoid parallel or anti - parallel directions in the case of p incidence waves and s part of far field patterns (ps case) or s incidence waves and p part of far field patterns (sp case), we choose the incidence and observation angles as follows (fig., the minimum angle between the incidence and observation angles is 4nd. in the following examples, the parameters are chosen as =2, =1 and k=2 resulting in kp= and ks=2. let us first consider four point - like scatterers located at y1=(0,0), y2=(0,0.5), y3=(1,1) and y4=(1,1). since the music algorithm is an exact method, see theorem 3.1 (see also), the reconstruction is very accurate if there is no noise in the measured data. in this paper, we concentrate on the resolution of the algorithm in the case of noisy data. to analyze the effect of noise level on the resolution of the algorithm, 24 show the pseudo spectrum of the scatterers with 1%, 5% and 10% random noise in the measured far field patterns, respectively. we should emphasize that, by converting from the vector far field patterns to the scalar one as in (17), the noise in the measured far field patterns is amplified in the modified multistatic response matrix resulting worse results than the scalar case. 2 shows good reconstructions for all scatterers in both pp and sp cases even though in the latter case the peaks are a bit sharper at the locations y1 and y2. in fig. 3, with 5% noise in the data, the two scatterers at y1 and y2 are not well separated anymore in the pp case but they are still very well separated in the sp case. here the two close scatterers are still clearly visible in the sp case but not anymore distinguishable in the pp case. these results show that using the s incident waves we can obtain better resolution with the music algorithm than using the p incident waves. 5 shows that, with nine scatterers, the scatterers close to each other (at the distance of about one fourth of the wavelength) are hardly or even impossible to be separated in the pp case although the noise level is only 5%, but they are still distinguishable in the sp case. however, even in the sp case, the result is less accurate than the previous example of four scatterers. actually, the higher the number of scatterers, the lower the accuracy in both cases. finally, we should mention that the reconstruction results depend on the choice of the signal and noise subspaces of the multistatic response matrix. for small measurement noise, these two subspaces are easy to choose since there is a clear cut in the distribution of the singular values of the multistatic response matrix. however, for large noise, the distribution of the singular values are smooth and it becomes more difficult to separate the singular values of the signal and noise subspaces ; see fig. 6 for the pp case with four scatterers. in this paper, since we want to compare the pp and sp cases, the singular values were separated manually which is based on the true number of point - like scatterers. as a conclusion, we can say that using the s incident waves provides more accurate reconstruction of the locations of point - like scatterers using the music algorithm compared to the p incident waves. moreover, the larger the lam parameter, the better the reconstruction with the s incident waves compared to the p incident waves since, in this case, the wavelength of the p - incident wave is much larger than the one of the s - incident wave. it is worth mentioning that from the numerical tests, we observed that the p and s parts of far field patterns, for a given incident wave, provide almost the same resolution. however, the magnitudes of the peaks may be different using p or s parts of the far field patterns. this may result in a better or worse reconstruction quality | in this paper, we are concerned with the detection of point - like obstacles using elastic waves. we show that one type of waves, either the p or the s scattered waves, is enough for localizing the points. we also show how the use of s incident waves gives better resolution than the p waves. these affirmations are demonstrated by several numerical examples using a music type algorithm. |
patients with liver cirrhosis (lc) induced by chronic hepatitis b (chb) are at high risk of developing hepatocellular carcinoma (hcc) [13 ]. the proportion of people chronically infected with hepatitis b virus (hbv) is about 350 million people worldwide. the lifetime risk of hbv carriers to develop cirrhosis is estimated to be more than 15%. at present, liver biopsy is still the golden standard for the evaluation of liver fibrosis and cirrhosis. although histological assessment provides valuable information on the degree of necroinflammation and fibrosis in such patients, it is an invasive procedure associated with a finite albeit small risk of severe complications of 0.5%, patient discomfort, and expense. moreover, a liver biopsy does not provide information regarding the balance between production and destruction of the extracellular matrix (ecm) or the rate of progression to cirrhosis. over the past decade, attempts have been made to develop noninvasive methods to assess lc, including physical approaches and biological approaches. transient elastography (te), a recently developed noninvasive technique based on physical approach, has proved to have high diagnostic accuracy for lc [9, 10 ]. however, the accuracy of te is highly dependent on the experience of operators and clinicians, and its applicability is not as good as that of serum biomarkers with limitation in some patients, such as pacemakers, defibrillators, or overweight patients. up to now, about 20 numerical scores or indices are reported mostly based on the routine laboratory parameters. some models such as fibrotest and ast to platelet ratio index (apri) have been proposed for clinical application in patients with chronic hepatitis c (chc) [12, 13 ]. however, some models based on chc patients may not be suitable for predicting significant fibrosis and cirrhosis in hepatitis b - related fibrosis and can not reduce the number of liver biopsies. recently, a validation study which examined 13 panels of indirect blood markers in chb patients, including fib-4, apri, and forns, demonstrated that the performance for predicting liver fibrosis in chb patients had yet to be improved. consequently, the objective of this work was to construct and evaluate a new classifier for predicting liver cirrhosis in chb patients using supervised machine learning methods based on the routine clinical parameters. from october 2010 to march 2013, a total of 239 subjects were collected, comprising 124 lc patients with chb and 115 patients with chb. all patients were admitted to jinan military general hospital and changhai hospital of second military medical university. this study was conducted in accordance with the declaration of helsinki and approved by the ethical committees of the hospitals mentioned above. both hbv - induced chronic hepatitis and liver cirrhosis after hbv infection were diagnosed depending on the criteria established by the chinese medical association (chinese society of hepatology and chinese society of infectious diseases). all the participants were not coinfected with hiv or hepatitis c. patients were excluded if they consumed > 5 g of alcohol per day on average or were taking intravenous drugs or antihypertensive medications. in addition, patients with evidence of a concurrent liver disorder such as primary biliary cirrhosis, autoimmune hepatitis, hcc, and wilson 's disease were excluded from this study. an abdominal ultrasound scan was carried out on these patients to confirm normal hepatobiliary anatomy and to exclude biliary obstruction and hepatic space - occupying lesions. clinical characteristics and laboratory parameters of patients were documented, including age, gender, total bilirubin (tbil), creatinine (cre), prothrombin time (pt), albumin (alb), platelet count (plt), alanine transaminase (alt), aspartate aminotransferase (ast), alkaline phosphatase (akp), red cell distribution width (rdw), hemoglobin (hgb), and mean corpuscular volume (mcv). for the patients with liver cirrhosis, the child - pugh score was calculated, as previously described. only the clinical characteristics and laboratory parameters of the first admission were recorded for the patients receiving hospital care more than once between october 2010 and march 2013. similarly, the results of the first measurement were accepted regarding serial laboratory tests during hospitalization. the aim of this study was to construct and evaluate the classifiers for prediction of liver cirrhosis with chronic hepatitis b based on the mentioned 13 routinely available clinical parameters. the training set consisting of 120 patients (50.2%) was used to build the classifier. the remaining 119 patients (49.8%) were used to construct the test set for validation. waikato environment for knowledge analysis (weka) was employed for feature selection and classifier construction. based on the training set, variable selection was performed by using the geneticsearch - based strategy in weka. a list of clinical parameters sorted along the statistical difference between the two classes (e.g., lc and chb) was obtained, which was used for classifier construction. classifiers were constructed based upon the training set using multilayered perceptron (mlp) and nave bayes (nb) method in weka. a 10-fold cross - validation was performed to avoid model - specific overfitting, as previously described. briefly, all the entries were randomly divided into ten parts ; nine sets were used for training and the remaining one for testing. the process was repeated ten times and the accuracy for true, false, and total accuracy calculated. the final accuracy is the average of the accuracy in all ten tests. to evaluate the generalization performance of the two classifiers, in the next step accuracy (acc), sensitivity (se), specificity (sp), positive and negative predictive values, and likelihood ratios were calculated. to further evaluate our classifier, the classifier with optimal cutoff was compared to two reported noninvasive indices using the test set : the apri and the fib-4 index. the apri was calculated using ast [u / l]/(uln of ast)/plt [10/l ] 100. the fib-4 index was calculated using (age [yr ] ast [u / l])/((plt [10/l ]) (alt [u / l ])). student 's t - test or mann - whitney u test was used to test the difference between mean or median values. to compare the performance of a range of algorithms, star was used to plot receiver operator curves (roc) and statistical comparison of area under curve (auc) of each roc. all statistical analyses were performed using spss version 17.0 (spss inc., chicago, il, usa) and graphpad prism software (version 5.0). a total of 239 patients were enrolled in this study. clinical characteristics and laboratory findings from individuals with there were significant differences between the two groups with respect to age, alp, albumin, tbil, pt, hb, mcv, rdw, plt, and cre. the child - pugh scores of lc patients were as follows : a = 50 (40.3%), b = 45 (36.3%), and c = 29 (23.4%). to construct and evaluate the classifier, the 239 patients were divided into training set (n = 120) and test set (n = 119). details of patients from the training set and test set in this study were given in table 2. after data preprocessing, feature variables were evaluated by using the geneticsearch - based strategy. a panel of seven features was selected for classifier construction based upon the training set, including age, alt, ast, pt, plt, hgb, and rdw. to construct classifiers for predicting lc with chb, a preliminary test by tenfold cross - validation on the training set (table 3) was carried out to evaluate the performance of the two classifiers. mlp showed better results with acc of 82.2% and se of 80.6% but gave a poorer sp of 77.6%. nb gave better results on specificity (acc, 77.5%, se, 67.7%, and sp, 87.9% for nb). the sensitivity of nb was not as high as expected. meanwhile, the auc of mlp was higher than that of nb (p = 0.0125). the ability of a classifier to discriminate data correctly in the test set is known as its generalization performance. the mlp classifier (table 4) gave better results on the test set, with se of 85.5% and sp of 89.5% (overall accuracy 87.4%). the auc of the mlp classifier was significantly better than that of nb (p = 0.0133). the mlp classifier was then compared with two previously published noninvasive indices, including apri and fib-4 index. the auc for predicting liver cirrhosis on the test set for all three algorithms is shown in figure 1. the aucs of the mlp classifier, apri, and fib-4 index were 0.942, 0.817, and 0.726, respectively (table 5). the auc of the mlp classifier was significantly better than those of fib-4 (p = 0.0003) and apri (p 2,000 iu / ml). although liver biopsy has been the golden standard for evaluation of stage of liver fibrosis and cirrhosis, it is limited as it is an invasive procedure with significant expense, manpower issues, and some risks. therefore, there is a need for a simple, reliable, and noninvasive alternative method for regular monitoring of disease progression. in this study, we filtered out seven routine clinical parameters for the prediction of hbv - induced liver cirrhosis by statistical comparison of those of lc and chb, including age, alt, ast, pt, plt, hgb, and rdw. we then investigated two supervised machine learning methods for predicting liver cirrhosis with these seven parameters. we found that mlp gave better results (auc = 0.942) between the two classifiers. when compared to the two reported noninvasive algorithms using routine clinical parameters, our results indicate that the mlp was superior to them in the independent test set. the mlp classifier with optimal cut - off gives better accuracy (89.9%), higher sensitivity (95.2%), and acceptable sensitivity (84.2%) for predicting hbv - induced liver cirrhosis. a diagnostic model is considered as good if the auc is greater than 80% and excellent if the auc is greater than 90%. therefore, we concluded that mlp was an excellent tool for hbv - induced liver cirrhosis prediction. moreover, our finding confirmed that predicted probability value of mlp was increased with the raising of child - pugh scores in hbv - related liver cirrhosis patients. since child - pugh score is a well - recognized prognostic index for liver diseases, our result suggests that mlp has potential prognostic value for liver disease. first, mlp was based on seven routine clinical and laboratory parameters without any additional costs. therefore, mlp represents a cost - effective tool for hbv - induced liver cirrhosis prediction. second, the parameters in mlp are easily acquired in clinical practice, even in community hospitals. these advantages may facilitate the clinical utility of the mlp classifier for predicting liver cirrhosis in chb patients and reduce the number of liver biopsies. apri and fib-4 are two widely used, noninvasive and inexpensive tools to predict liver cirrhosis. it has satisfactory sensitivity and specificity together with a high predictive value for reducing the frequency with which biopsies need to be carried out to monitor the evolution of chc. auc values of apri in chb fibrosis - related studies range between 0.541 and 0.86 [2633 ]. in a study of meta - analysis, the summary auc of apri was 0.75 with regard to hbv - related liver cirrhosis. it was consistent with our result (auc = 0.726), which suggested that the diagnostic adequacy of apri was limited as a marker of liver fibrosis in chb patients compared to the patients with chc. in sterling 's report, fib-4 index was first created for predicting significant fibrosis in patients with hiv / hcv coinfection with an auc value of 0.737. in our study, the auc of fib-4 was greater than apri in the test set which was consistent with the study of erdogan.. in order to clarify whether mlp was better than apri and fib-4 for the prediction of lc with chb, we compared these tools in a head - to - head manner. the results showed that the mlp classifier had higher auc when compared with apri or fib-4. therefore, we concluded that the mlp classifier had superior diagnostic efficiency than apri or fib-4, two the most widely used noninvasive and inexpensive tools for liver cirrhosis prediction. first used machine learning methods in predicting cirrhosis in patients with chc based on viral and clinical factors. in cazzaniga 's work, they obtained better auc for predicting cirrhosis in chc patients using artificial neural networks. recently, wang and his colleagues predicted significant liver fibrosis of chb patients using an artificial neural network based upon routine and serum markers. in their work, the aucs of training, validation, and test set were 0.883, 0.884, and 0.920, respectively. although 455 patients were enrolled in their study, there were 27.7% patients with significant liver fibrosis, which might generate predictive bias during modeling. moreover, it requires further validation for predicting liver cirrhosis in chb patients because only 9 cirrhosis patients were included in their study. in our study, seven common clinical parameters were selected to build the mlp classifier, including age, alt, ast, pt, plt, hgb, and rdw. among the seven routine clinical parameters, age, plt, ast, alt, pt, and hgb had been reported for predicting significant liver fibrosis [6, 30, 36, 37 ]. in our published study, we found that rdw was increased with the worsening of hbv - related liver disease. first, increased rdw was potentially associated with inflammation response during the process of liver fibrosis [40, 41 ]. second, the prevalence of renal failure is higher in patients with liver cirrhosis than in the general population, while increased rdw is related to impaired renal function. in our study, similar result was observed that the levels of serum creatinine and rdw were higher in liver cirrhosis patients. firstly, only chinese chb patients were included in our study. due to small sample size, verification of the mlp classifier with accuracy, sensitivity, and specificity in more large population from different race and regions would be important before considering clinical use. secondly, our work was a retrospective study, and some clinical details of participants such as body mass index (bmi), cholesterol levels, alpha - fetoprotein (afp), inr, and duration of disease were not available in this study. so it remains unclear whether including these clinical details in our mlp classifier will improve the diagnostic accuracy. in addition, this deficiency also made us unable to compare our classifier with some reported algorithms, such as forns index, apga, and papas. however, in validation studies reported recently [6, 33, 46 ], fib-4 index gave the best diagnostic accuracy for the evaluation of hepatic fibrosis in patients with chb among these models, including forns index, apga, papas, and apri. likewise, fib-4 index showed better auc than that of apri with optimal cutoff value in our work. the main findings of this work are listed as follows. (1) the mlp classifier was developed for predicting liver cirrhosis in patients with chb using seven routine clinical parameters which are of low cost and easily implemented, even in community hospitals. (2) high auc of the mlp classifier which was superior to the two reported models could reduce the number of liver biopsies in clinical practice. (3) the mlp classifier has potential prognostic value for liver disease, especially in hbv - related liver cirrhosis patients. in conclusion, we describe an mlp classifier, a noninvasive, accurate, inexpensive, and easily acquired tool for predicting and evaluating liver cirrhosis in chb patients. other studies are necessary for further verification of its accuracy, sensitivity, and specificity in a larger population from different races and regions. | background. liver cirrhosis (lc) is the final stage of most of chronic liver diseases and is almost caused by chronic hepatitis b (chb) in china. liver biopsy is the reference method for the evaluation of liver cirrhosis. however, it is an invasive procedure with inherent risk. the aim of this study was to construct a new classifier based on the routine clinical markers for the prediction of hbv - induced lc. subjects and methods. we collected routine clinical parameters from 124 lc patients with chb and 115 with chb. training set (n = 120) and test set (n = 119) were built for model construction and evaluation, respectively. results. we describe a new classifier, mlp, for prediction of lc with chb. mlp was built with seven routinely available clinical parameters, including age, alt, ast, pt, plt, hgb, and rdw. with optimal cutoff, we obtained a sensitivity of 95.2%, a specificity of 84.2%, and an overall accuracy of 89.9% on an independent test set, which were superior to those of fib-4 and apri. conclusions. our study suggests that the mlp classifier can be implemented for discriminating lc and non - lc cohorts by using machine learning method based on the routine available clinical parameters. it could be used for clinical practice in hbv - induced lc assessment. |
little dispute exists that special training is essential for pediatric surgeons before starting endoscopic surgery in children ; however, a structured and readily available training program for them has not been established. we developed a basic modular training program (mtp), specially designed for pediatric surgeons. the aim of this study was to investigate whether our current mtp is effective for pediatric surgeons and whether their previous laparoscopic experience affects the training results. nine surgeons who participated in mtp (7 males, 2 females, mean age 36.85.8 years, range 30 to 45 years) were assigned to this study. they were qualified by the japanese surgical society after 4 years of residency in general surgery, and they had been in pediatric surgery practice for 1 to 15 years (mean 7.3 years). they were divided into 2 groups according to their clinical experience in laparoscopic surgery prior to mtp : group a (n=4), surgeons who had experienced more than 10 cases of laparoscopic surgery ; group b (n=5), those who had experienced fewer than 10 cases. because surgeons in group b had completed their surgical residency before the laparoscopic era, their age and postgraduate years were significantly higher than those of surgeons in group a (table 1). profile of modular training program participants each surgeon participated in the standardized 3-hour - mtp workshop, which consisted of an introductory video, a didactic session about instrumentation, and 2 see - through and 3 laparoscopic tasks. see - through tasks were done as follows in an original training box made of acrylic acid resin, which mimicked the small abdomen of pediatric patients : with the use of 2 pegboards and 10 pegs, each examinee was required to lift each peg from 1 pegboard with the left hand, transfer it to the right hand, and place it on the other pegboard (figure 1a). modular training units : a. peg transferring task in see - through module ; b. pattern cutting task in see - through module ; c. laparoscopic training box, laparotrainer, with built - in light source, trocars, scope, and videomonitor in place. this task involved cutting a 6-cm diameter circular pattern out of a 10 x 10-cm piece of white paper placed in the box (figure 1b). the aim was to use the grasper in 1 hand, placing the material under tension while cutting with the endoscopic scissors in the other hand. after completing the see - through tasks, the participants then proceeded to the laparoscopic session. the tasks were done in a laparoscopic training box (laparo trainer, nippon stryker, japan) covered by an opaque membrane (figure 1c). two 12-mm trocars were placed through the membrane at convenient working angles on either side of a 10-mm zero - degree rigid scope, which was mounted on a stand at a fixed focal length. the following 3 tasks were done laparoscopically by displaying the modules on a high - resolution video monitor (vt-21g1, sharp, japan) placed in line with the operator. the examinee was required to perform the peg transferring in the same way as in the see - through session. the aim was to get eye - hand coordination and ambidexterity under laparoscopic visual control. this task involved placing 3 hemostatic clips on a tubular foam structure with appropriate margins and then cutting halfway between the clips. each operator was required to twist the metal wire placed through a sucking disc with 2 hands. this task was designed to develop the right and left hand coordination that is necessary for manual knot tying. each participant 's performance was objectively evaluated before and after mtp with the computer - generated virtual simulator (mist vr, virtual presence inc., uk), which has recently been reported as a confidential tool for objective assessment of psychomotor skills in laparoscopic surgery (figure 2a). participants were required to perform a simple virtual task that involved picking up a virtual ball, placing it in a virtual box, and then releasing it (figures 2b, 2c, 2d). path lengths, path velocities and distances to target profiles were recorded, and the following 3 scores were obtained : (1) a number of mispointing target balls (as an indicator of movement precision) ; (2) an actual / ideal path length ratio of the movements (as movement efficiency) ; (3) a time taken to complete the virtual task (as speed). computer - generated virtual simulator, mist vr : a. whole view of system with interface ; b. starting task by picking up a virtual ball on pc screen ; c. transferring the ball into a box ; d. terminating task by releasing the target. the data were analyzed with the student paired t test to test differences in performance between before and after mtp and the unpaired t test between groups a and b. a value of p<0.05 was considered statistically significant. nine surgeons who participated in mtp (7 males, 2 females, mean age 36.85.8 years, range 30 to 45 years) were assigned to this study. they were qualified by the japanese surgical society after 4 years of residency in general surgery, and they had been in pediatric surgery practice for 1 to 15 years (mean 7.3 years). they were divided into 2 groups according to their clinical experience in laparoscopic surgery prior to mtp : group a (n=4), surgeons who had experienced more than 10 cases of laparoscopic surgery ; group b (n=5), those who had experienced fewer than 10 cases. because surgeons in group b had completed their surgical residency before the laparoscopic era, their age and postgraduate years were significantly higher than those of surgeons in group a (table 1). profile of modular training program participants each surgeon participated in the standardized 3-hour - mtp workshop, which consisted of an introductory video, a didactic session about instrumentation, and 2 see - through and 3 laparoscopic tasks. see - through tasks were done as follows in an original training box made of acrylic acid resin, which mimicked the small abdomen of pediatric patients : with the use of 2 pegboards and 10 pegs, each examinee was required to lift each peg from 1 pegboard with the left hand, transfer it to the right hand, and place it on the other pegboard (figure 1a). modular training units : a. peg transferring task in see - through module ; b. pattern cutting task in see - through module ; c. laparoscopic training box, laparotrainer, with built - in light source, trocars, scope, and videomonitor in place. this task involved cutting a 6-cm diameter circular pattern out of a 10 x 10-cm piece of white paper placed in the box (figure 1b). the aim was to use the grasper in 1 hand, placing the material under tension while cutting with the endoscopic scissors in the other hand. after completing the see - through tasks, the participants then proceeded to the laparoscopic session. the tasks were done in a laparoscopic training box (laparo trainer, nippon stryker, japan) covered by an opaque membrane (figure 1c). two 12-mm trocars were placed through the membrane at convenient working angles on either side of a 10-mm zero - degree rigid scope, which was mounted on a stand at a fixed focal length. the following 3 tasks were done laparoscopically by displaying the modules on a high - resolution video monitor (vt-21g1, sharp, japan) placed in line with the operator. the examinee was required to perform the peg transferring in the same way as in the see - through session. the aim was to get eye - hand coordination and ambidexterity under laparoscopic visual control. this task involved placing 3 hemostatic clips on a tubular foam structure with appropriate margins and then cutting halfway between the clips. each operator was required to twist the metal wire placed through a sucking disc with 2 hands. this task was designed to develop the right and left hand coordination that is necessary for manual knot tying. each participant 's performance was objectively evaluated before and after mtp with the computer - generated virtual simulator (mist vr, virtual presence inc., uk), which has recently been reported as a confidential tool for objective assessment of psychomotor skills in laparoscopic surgery (figure 2a). participants were required to perform a simple virtual task that involved picking up a virtual ball, placing it in a virtual box, and then releasing it (figures 2b, 2c, 2d). path lengths, path velocities and distances to target profiles were recorded, and the following 3 scores were obtained : (1) a number of mispointing target balls (as an indicator of movement precision) ; (2) an actual / ideal path length ratio of the movements (as movement efficiency) ; (3) a time taken to complete the virtual task (as speed). computer - generated virtual simulator, mist vr : a. whole view of system with interface ; b. starting task by picking up a virtual ball on pc screen ; c. transferring the ball into a box ; d. terminating task by releasing the target. the data were analyzed with the student paired t test to test differences in performance between before and after mtp and the unpaired t test between groups a and b. a value of p<0.05 was considered statistically significant. table 2 summarizes the training results of overall participants. the time taken to complete tasks was significantly decreased after mtp, indicating the increased speed with which the participants handled the virtual tasks. no significant differences occurred in the number of mispointings and the actual / ideal path ratio of both dominant and nondominant hands before and after mtp. participants ' overall performance table 3 shows the changes of performance before and after mtp in each group and the comparison of performance between 2 groups at each time point. performance of each group p < 0.05 vs before modular training program in group a. p < 0.05 vs before modular training program in group b. in group a, no significant differences occurred in the number of mispointings, the path ratio and the time before and after mtp. in group b, however, the path ratio of dominant hands was significantly decreased after mtp, indicating the significantly higher movement efficiency. the time was also decreased significantly after mtp, indicating the increased speed in this group. before mtp, the path ratio of dominant hands was significantly higher in group b than in group a, indicating the significantly lower movement efficiency in group b. after mtp, a significant difference no longer existed in the path ratio between the 2 groups. table 2 summarizes the training results of overall participants. the time taken to complete tasks was significantly decreased after mtp, indicating the increased speed with which the participants handled the virtual tasks. no significant differences occurred in the number of mispointings and the actual / ideal path ratio of both dominant and nondominant hands before and after mtp. table 3 shows the changes of performance before and after mtp in each group and the comparison of performance between 2 groups at each time point. performance of each group p < 0.05 vs before modular training program in group a. p < 0.05 vs before modular training program in group b. in group a, no significant differences occurred in the number of mispointings, the path ratio and the time before and after mtp. in group b, however, the path ratio of dominant hands was significantly decreased after mtp, indicating the significantly higher movement efficiency. the time was also decreased significantly after mtp, indicating the increased speed in this group. before mtp, the path ratio of dominant hands was significantly higher in group b than in group a, indicating the significantly lower movement efficiency in group b. after mtp, a significant difference no longer existed in the path ratio between the 2 groups. despite the wide acceptance of laparoscopic surgery among general surgeons, most pediatric surgeons remain unconvinced that laparoscopic surgery can be a valuable modality for pediatric surgical disorders. the major reasons are the lack of proven long - term benefits, the question of increased complication rates, the longer operating time and the higher cost of equipment. another reason is that only a few pediatric surgeons have the opportunity to obtain endoscopic skills adequately and appropriately. the most natural place to learn surgical skills is in the operating theater ; however, it is becoming increasingly difficult for present and future surgeons to acquire all surgical skills in the operating room, largely because of concern about safety, time, and cost. it is therefore considered essential to establish a structured endoscopic training program outside the operating rooms, but to our knowledge, no previous study has been conducted regarding the development of such training courses for pediatric surgeons. our current mtp regimen includes 5 standard training tasks, 2 of which were done under 3-dimensional visualization in an original, transparent training box. the box has 15- to 20-cm widths and 10- to 20-cm depths, simulating the insufflated abdomen of infants. two small holes drilled on the surface of the box mimic the surgical ports ; thus, each trainee can realize the actual feeling of the fulcrum effect. through repeated simple tasks, participants can gradually feel familiar with the unique action of delicate and long - handled endoscopic instruments.. the participants could complete these 5 modules step by step at their own pace, being mentored by experienced endoscopic instructors. our mtp did improve the psychomotor skills of pediatric surgeons who had been less exposed to laparoscopic surgery. their movement efficiency and speed were significantly improved after mtp, indicating the validity of our current curriculum. on the contrary, surgeons who had already been exposed to laparoscopic surgery could not gain further development of their skills through mtp. these results suggest that our current basic program is effective for initiating the nonlaparoscopic pediatric surgeons, but is not entirely satisfactory for laparoscopic surgeons. it has been a commonly held assumption that younger surgeons have a natural advantage in the development of laparoscopic skills. our present study showed, however, that age did not appear to play a dominant role in the outcome. senior pediatric surgeons who completed mtp could maintain their initial enthusiasm by being convinced that this is partly because our mtp did not have proper modules to develop precise motion. another explanation is that it may take more time to learn precise motion than effective and speedy motion. further modifications are necessary to make mtp more effective in allowing surgeons to learn delicate, not merely quick motion, which seems more essential in clinical settings of pediatric laparoscopic surgery. our training program was effective for pediatric surgeons who have less experience in clinical laparoscopic surgery. a simple, nontime - consuming, and inexpensive training course is an attractive method for nonlaparoscopic pediatric surgeons to obtain basic endoscopic skills and to overcome their mental reservations. | objectives : a structured endoscopic training program for pediatric surgeons has not yet been established. this study was conducted to develop a modular training program (mtp) for pediatric surgeons and to evaluate its effectiveness for surgeons with and without previous experience in laparoscopic surgery.methods:nine pediatric surgeons participated in the study. they were divided into 2 groups : group a (n=4), surgeons who had experienced more than 10 cases of laparoscopic surgery prior to mtp ; group b (n=5), those who had experienced fewer than 10 cases. they participated in a standardized mtp workshop, which consisted of 2 see - through and 3 laparoscopic tasks. each participant 's psychomotor skills were evaluated objectively before and after mtp with a computer - generated virtual simulator and were evaluated for precision, efficiency, and speed.results:in participants, speed was significantly enhanced after mtp. in group a, no differences were observed after mtp, whereas significant improvements were noted in efficiency and speed after mtp in group b. before mtp, efficiency was significantly higher in group a than in group b ; however, no difference remained between the 2 groups after mtp.conclusions:mtp is effective for nonlaparoscopic pediatric surgeons to become familiar with basic endoscopic skills. |
we report the case of one patient undergoing routine gastric bypass who experienced major complications. a 56 year old female was referred from a district general hospital after a protracted history of abdominal pain and vomiting. past medical history included osteoarthritis and hypertension, and a past surgical history of laparoscopic roux - en - y gastric bypass in 2008 (pre - operative bmi of 48 kg / m2). a laparotomy and revision of roux - en - y gastric bypass was undertaken with post operative management on hdu, but the patient had ongoing problems with pain and wound discharge. a ct scan revealed a 7 cm collection adjacent to the alimentary limb which was percutaneously drained under ultrasound guidance. due to clinical deterioration, a further ct was performed which demonstrated a large communicating collection filling the paracolic gutters and pelvis, with a multiloculated left lobe liver abscess, and a smaller abscess in the right lobe (figure 1). the patient was discharged home. multiloculated abscess within the left lobe of liver the patient re - presented 2 weeks later with increasing epigastric pain and malaise. imaging showed a persistent collection in the pelvis and liver, and a further two percutaneous procedures were performed. during inpatient stay, she became haemodynamically unstable with spiking temperatures and an elevating white cell count. she also complained of bilateral discolouration of her toes and examination revealed increasing oedema with diminished pedal pulses. a contrast ct scan of the abdomen confirmed ongoing collections with evidence of an aortic thrombus extending into the left iliac artery, presumed to be the embolic source for her vascular findings (figure 2 & 3). there was resolution of vascular symptoms and evidence of improvement in peripheral perfusion postoperatively, and the patient was eventually discharged. thrombus within the distal aorta shown by a filling defect (red arrow) extension into the left iliac artery shown by a filling defect (red arrow) surgery has inherent risks and complications, which may be further be potentiated by the high risk nature of bariatric patients. a systematic review of the literature concluded that peri - operative complications were encountered more frequently in bypass patients compared to those undergoing laparoscopic gastric banding (9% and 5%), although long - term re - operation rates were lower (16% and 24% respectively) (1). the mortality of both bypass and band operations were low (0.17% and 0.06%). further systematic reviews have reported major early complications rates of gastric bypass surgery as 6.3%, with major complications including death (0.2%), perforation (0.5%), venous thromboembolism (0.3%), significant postoperative bleed (0.9%), postoperative infection (2.9%) and anastomotic leaks (0.9%) (1). despite these risks gastric bypass is an effective operation for obesity ; one study reported 76% excess body weight loss and a 78% resolution of diabetes at 1 year in patients undergoing bypass compared to 48% weight loss and 50% diabetes resolution in patients undergoing banding (2). generally high levels of patient satisfaction are recorded, estimated to approach 80% (3). venous thrombosis is well documented in the literature as a complication of obesity and bariatric surgery. one study reported an incidence of deep vein thrombosis in the group of 500 consecutive patients to be 0.2% (4). however, there is little data regarding arterial thrombosis as a complication of bariatric surgery. bariatric patients have significant risk factors for atherosclerotic disease, both due to obesity, and to secondary diseases such as hypertension and diabetes, which may develop as part of the metabolic syndrome. however, with this patient there was no evidence of aortic thrombosis on previous imaging, and a new presentation of clinical signs and symptoms would be in keeping with a new onset thrombosis. there are also factors relating to a hypercoagulable state, which in this patient could be numerous. this patient had recurrent episodes of sepsis, which is well known to precipitate hypercoagulability, which at the severe end of the spectrum, exists disseminated intravascular coagulation (dic). sepsis mediated hypercoagulability occurs as a result of the activation of inflammatory and coagulation cascades (5). dehydration secondary to both poor intake and insensible losses through either infection or surgery may also cause an increased coagulable state. inflammatory and hypercoagulation may be due to the direct effects of surgery through the stress response (6). roux - en - y gastric bypass is deemed a safe operation in the management of obesity. we present a case complicated postoperatively by recurrent intra - abdominal collections and sepsis, and aortic thrombosis with distal embolisation. | laparoscopic gastric bypass is becoming a frequently performed bariatric operation for patients with obesity. however, bariatric surgery has inherent risks and complications, which are further potentiated by the high risk nature of bariatric patients. these complications can be either site specific i.e. anastomotic leaks, or systemic i.e. venous thromboembolism, however they can intimately related. we present the case of one patient undergoing routine gastric bypass complicated postoperatively by recurrent intra - abdominal collections and sepsis, and aortic thrombosis with distal embolisation. |
intellectual and developmental disabilities are lifelong chronic conditions, with multiple etiologies. further, intellectual and developmental disabilities (i / dd) can affect anyone, that is, from any ethnic or socioeconomic background. they are characterized by limitations in both intellectual functioning and adaptive skills, including learning, language, self - care, and capacity for independent living. prevalence rates for i / dd vary from 1.5% to 2.5% of the us population [2, 3 ]. this means that one in ten families, or roughly 30 million americans, will at some point in their lifetime be directly affected by a person with an i / dd. unfortunately individuals with i / dd are at increased risk of certain chronic health conditions including seizures, mental health issues, obesity, heart disease, diabetes, osteoarthritis, osteoporosis, vision abnormalities, and poor oral health [57 ]. they often experience disparities in health care including opportunities for health screening and health promotion. if health promotion programs exist, they often are not targeted to meet the specific needs of this population. in december of 2011 those with i / dd were designated a medically underserved population by the american medical association. therefore, finding a medical home is difficult and a critical need when individuals age out of pediatric practices, and there is evidence that many are followed well into their adult lives by pediatrician and pediatric specialists. educational experiences can be challenging and limited as well, and opportunities for employment upon completion of primary and/or secondary schooling (if successful) are also limited. employment opportunities are so limited that it is rare that one with i / dd would earn enough money to be self - sufficient. for example, in academic year 2007 - 2008, only 34% of students with intellectual disabilities graduated from high school with a regular diploma. sadly, even with their diplomas, those with intellectual disability were among the lowest paid of graduates with disabilities 1 to 4 years after graduating high school. all of these challenges contribute to significant health and social disadvantages when compared to the general population and highlight the complex planning and coordination needs at both the individual and system level to promote optimal health and quality of life. consequently, the majority of individuals with i / dd will need varied and individualized lifelong support and services in the communities where they live. with an increasing number of americans turning to the internet for general and health information, we saw a need to evaluate this resource for persons with i / dd. this study was undertaken utilizing the lens of the individual family caregiver to assess and compare programs identified on e - government websites as resources for persons with i / dd. once the needs for housing and health care have been ensured, a focus on quality of life and socialization becomes paramount for the individual and the family. another focus of this study was to identify unique services in individual states that could serve as a benchmark for other states. a descriptive, cross - sectional assessment to identify state services and available programs for the adult with i / dd was conducted by three researchers. our team collectively assessed all fifty states and the district of columbia on the use of their respective government - run websites (.gov or.us) and program links found on their respective pages to communicate to the public which services are offered by the state for adults with i / dd. next we assembled data by state, and each researcher explored 17 states using the questions identified in the list below. the team met for peer review and presented and assembled lists of resources as a group. data collection and analysis was accomplished in the summer of 2011, with additional assessments of targeted e - government websites completed in the fall / winter of 2012 - 2013. we used the widely known search engine, google, and a combination of simple search times (i.e., [state ] intellectual disability resources and i / dd programs in [state ]). criteria used to analyze each e - government website were as follows.does the website offer state - supported respite care?does the website offer state - supported transportation services?does the website offer state - supported residential services?does the website have specific services for the transition period into adulthood?does the website recognize the aging i / dd population and offer unique services targeted towards the special needs of this group?does the state offer assistance in purchasing or renting assistive technologies and medical equipment?what difficulties, if any, were encountered?what unique features or programs did the website offer?was any contact information listed ? does the website offer state - supported respite care ? does the website offer state - supported transportation services ? does the website offer state - supported residential services ? does the website have specific services for the transition period into adulthood ? does the website recognize the aging i / dd population and offer unique services targeted towards the special needs of this group ? does the state offer assistance in purchasing or renting assistive technologies and medical equipment ? what difficulties, if any, were encountered ? what unique features or programs did the website offer ? our focus of e - government sites was deemed as appropriate websites for state services and provided uniform comparison among states. specifically, available information on services was as follows : residential 100% (51/51), respite 96% (49/51), transportation 88% (45/51), medical equipment 57% (29/51), aging 55% (28/51), and transitional 47% (24/51). figure 1 represents the total number of e - government sites (out of 51 evaluated) that had easily identifiable information on specific types of disability service. for contact information, a phone number was listed 96% (49/51), a phone number with a local area code 65% (33/51), an email 45% (23/51), and an inquiry form 6% (3/51), while only 45% included an email address. four states replied within two days (arizona, delaware, illinois, and iowa), while the other 4 states never replied to a request for more information on disability services (alabama, arkansas, alaska, and connecticut). unique programs identified and the states they are found in include the wyoming institutes for disability (wind) assistive technology resources (wyoming), meaningful day initiative (new mexico), clyde 's karate (west virginia), vanderbilt kennedy center next steps (tennessee), parent empowerment network (west virginia), and green mountain self advocates (vermont) ; each is described next. in our search, we came across dozens of unique programs on different e - government sites, many of which would benefit implementation nationwide or internationally. a few of these programs are highlighted here, offering a template for states to decrease the nationwide disparities in meaningful care. we selected four that demonstrate distinguishing services that enhance the quality of life for those with i / dd. the programs included for discussion were also selected for their innovation and ability to serve as models for programs in other states. wyoming, for example, offers the intellectually disabled population one rather intriguing and unique service. on the state 's website wyoming institute for disabilities (wind) assistive technology resources, which acts as an intermediary for the exchange of medical assistive technologies. the program helps locate used adaptive equipment, which is often donated or loaned through the program. loans for equipment can last years and are often done cheaply or free of charge. the site operates on a pay - it - forward type of platform, which allows users to sign up and both exchange their old medical equipment and locate new equipment they may need. the site currently has over 1,500 items listed, which range from products like walkers to car modifications to electronic devices. the new mexico state - run website for developmental disabilities had a subpage dedicated to the this initiative encapsulates many of the self - autonomous goals of individuals with i / dd. the program promotes freedom of choice, allowing the individual to play a crucial role in the support process. meaningful day is intended to help build relationships, provide resources for challenging employment, ensure housing in a safe, suitable environment, assist in accessing health care, promote engaging activities, and improve quality of life. furthermore, this initiative intends to prevent or limit isolation and isolating activities, replacing them instead with engaging activities that would be used by the general population as well. the program has specific guidelines for the retired and elderly individual with i / dd and reflects the importance of an evolving lifelong individualized program of care. this goal is consistent for all individuals with i / dd, regardless of whether they are using waivered state funds. the west virginia developmental disability council funded clyde 's karate to provide recreational services, and they developed an instructional dvd of their martial arts classroom. the dvd realistically and honestly profiles the added benefits realized for individuals, their peers, and their parents, as well as the instructor of the class. for example, one student of this program, sean, a young man who has down syndrome, has had many gains. sean lost 20 pounds and his coordination, strength, breathing, and endurance improved. most importantly, he increased his self - discipline, self - esteem, respect, and brotherhood and camaraderie with peers both with and without developmental disabilities. one student of this program, sean, a young man who has down syndrome, has had many gains. sean lost 20 pounds and his coordination, strength, breathing, and endurance improved. most importantly, he increased his self - discipline, self - esteem, respect, and brotherhood and camaraderie with peers both with and without developmental disabilities. in tennessee, a task force to develop a pilot program in postsecondary education for individuals with i the following year, the future program at the university of tennessee opened its doors for students with i / dd who wished to further their education beyond high school. both programs are two years in length and award certificates through individualized and person centered planning programs. the us department of education in 2012 awarded funding to 27 institutions of higher education to either create or expand high quality transition and postsecondary programs for individuals with intellectual disability. our team took notice of numerous recurring problems the average proxy caregiver or person with i / dd might face during their search for state resources. common problems encountered while searching for available resources included the fact that certain links led to unavailable pages, invalid or nonactive phone numbers and email addresses, no search feature, and/or search features that did not provide relevant results. quite often, state - run websites contained dead links, or links that when clicked did not access a valid webpage. two states were noted for their convenient and helpful search options : idaho and kansas. while a few states had definitions for terms like intellectual disability, on rare occasions the words mental retardation still appeared in place of the more current and accepted term intellectual disability. finding contact information for a representative was often challenging, and in certain instances, our researchers could not find an email or phone number for a representative to contact specifically on i / dd services ; for example, 98% of states had a phone number listed, while only 45% included an email address. of the 8 states chosen at random for an email inquiry, only 4 responded. furthermore, after attempting to contact a number of representatives from various states, we found inactive phone numbers and emails. all of these barriers pose a potential source of frustration for individuals seeking more information on services for persons with i / dd, as they may have a difficult time finding reliable information, a representative to contact, and a response, depending on the state 's website they are researching and contacting. in addition to aging services, aging disability resource centers (adrcs), links were found on a number of state websites and we suggest that all e - government i / dd sites should provide links to their local adrcs (if applicable). knowledge of age - specific services is critically important to our seniors with disability to successfully age in place. quality of life concerns and the need to establish networks were obvious on a number of websites that included programs (e.g., best buddies) to address the isolation and lack of socialization that can often accompany this developmental disability. for many families, a circle of friends beyond relatives is nonexistent for their loved ones with i / dd. the need for connection by both caregiver and individual was apparent and is identified in the number of programs emerging to connect people, both nationally and internationally. the parent empowerment network (pen), for example, in west virginia, serves to establish links with carers. green mountain self in vermont is operated and run by people with intellectual disability. how to give a speech and how to educate yourself widespread use of the internet has led an increasing number of americans to access medical information online. reported findings range vastly [2022 ], but the data suggests that up to 80% of citizens have turned to the internet for information on their health or for the health of their loved ones, which is roughly the percentage of adults that access the internet. half of the health - related internet inquiries for health - related issues are on behalf of someone else. concerns have been raised over the quality of information and accessibility of consumer health information on the internet, as well as the implications of its increasingly prevalent use for health - related inquiries. with the expanding reliance on the internet for information, numerous researchers have conducted studies evaluating website characteristics such as reliability, usability, veracity, and accessibility [2426 ]. while several studies have had a focus on domestic us websites, some studies have evaluated internet sources from an international perspective [2528 ]. one study developed a website evaluation questionnaire for nursing websites that was tested in both the usa and taiwan, while newby and groom evaluated the usability of a uk rehabilitative site and noted room for improvement, specifically in relation to legibility, layout, writing style, and the need for more information. another study assessed the quality and readability of internet information for adults with hearing impairment and their caregivers in five different countries and offered a number of suggestions for improvement based on the disparities they found. the literature suggests that readability, reliability, and ease of access are among the commonly found issues in accessing reliable health information online both in the united states and in other countries. while nearly all states have at least one aging and disability resource center (adrc), these centers do not specifically focus on i / dd. all us states have a department or division that provides i / dd services, but there are numerous discrepancies between each state. consequently, we need to focus attention on resources that provide both formal and informal services that can support a quality of life to age in place, as individuals with i / dd are aging and will outlive family caregivers, creating uncertainty for future care giving, health care, and housing needs. families are struggling to find adequate lifelong care and services, especially those that enhance or support quality of life needs [10, 30 ]. the national committee on quality assurance (ncqa) in the united states annually analyzes the performance and service of individual states ' medicare and medicaid health programs. using over 100 data measures, their findings ranked all fifty states and the district of columbia from best to worst, identifying both changes in performance and a model for other states to implement improved delivery of service [13, 14 ]. while access and availability to services differs across state lines, current literature offers very little insight into the actual experience of identifying and accessing developmental disability services. our approach was to consider the caregiver, prior to the actual utilization of services with a goal of analyzing the ease or difficulty in finding or identifying available resources for individuals with i / dd. this approach was considered important, as decision making for care and services is commonly done by proxy, that is, by family members or caregivers [15, 32 ]. website domains signify the level of restriction, if any, one faces when registering for a webpage. any individual can apply for a.com or.org domain name, and while these links may be helpful, the user needs to be aware that reliability and authenticity of these sites can vary. unlike general domains, domains that have restrictions on who can register for them include.edu (reserved mainly for us - affiliated institutions of higher learning),.us (reserved mainly for us state and local governments), and one study notes the following : given the multitude of federal laws and policies, one might expect a relatively uniform and high level of accessibility to state web sites. furthermore, research has indicated that individuals with a disability may encounter even greater difficulties in accessing information via e - government websites, as there exists a gap between policy and practice, noting that policy alone is insufficient in adequate action and implementation by state governments. while the internet is not immune to limitations and inadequacies, research suggests it can be a promising source and effective platform for health information, communication, and education, and it is an empowering way to put more control in the hands of the public. in our findings, we note that our inability to locate specific services in a state does not conclude nor should it imply that the state does not offer these services. rather, it can portray the difficulties a caregiver or individual with disabilities may experience in their own efforts to locate state services and resources. in reality, we believe that a higher percentage of states offer the services we screened for. our experience highlighted that there are numerous disparities across e - government websites and that further research should be conducted to highlight specific difficulties caregivers perceive in their efforts to access services for individuals with i / dd. although our analysis of websites was uniform and our team used the same combination search terms for all states, it is possible that information was overlooked for any number of the states. we attribute any information overlooked as typical of the challenges an average caregiver or person with i / dd may experience in identifying resources and services. further, the scope of our analysis did not include any criteria to determine whether the websites evaluated were disability - friendly and easily navigable by an individual with i / dd. websites are always changing, and sites may contain out of date or inaccurate information. resources, even if identified, are not always properly funded or available, and thus, identification is simply the first step of several necessary to benefit from state services. while this study focused solely on e - government sites and the links contained in them, we do contend that caregivers may not distinguish between the government - run website and non - e - government websites. nongovernment sites were only mentioned if they had been linked on e - government sites. this narrow focus is a limitation in the fact that this study 's findings may not be applicable to the panoply of private (nongovernmental) or local government sites that can offer a wide array of i / dd services. the internet plays a significant role in our society and increasingly americans are using the internet to identify health services and programs and for health information. we highlighted some unique programs that could serve as models for other states to enhance quality of life for those with i / dd. our study suggests that finding information on specific i / dd support services through state websites might prove to be difficult, as availability of information was variable across the united states. further, this study reveals an appreciable gap in contact information and response from state representatives in regard to inquiring for further i / dd resource information. the convenience of services offered by an individual state can be diminished by limitations in that state 's ability to relay reliable information through their internet webpages to potential recipients in need of such services. we affirm the call for increased readability, reliability, and presentation of information on health - related websites, specifically for i / dd resource websites in the united states. | this descriptive cross - sectional study identified resources and programs that are available nationwide on the internet to support individuals and families with intellectual and developmental disabilities (i / dd), with a focus on intellectual disability. this evaluation included easily identifiable information on specific resources and highlighted unique programs found in individual states that were linked from e - government websites. researchers documented the ease of access and available information for all 50 states and the district of columbia. a number of disparities and areas for improvement were recorded for states and i / dd websites. the researchers conclude that a number of additional health and support services will be needed to address the growing needs of this vulnerable population. |
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