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a great majority of phenotypes observed in animals, plants and humans are both quantitative and longitudinal, i.e. they attribute variation on a continuous scale and can be recorded several times during an individual s lifetime or physiological cycle. the evidence of variation of genetic effects over time is observed in experimental organisms (leips. 2006), as well as in livestock (e.g. schaeffer and dekkers 1994). in the former study, time - dependent quantitative trait loci (qtl) affecting fecundity in drosophila melanogaster are identified, while the latter study provides evidence that the additive genetic variance underlying milk production traits in dairy cattle is not constant over time. most statistical applications aiming to model growth traits either assume that the underlying genetic background is constant in time (e.g. corva and medrano 2001), or that the changes of all gene effects are the same throughout the whole growth period (e.g. jaffrzic. 2004). the functional relationship between genetic parameters and time - dependent variables are described by orthogonal polynomials, which were first introduced by schaeffer and dekkers (1994) to model a joint additive effect of all genes (a so - called polygenic effect) for milk yield in dairy cattle. modelling effects of particular genes as variable over time is known in the literature as functional gene mapping. the changes of a gene effect over time are described by the logistic function (ma. 2002), legendre orthogonal polynomials (yang. 2006, 2007) or b - splines (yang. however, none of the applications tackles the problem of the prediction of future trait values. the major goal of this study is an application of a model in which effects of single - nucleotide polymorphisms (snps) representing genetic factors, as well as a permanent environmental effect, are assumed to be variable over time. such modelling is especially useful for association studies in the situation when genetic effects underlying an snp affect only some, but not all, time periods of a longitudinal trait. when a model with time - independent effects is applied to such a genetic background, the snp effect is averaged over the whole time period and may, thereby, be difficult, or even impossible, to be detected. the longitudinal estimators optimally use the information contained in the whole analysed period and are, thus, robust towards the variation of genetic effects over time. for the detection of an association between snps and a longitudinal trait, a single snp random regression model proposed by yang. (2006) is used. in this model, all time - dependent parameters (both fixed and random) are described by legendre polynomials of order 3. this random regression model is an extension of the following single snp linear mixed model : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { y_i}(t) = \mu (t) + { x_i}\alpha (t) + { \xi_i}(t) + { \varepsilon_i } $ $ \end{document}where yi(t) is a phenotypic trait of individual i at time t, (t) is a population mean at time t, xi { 1, 0, 1 } is an snp genotype indicator variable for the ith individual and (t) is a fixed additive snp effect at time t. it is assumed that i(t) is a time - dependent random permanent environmental effect with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n(0,\sigma_{\xi } ^2(t)) $ $ \end{document } distribution and i is a time - independent residual term with n(0,) distribution. the genetic variance at time t for model 1 is expressed as : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_g^2(t) = \sigma_x^2{\alpha^2}(t) = 2p(1 - p){\alpha^2}(t) $ $ \end{document}where p is the frequency of allele 1 for a given snp. the total phenotypic variance at time t has the form : 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_p^2(t) = \sigma_g^2(t) + \sigma_{\xi } ^2(t) + { \sigma^2 } = 2p(1 - p){\alpha^2}(t) + \sigma_{\xi } ^2(t) + { \sigma^2 } $ $ \end{document}note that it is a single snp model, so the genetic and phenotypic variances are calculated separately for each snp. since phenotypic values for each individual are expressed as a longitudinal trait, measured at n + 1 time points, t = [t0, t1,..., tn ], legendre orthogonal polynomials can be used for describing time - dependent effects. these polynomials are defined on an interval [1, 1 ], therefore, the original time points t are recoded into using the following formula : 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \tau = 2 \cdot \frac{{t - { t_0}}}{{{t_n } - { t_0 } } } - 1 $ $ \end{document}where t0 and tn are the extreme time values. for modelling three time - dependent effects ((), (), i()) in model 1, legendre polynomials of order 3 are used. namely, a time - dependent parameter () can be described as a linear combination of (), where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{\psi } } (\tau) = [{ \psi_0}(\tau), { \psi_1}(\tau), { \psi_2}(\tau), { \psi_3}(\tau) ] $ $ \end{document } are coefficients of the polynomial and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{\mu } } = { [{ \mu_0},{\mu_1},{\mu_2},{\mu_3}]^t } $ $ \end{document } is a vector of the time - independent population means. the polynomial coefficients have the following form : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \psi_0}(\tau) = 1,\;{\psi_1}(\tau) = \tau, \;{\psi_2}(\tau) = \frac{1}{2}(3{\tau^2 } - 1),\;{\psi_3}(\tau) = \frac{1}{2}(5{\tau^3 } - 3\tau) $ $ \end{document}the remaining time - dependent fixed and random terms (() and i()) are described in the same way, using linear combinations of () and ()i. the third order of polynomials is chosen because linear or quadratic curves (polynomial order 2) usually exhibit very poor fit to growth curves. on the other hand, since, for each individual, several observations are available, it would be impossible to fit a higher order of polynomials. it is assumed that random regression coefficients for a permanent environmental effect \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \mathbf{\xi } } _ i } = { [{ \xi_{{i0}}},{\xi_{{i1}}},{\xi_{{i2}}},{\xi_{{i3}}}]^t } $ $ \end{document } are normally distributed with mean 0 and covariance matrix. under the above parameterisation, the model 1 can be transformed into a random regression model, which has the following form : 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { y_i}(\tau) = { \mathbf{\psi } } (\tau) { \mathbf{\mu } } + { x_i}{\mathbf{\psi } } (\tau) { \mathbf{\alpha } } + { \mathbf{\psi } } (\tau) { { \mathbf{\xi } } _ i } + { \varepsilon_i } $ $ \end{document}the genetic variance for model 6 is given by : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_g^2(\tau) = 2p(1 - p){\alpha^2}(\tau) = { \mathbf{\psi } } (\tau) [2p(1 - p){\mathbf{\alpha } } { { \mathbf{\alpha } } ^t}]{{\mathbf{\psi } } ^t}(\tau) $ $ \end{document}and the total phenotypic variance is expressed as : 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_p^2(\tau) = \sigma_g^2(\tau) + \sigma_{\xi } ^2(\tau) + { \sigma^2 } = { \mathbf{\psi } } (\tau) [2p(1 - p){\mathbf{\alpha } } { { \mathbf{\alpha } } ^t } + { \mathbf{\sigma } } ] { { \mathbf{\psi } } ^t}(\tau) + { \sigma^2 } $ $ \end{document}the estimation of all unknown parameters (,,,) in model 6 is based on the expectation maximisation (em) algorithm as described by yang. (2006). for testing the association between an snp and a phenotype, the likelihood ratio test can be used. the corresponding hypotheses are : h0 : = 0 vs. h1 : i { 0, 1, 2, 3 } (i 0). under h0, the test statistic asymptotically follows the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \chi_{{4df}}^2 $ $ \end{document } distribution. since a single snp is tested at a time, a multiple testing problem arises. second, we used a pairwise linkage disequilibrium measure between all snp pairs (r) for dropping one of the two snps, which remained in strong linkage disequilibrium. the model for the prediction of future yields at time point t > tn is based on the linear regression, where the population mean () and the fixed additive effects of snps () are described using legendre polynomials of order 3. first, estimators of (,) based on the data set consisting of phenotyped animals are obtained using the following model : 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{y}}(\tau) = { \mathbf{\psi } } (\tau) { \mathbf{\mu } } + \left [{ { { \mathbf{x}}_1 } \otimes { \mathbf{\psi } } (\tau) } \right]{\mathbf{\alpha } } + { \mathbf{\varepsilon } } $ $ \end{document}where y() is a vector of trait values for all phenotyped individuals at time point, x1 is an incidence matrix of snp genotypes for phenotyped animals and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \otimes $ $ \end{document } is the kronecker product. then, using \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{{\mathbf{\mu } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{{\mathbf{\alpha } } } $ $ \end{document }, future yields at time point t for unphenotyped animals are calculated using : 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \widehat{y}_{i}}(1) = { \mathbf{\psi } } (1)\widehat{{\mathbf{\mu } } } + \left [{ { { \mathbf{x}}_{{2i } } } \otimes { \mathbf{\psi } } (1) } \right]\widehat{{\mathbf{\alpha } } }, $ $ \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}$$ { \widehat{y}_{i}}(1) $ $ \end{document } is the predicted future phenotypic value for the ith unphenotyped animal at time point t and x2i is an incidence matrix of snp genotypes for the ith unphenotyped animal. note, now, that n + 2 (not n + 1) time points are available and the corresponding formula for the transformation of time points t into has the following form : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \tau = 2 \cdot \frac{{t - { t_0}}}{{{t^ { } } - { t_0 } } } - 1 $ $ \end{document}note that prediction is performed for two data sets : the nominal data set with all available markers and the selected data set with only snps significant for model 6. the analysed data set was generated using monte carlo simulations for the 13th qtl - mas workshop (wageningen, the netherlands, april 2009). it consists of 2,025 genetically related individuals from two generations : 25 individuals represent the parental generation (20 females and 5 males) and the remaining 2,000 individuals are offspring (100 full sib families, one from each combination of a male and female parent). all individuals have complete marker information consisting of 453 biallelic markers represented by snps, which are randomly distributed over five chromosomes, each of approximately 1 morgan in length. chromosome two is the most densely covered by snps (99 snps), while chromosome five is the most sparsely covered (86 snps). quantitative phenotypes are available for 50 full sib families, while the other 50 full sib families have unknown phenotypes. phenotypes were generated using a logistic growth curve and recorded at five different time points, denoted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{t } } = [{ t_0},{t_1},{t_2},{t_3},{t_4 } ] = $ $ \end{document}. the asymptotic values of individuals yield range from 14 to 66. the phenotyped full sib families are selected such that each female parent has at least 40 phenotyped offspring, while each male parent has 100 phenotyped offspring. three on the first and fifth chromosomes and four on the second, third and fourth chromosomes. for the detection of an association between snps and a longitudinal trait, a single snp random regression model proposed by yang. (2006) is used. in this model, all time - dependent parameters (both fixed and random) are described by legendre polynomials of order 3. this random regression model is an extension of the following single snp linear mixed model : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { y_i}(t) = \mu (t) + { x_i}\alpha (t) + { \xi_i}(t) + { \varepsilon_i } $ $ \end{document}where yi(t) is a phenotypic trait of individual i at time t, (t) is a population mean at time t, xi { 1, 0, 1 } is an snp genotype indicator variable for the ith individual and (t) is a fixed additive snp effect at time t. it is assumed that i(t) is a time - dependent random permanent environmental effect with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n(0,\sigma_{\xi } ^2(t)) $ $ \end{document } distribution and i is a time - independent residual term with n(0,) distribution. the genetic variance at time t for model 1 is expressed as : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_g^2(t) = \sigma_x^2{\alpha^2}(t) = 2p(1 - p){\alpha^2}(t) $ $ \end{document}where p is the frequency of allele 1 for a given snp. the total phenotypic variance at time t has the form : 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_p^2(t) = \sigma_g^2(t) + \sigma_{\xi } ^2(t) + { \sigma^2 } = 2p(1 - p){\alpha^2}(t) + \sigma_{\xi } ^2(t) + { \sigma^2 } $ $ \end{document}note that it is a single snp model, so the genetic and phenotypic variances are calculated separately for each snp. since phenotypic values for each individual are expressed as a longitudinal trait, measured at n + 1 time points, t = [t0, t1,..., tn ], legendre orthogonal polynomials can be used for describing time - dependent effects. these polynomials are defined on an interval [1, 1 ], therefore, the original time points t are recoded into using the following formula : 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \tau = 2 \cdot \frac{{t - { t_0}}}{{{t_n } - { t_0 } } } - 1 $ $ \end{document}where t0 and tn are the extreme time values. for modelling three time - dependent effects ((), (), i()) in model 1, legendre polynomials of order 3 are used. namely, a time - dependent parameter () can be described as a linear combination of (), where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{\psi } } (\tau) = [{ \psi_0}(\tau), { \psi_1}(\tau), { \psi_2}(\tau), { \psi_3}(\tau) ] $ $ \end{document } are coefficients of the polynomial and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{\mu } } = { [{ \mu_0},{\mu_1},{\mu_2},{\mu_3}]^t } $ $ \end{document } is a vector of the time - independent population means. the polynomial coefficients have the following form : 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \psi_0}(\tau) = 1,\;{\psi_1}(\tau) = \tau, \;{\psi_2}(\tau) = \frac{1}{2}(3{\tau^2 } - 1),\;{\psi_3}(\tau) = \frac{1}{2}(5{\tau^3 } - 3\tau) $ $ \end{document}the remaining time - dependent fixed and random terms (() and i()) are described in the same way, using linear combinations of () and ()i. the third order of polynomials is chosen because linear or quadratic curves (polynomial order 2) usually exhibit very poor fit to growth curves. on the other hand, since, for each individual, several observations are available, it would be impossible to fit a higher order of polynomials. it is assumed that random regression coefficients for a permanent environmental effect \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \mathbf{\xi } } _ i } = { [{ \xi_{{i0}}},{\xi_{{i1}}},{\xi_{{i2}}},{\xi_{{i3}}}]^t } $ $ \end{document } are normally distributed with mean 0 and covariance matrix. under the above parameterisation, the model 1 can be transformed into a random regression model, which has the following form : 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { y_i}(\tau) = { \mathbf{\psi } } (\tau) { \mathbf{\mu } } + { x_i}{\mathbf{\psi } } (\tau) { \mathbf{\alpha } } + { \mathbf{\psi } } (\tau) { { \mathbf{\xi } } _ i } + { \varepsilon_i } $ $ \end{document}the genetic variance for model 6 is given by : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_g^2(\tau) = 2p(1 - p){\alpha^2}(\tau) = { \mathbf{\psi } } (\tau) [2p(1 - p){\mathbf{\alpha } } { { \mathbf{\alpha } } ^t}]{{\mathbf{\psi } } ^t}(\tau) $ $ \end{document}and the total phenotypic variance is expressed as : 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \sigma_p^2(\tau) = \sigma_g^2(\tau) + \sigma_{\xi } ^2(\tau) + { \sigma^2 } = { \mathbf{\psi } } (\tau) [2p(1 - p){\mathbf{\alpha } } { { \mathbf{\alpha } } ^t } + { \mathbf{\sigma } } ] { { \mathbf{\psi } } ^t}(\tau) + { \sigma^2 } $ $ \end{document}the estimation of all unknown parameters (,,,) in model 6 is based on the expectation maximisation (em) algorithm as described by yang. (for testing the association between an snp and a phenotype, the likelihood ratio test can be used. the corresponding hypotheses are : h0 : = 0 vs. h1 : i { 0, 1, 2, 3 } (i 0). under h0, the test statistic asymptotically follows the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \chi_{{4df}}^2 $ $ \end{document } distribution. since a single snp is tested at a time, a multiple testing problem arises. second, we used a pairwise linkage disequilibrium measure between all snp pairs (r) for dropping one of the two snps, which remained in strong linkage disequilibrium. the model for the prediction of future yields at time point t > tn is based on the linear regression, where the population mean () and the fixed additive effects of snps () are described using legendre polynomials of order 3. first, estimators of (,) based on the data set consisting of phenotyped animals are obtained using the following model : 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{y}}(\tau) = { \mathbf{\psi } } (\tau) { \mathbf{\mu } } + \left [{ { { \mathbf{x}}_1 } \otimes { \mathbf{\psi } } (\tau) } \right]{\mathbf{\alpha } } + { \mathbf{\varepsilon } } $ $ \end{document}where y() is a vector of trait values for all phenotyped individuals at time point, x1 is an incidence matrix of snp genotypes for phenotyped animals and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \otimes $ $ \end{document } is the kronecker product. then, using \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{{\mathbf{\mu } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widehat{{\mathbf{\alpha } } } $ $ \end{document }, future yields at time point t for unphenotyped animals are calculated using : 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \widehat{y}_{i}}(1) = { \mathbf{\psi } } (1)\widehat{{\mathbf{\mu } } } + \left [{ { { \mathbf{x}}_{{2i } } } \otimes { \mathbf{\psi } } (1) } \right]\widehat{{\mathbf{\alpha } } }, $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \widehat{y}_{i}}(1) $ $ \end{document } is the predicted future phenotypic value for the ith unphenotyped animal at time point t and x2i is an incidence matrix of snp genotypes for the ith unphenotyped animal. note, now, that n + 2 (not n + 1) time points are available and the corresponding formula for the transformation of time points t into has the following form : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \tau = 2 \cdot \frac{{t - { t_0}}}{{{t^ { } } - { t_0 } } } - 1 $ $ \end{document}note that prediction is performed for two data sets : the nominal data set with all available markers and the selected data set with only snps significant for model 6. the analysed data set was generated using monte carlo simulations for the 13th qtl - mas workshop (wageningen, the netherlands, april 2009). it consists of 2,025 genetically related individuals from two generations : 25 individuals represent the parental generation (20 females and 5 males) and the remaining 2,000 individuals are offspring (100 full sib families, one from each combination of a male and female parent). all individuals have complete marker information consisting of 453 biallelic markers represented by snps, which are randomly distributed over five chromosomes, each of approximately 1 morgan in length. chromosome two is the most densely covered by snps (99 snps), while chromosome five is the most sparsely covered (86 snps). quantitative phenotypes are available for 50 full sib families, while the other 50 full sib families have unknown phenotypes. phenotypes were generated using a logistic growth curve and recorded at five different time points, denoted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathbf{t } } = [{ t_0},{t_1},{t_2},{t_3},{t_4 } ] = $ $ \end{document}. the asymptotic values of individuals yield range from 14 to 66. the phenotyped full sib families are selected such that each female parent has at least 40 phenotyped offspring, while each male parent has 100 phenotyped offspring. three on the first and fifth chromosomes and four on the second, third and fourth chromosomes. 1. it was evident that, even after bonferroni correction was applied, many snps significantly affected the trait. in total, 207 snps were significant at the 0.05 level (all snps above the black line in fig. 1). additionally, one of the two snps which remain in strong pairwise linkage disequilibrium (r > 0.8) were excluded from the data set. after applying both selection criteria, the final number of selected snps was 179. in order to check how well the set of significant snps covered the positions of simulated qtl an average distance between two snps ranged between 0.999 and 1.206 cm. as a result, 16 out of 18 qtl (88.89%) were located within the confidence intervals. 1p - values after bonferroni correction for all 453 single - nucleotide polymorphisms (snps). p - values are after logarithm transformation p - values after bonferroni correction for all 453 single - nucleotide polymorphisms (snps). p - values are after logarithm transformation figure 2 illustrated an advantage of a longitudinal model, which was capable of describing the genetic background of a longitudinal trait in a more flexible way than models assuming a genetic effect constant over time. effects of the selected snps during the whole growth period changed between the early and the late growth stage. for example, the effect of snp 48 was low until approximately the 120th day, and then it increased towards the terminal day of the growth period, with an especially rapid change between days 120 and 300. on the other hand, snp 198 had an opposite effect. its influence on the genetic variance was observed mainly at the beginning of the growth phase, between days 1 and 200. an example of a locus with a relatively constant effect was snp 89 (fig. 2a percentage of contributed variance of each significant snp on chromosome one in five different time points. 3percentage of contributed variance of three different snps a percentage of contributed variance of each significant snp on chromosome one in five different time points. b percentage of variance contributed by sn p37 percentage of contributed variance of three different snps the results for the prediction of future yields at time 600 are shown in fig. note that, when all snps were used, some of the individuals had predicted phenotypic values which exceed the trait limit predefined in the simulation. when only the selected snps were used, no individual exceeded the trait limit and the correlation of 0.84 was reached. phenotypes predicted based on the selected snp data set were less variable (standard deviation 6.20) than the ones predicted using all snps (8.10), while the standard deviation of the true values of trait at time point 600 was 5.03. b for the data set with 179 selected significant snps predicted trait value at time point 600 for non - phenotyped animals. in the context of interval qtl mapping, using simulation, yang. (2007) observed that models with time - dependent gene effects provide accurate estimates of qtl positions and effects. they allow for a more precise description of the variability of the dependent variable and require only a few parameters for this purpose. the present analysis demonstrates that the parameterisation of each snp in a longitudinal data context gives a model with a very high flexibility. (2006), in model 6, only additive effects of snps were taken into account. such parsimonious parameterisation allowed us to fit a multiple snps model (model 9). (2006) is extended so that it was not only used for qtl detection, but also for the prediction of future yields. this may play a key role in the pre - selection of animals for breeding. the method proposed for the prediction of future yields is based on the linear regression with snp effects modelled as time - dependent variables. a disadvantage of the proposed prediction approach is that it takes no account of the relationship between individuals. its advantage lies in its computational speed, which might be a critical issue for practical breeding with large data sets and demand for frequent evaluations (e.g. daily, as it is the case in poultry or pig breeding). the same simulated data set was also analysed with other approaches. bayesian methods (bayes a and bayes b) provided the best accuracy of prediction at time 600, ranging between 0.86 and 0.95 (bastiaansen. 2010), whereas the lowest accuracy of 0.65 was obtained using a blup model. a comparison of these results indicates that the snp selection approach plays an important role for prediction purposes, even for a quantitative trait determined by only several (18) qtl. rapid advances in high - throughput genotyping technologies in livestock make large amounts of genotypic data (thousands of snps) available widely with reasonable costs. on the other hand, such rich sources of information can be utilised not only for the prediction of future yields, but also for predicting the genetic value of young selection candidates (schaeffer 2006). that is why models capable of incorporating time - dependent effects are going to gain importance in practical applications in the near future. | in statistical modelling, the effects of single - nucleotide polymorphisms (snps) are often regarded as time - independent. however, for traits recorded repeatedly, it is very interesting to investigate the behaviour of gene effects over time. in the analysis, simulated data from the 13th qtl - mas workshop (wageningen, the netherlands, april 2009) was used and the major goal was the modelling of genetic effects as time - dependent. for this purpose, a mixed model which describes each effect using the third - order legendre orthogonal polynomials, in order to account for the correlation between consecutive measurements, is fitted. in this model, snps are modelled as fixed, while the environment is modelled as random effects. the maximum likelihood estimates of model parameters are obtained by the expectation maximisation (em) algorithm and the significance of the additive snp effects is based on the likelihood ratio test, with p - values corrected for multiple testing. for each significant snp, the percentage of the total variance contributed by this snp is calculated. moreover, by using a model which simultaneously incorporates effects of all of the snps, the prediction of future yields is conducted. as a result, 179 from the total of 453 snps covering 16 out of 18 true quantitative trait loci (qtl) were selected. the correlation between predicted and true breeding values was 0.73 for the data set with all snps and 0.84 for the data set with selected snps. in conclusion, we showed that a longitudinal approach allows for estimating changes of the variance contributed by each snp over time and demonstrated that, for prediction, the pre - selection of snps plays an important role. |
collagenous colitis is an inflammatory bowel disease characterized by normal macroscopic morphology of the colonic mucosa but having a specific histological feature : a collagen band deposition in the lamina propria. although collagenous colitis has been linked to several neutrophilic dermatoses, we have found no reports in the literature associating the disorder with sweet s syndrome and/or neutrophilic panniculitis. we describe the case of a woman who developed sweet s syndrome and a neutrophilic infiltrate, mainly confined to the panniculus, during periods of collagenous colitis activity. a 75-year old woman presented with erythematous and edematous plaques on the interciliary region in december 2005. her medical history included a collagenous colitis diagnosed in 1990 from multiple biopsies of the rectum, colon and cecum during the workup for chronic watery diarrhea. moderate chronic inflammation of the lamina propria and irregular thickening of the basal membrane were observed. the patient did not undergo regular check - ups at our center and chronic diarrhea continued. she first visited our dermatology department in april 2002 when she presented oral ulcers and erythematous papules and vesicles on her lower limbs and trunk. she complained of malaise, arthralgias, fever, diarrhea and progressive weight loss in the previous two weeks. a skin punch biopsy showed acute and chronic perivascular, superficial and diffuse dermatitis with numerous polymorphonuclear lymphocytes (figures 1 and 2). sweet s syndrome was diagnosed and she was admitted to hospital. she was put on oral prednisone at 1mg / kg / day and proctitis symptoms, cutaneous and oral lesions resolved within a week. prednisone was gradually tapered off (10 mg every five days) until complete resolution. figure 1histopathological study of skin biopsy showing a perivascular and interstitial inflammatory infiltrate composed by lymphocytes, histiocytes and abundant neutrophils (he100). histopathological study of skin biopsy showing a perivascular and interstitial inflammatory infiltrate composed by lymphocytes, histiocytes and abundant neutrophils (he100). detail of the lesion, showing dense neutrophilic infiltrate (he400). in december 2005 she complained of fever, oral ulcers and erythematous plaques on periorbital and eyelid regions of 30 days duration. a course of 0.5 mg / kg / day oral prednisone was prescribed and skin lesions improved. when steroids were discontinued, the lesions relapsed and diarrhea and fever reappeared. the patient returned to our dermatology unit one month later and clinical examination revealed erythematous, edematous and well - defined plaques around the eyes (figure 3) and oral ulcers (figure 4). she did not respond to co - amoxiclav 500 mg/8h and was put on prednisone at 20 mg / day. multiple rectum, sigmoid, colon and cecum biopsies were consistent with collagenous colitis. figures 3 and 4 clinical aspect of the facial and oral lesions. clinical aspect of the facial and oral lesions. figures 3 and 4 clinical aspect of the facial and oral lesions. clinical aspect of the facial and oral lesions. it showed a septal and lobular pattern, and the inflammatory infiltrate was composed of lymphocytes, histiocytes and neutrophils. there was a mild perivascular and interstitial infiltrate with neutrophils and lymphocytes in the reticular dermis. treatment was slowly tapered (approximately 5 mg every ten days) and the patient is currently on prednisone at 2.5 mg / day.. figures 5 and 6 histopathological study of skin lesion showing a neutrophilic infiltrate in the subcutaneous fat between septa and adipocytes (he100, he400). histopathological study of skin lesion showing a neutrophilic infiltrate in the subcutaneous fat between septa and adipocytes (he100, he400). figures 5 and 6 histopathological study of skin lesion showing a neutrophilic infiltrate in the subcutaneous fat between septa and adipocytes (he100, he400). histopathological study of skin lesion showing a neutrophilic infiltrate in the subcutaneous fat between septa and adipocytes (he100, he400). we consider that the two forms of neutrophilic dermatoses in this patient with collagenous colitis were reactive to colitis flares. the first skin eruption was diagnosed as sweet s syndrome on the basis of clinical and histological presentation. there was a temporal correlation between cutaneous and digestive symptoms since all skin eruptions were accompanied by diarrhea and all intestinal biopsies performed during these periods were consistent with collagenous colitis. the most common pattern is a septal panniculitis, similar to that seen in erythema nodosum. it may appear as an isolated event or simultaneously with a classical sweet s syndrome. the second type is a pure lobular neutrophilic infiltration of subcutaneous fat (neutrophilic panniculitis). several reports have been published regarding neutrophilic dermatoses presenting as facial or periorbital edematous plaques, sometimes simulating facial cellulitis. we know of only three reports of cutaneous manifestations associated to collagenous colitis, and all three corresponded to pyoderma gangrenosum (one was a peristomal lesion). to the best of our knowledge, this is the first case of collagenous colitis associated to sweet s syndrome and neutrophilic panniculitis. most cutaneous manifestations of inflammatory bowel diseases lie within the spectrum of neutrophilic dermatoses : erythema nodosum, pyoderma gangrenosum, sweet s syndrome and neutrophilic pustulosis. there are several reports in the literature of inflammatory bowel disease patients who developed reactive lesions (erythema nodosum, pyoderma gangrenosum, generalized pustulosis) together with sweet s syndrome as in our case, but these did not occur simultaneously. when associated to inflammatory bowel disease, sweet s syndrome may be a marker of its activity, as it can run parallel to its clinical course. and it is important to note that the association between sweet s syndrome and inflammatory bowel diseases encompasses special features. in this context, the eruption shows a strong predilection for women, and all the reports of inflammatory bowel diseases associated to sweet s syndrome have colonic or perianal involvement. findings in our patient support these data. in conclusion, in view of the parallel course with the intestinal disease, the response to steroids, and the reports in the literature relating inflammatory bowel diseases with neutrophilic dermatoses, we consider that both the sweet 's syndrome and the neutrophilic panniculitis in the present patient were related to collagenous colitis. these findings suggest that collagenous colitis could be added to the list of inflammatory bowel diseases associated to neutrophilic dermatoses. | we report the case of a 75-year old woman with collagenous colitis who presented with erythematous and edematous plaques on the periorbital and eyelid regions, accompanied by oral ulcers. histopathology showed a dermal neutrophilic infiltrate plus mild septal and lobular panniculitis with lymphocytes, neutrophils and eosinophils. five years earlier she had presented a flare of papules and vesicles on the trunk, together with oral ulcers ; a skin biopsy revealed a neutrophilic dermal infiltrate and sweet s syndrome was diagnosed. both the neutrophilic panniculitis and the sweet s syndrome were accompanied by fever, malaise and diarrhea. cutaneous and intestinal symptoms disappeared with corticoid therapy. the two types of neutrophilic dermatoses that appeared in periods of colitis activity suggest that intestinal and cutaneous manifestations may be related. |
with the rapid increase in the proportion of elderly in japan, the proportion of adult diseases such as cancer, ischemic heart disease, cerebrovascular disease, and diabetes mellitus has also increased, now accounting for about 60% of the causes of mortality1, 2. exercise plays a crucial role in the prevention of adult disease and is recommended to maintain or improve health3, 4. it has been reported that a certain intensity of exercise is required for improvements in health and physical strength5,6,7. at least a moderate exercise intensity is required to improve the health of adults6, but comorbidities in the elderly may restrict their ability to exercise. therefore, exercise should be prescribed at a suitable intensity for the elderly, i.e., the intensity at which the benefits of exercise can be gained but risks of exercise avoided. the balance between benefit and safety varies with the physical condition of the individual but is considered to occur at a lower intensity in elderly people with existing disease than in healthy young adults. therefore, exercise intensity in the elderly must be carefully adjusted. anaerobic threshold (at) has been proposed as an objective criterion for precise adjustment of exercise intensity8. at is the exercise intensity just after which energy production by anaerobic metabolism is added to aerobic metabolic energy production during incremental exercise. it is estimated by the lactate threshold (lt), which is the workload at which the blood lactate concentration abruptly increases, and by the ventilatory threshold (vt), which is the workload at which the rate of pulmonary minute ventilation abruptly increases nonlinearly during a progressive exercise test9, 10. it has been reported that both thresholds are safe and effective criteria11, 12. however, both thresholds have disadvantages ; specifically, the gas analyzer used to measure expired gas for determination of vt is expensive and is limited to use in a laboratory setting, and blood collection for determination of lt is invasive. one is the double - product breakpoint (dpbp)13, or the point at which double product (dp), the product of heart rate and systolic blood pressure, abruptly increases during a progressive exercise test. the dpbp can be calculated from the intersection of two regression lines, and its validity has already been established based on the high correlation between dpbp and at (r = 0.87, p < 0.001)14,15,16. another method for estimating at is heart rate threshold (hrt), which uses only heart rate17, 18. hrt is obtained from the relationship between running speed and heart rate. the running speed heart rate relationship is linear from low to submaximal speeds and curvilinear from submaximal to maximal speeds. the speed of transition from the linear to the curvilinear phase coincides with the beginning of a sharp accumulation of blood lactate18. however, it has been reported that the dpbp method is inferior to expired gas analysis in detecting at14 and that the hrt occurs at a higher workload than at19. for this reason, it is necessary to develop a more sensitive indicator or to create a method combining a number of indicators to improve the ability to detect at and thus prescribe suitable exercise to subjects with reduced exercise tolerance. both systolic blood pressure and heart rate, the components of dp, are controlled by the sympathetic nervous system. the plasma catecholamine concentration, which reflects sympathetic nervous system activity during incremental exercise, has been shown to start to rise abruptly at a workload similar to the lt. in addition, it has been reported that the plasma catecholamine concentration correlates with the blood lactate concentration20, 21. this suggests that dp may reflect changes in sympathetic nervous system activity and blood lactate concentration through the plasma catecholamine concentration. therefore, it is possible to detect the at more precisely if an index is used that is a more sensitive reflection of sympathetic nervous system activity. the proposed index of sympathetic nervous system activity is salivary -amylase, an enzyme in saliva that has been reported to be controlled by the sympathetic - adrenal - medullary system22, 23 and to be a sensitive indicator of the activity of the sympathetic nervous system24. therefore, it is possible to detect changes in blood lactate concentration indirectly by measuring salivary -amylase, a measurement that can be performed quickly under test conditions. investigated the relationship between the salivary threshold (tsa), the workload at which salivary -amylase abruptly increases, and the lt by measuring salivary -amylase and blood lactate concentrations during a multistage incremental exercise25. the authors concluded that tsa had a high correlation with the lt (r = 0.95, p < 0.001), and the detection rate was 80%. the results suggested that the high correlation between the tsa and lt, as well as the ease of measuring salivary -amylase, makes tsa a valid and useful estimate of at. moreover, no report has investigated the relationship between tsa and other methods of estimating at. determination of whether the detection rate is improved by combining tsa and dpbp would provide meaningful information for exercise prescription, particularly for elderly people with preexisting disease. the purpose of this study was to assess the validity of salivary -amylase as a method for estimating at quickly and to clarify the relationship between salivary -amylase and dpbp in order to establish a way to adjust exercise intensity to a safe and effective range. the subjects of this study were 11 healthy young adults (23.8 1.8 years, height 173 5.0 cm, body weight 64.6 6.3 kg, body mass index 21.7 1.3 none of the subjects reported any neurological or vestibular disorders, orthopedic conditions, or oral cavity diseases before participating in this study. we used an electrically braked cycle ergometer (aerobike 75xl, combi co. ltd., the timing of the experiment (6:00 p.m. to 8:00 p.m.) was chosen to minimize the influence of circadian variation in salivary -amylase26. since the activity of salivary -amylase can be influenced by alcohol, medications, food, and caffeine, all subjects were instructed not to drink any alcohol the day before measurement and not to consume food or caffeine 2 hours before measurement. after a rest period of 3 min on the ergometer, subjects were instructed to try to maintain a cadence of 50 rpm while the workload level was increased by 20 watts per 3 min beginning with 10 watts. the exercise test was continued until subjects could no longer maintain the prescribed cadence due to fatigue or until completion of 2 stages following that at which at was observed. oxygen consumption (vo2), carbon dioxide production (vco2), and ventilatory equivalent (ve) were measured using a breath - by - breath gas analyzer (ae-300, minato medical science, osaka, japan). systolic blood pressure and heart rate were measured during the last 30 seconds of each stage of the incremental exercise test. heart rate was measured using an electrocardiogram monitor (bsm-2401, nihon kohden, tokyo, japan). the dpbp was determined using a computer algorithm as follows : the linear regression lines of dp as a function of workload were calculated for all possible divisions of the data. dpbp was determined by choosing the intersection, or breakpoint, of the 2 lines representing the minimum residual sum of squares from among various intersections of the 2 lines. salivary -amylase was measured using a portable salivary amylase analyzer (salivary amylase monitor, nipro, osaka, japan). it was verified that within the analyzer s linear range (10230 ku / l), this handheld monitor s accuracy (r = 0.989), precision (coefficient of variation < 9%), and measurement repeatability (range 3.1% to + 3.1%) approached those of a more elaborate laboratory - based automated clinical chemistry analyzer (olympus america inc., center valley, pa, usa). salivary -amylase was measured from saliva samples collected during the last 30 seconds of each stage of an incremental exercise test by inserting the reagent test strip directly into the subject s oral cavity. the mask for expired gas analysis was briefly removed to obtain the saliva sample and then replaced after saliva collection. workload at the at, tsa, and dpbp were compared using one - way anova to investigate the difference in workload between each index. pearson s correlation coefficients were used to assess the relationship between workload at the at and tsa and between the at and dpbp. in addition, with tsa-1 (or dpbp-1) representing 1 workload prior to that corresponding to tsa (or dpbp), tsa-2 representing 2 workloads prior, tsa-3 representing 3 workloads prior, and so on, tsa, tsa-1, tsa-2, and tsa-3 and workloads for dpbp, dpbp-1, dpbp-2, and dpbp-3 were compared by one - way anova with repeated measures to identify the dynamics of salivary -amylase and dpbp associated with an incrementally increasing workload. subject characteristicsage (yr)23.8 1.8height (m)1.73 0.05weight (kg)64.6 6.3bmi (kg / m)21.7 1.3hr at rest (beats / min)70.0 11.4hr at at (beats / min)116.2 16.4vo2 at at (ml / min)1,170.2 189.3vo2/w at at (ml / minkg)18.1 2.3salivary -amylase at rest (ku / l)22.2 27.5salivary -amylase at tsa (ku / l)40.0 18.1dp at rest (mmhg beats / min)8,740.4 1,994.7dp at dpbp (mmhg beats / min)14,868.2 3,153.1data presented as mean sd. bmi, body mass index ; at, anaerobic threshold ; dp, double product ; dpbp, double - product breakpoint ; hr, heart rate ; tsa, salivary threshold ; w, watt. in the present study, at could be determined in all subjects (100%). however, tsa and dpbp could not be detected in 2 of the subjects (detection rate of 82% for both methods) because the increase was not abrupt enough to identify 2 regression lines clearly. in the following analysis, therefore, data for the subjects whose tsa and dpbp could not be detected were excluded. the results of one - way anova revealed that there was no significant difference among workloads at the at, dpbp, and tsa (table 2table 2. power output at at, tsa, and dpbpat (w)70.0 28.3tsa (w)61.1 28.5dpbp (w)65.6 21.9at, anaerobic threshold ; dpbp, double - product breakpoint ; tsa, salivary threshold ; w, watt). bmi, body mass index ; at, anaerobic threshold ; dp, double product ; dpbp, double - product breakpoint ; hr, heart rate ; tsa, salivary threshold ; w, watt at, anaerobic threshold ; dpbp, double - product breakpoint ; tsa, salivary threshold ; w, watt there were significant correlations between at and tsa (r = 0.951, p < 0.01) and between at and dpbp (r = 0.940, p < 0.01). the results of one - way anova with repeated measures identified the significant main effects of salivary -amylase (f1.58, 12.6 = 24.0, p < 0.01, partial = 0.75) and dp (f1.34, 10.3 = 44.7, p < 0.01, partial = 0.85), and post hoc analysis using bonferroni s test was executed. the results showed that there were significant differences between tsa and tsa-1 (p < 0.05), between tsa and tsa-2 (p < 0.01), and between tsa and tsa-3 (p < 0.01). however, there were no significant differences between tsa-1 and tsa-2, between tsa-1 and tsa-3, or between tsa-2 and tsa-3. the purpose of this study was to assess the validity of tsa as a workload index by clarifying the relationship between at, which was measured by analysis of expired gas, and tsa, which represented the workload at which salivary -amylase abruptly increased. we also investigated the relationship between dpbp, which has been established as a method for quickly estimating at, and tsa in order to assess the usability of tsa as another method for quickly estimating at. the results of this study indicate significant correlations between at and tsa (r = 0.951, p < 0.01). furthermore, a significant difference was not observed in the workload at the at and tsa. this result suggests that tsa has validity as a method for estimating at quickly, which confirms results reported by calvo. yamamoto. investigated the activity of the parasympathetic and sympathetic nervous systems during exercise using heart rate variability. the authors revealed that the indicator of parasympathetic nervous system activity decreased progressively from rest to a workload equivalent to 60% vt and that the indicator of sympathetic nervous system activity increased only when exercise intensity exceeded vt28. these results suggest that the activity of the sympathetic nervous system changed at the vt, which was equivalent to the at, and that at might be estimated by monitoring the activity of the sympathetic nervous system. moreover, it has been reported that the salivary -amylase concentration increases with increased physical activity, such as treadmill exercise29, running30, 31, and cycle exercise24, 32.. investigated salivary -amylase during walking, jogging, and running and reported that jogging and running increased salivary -amylase but that walking did not affect salivary -amylase24. this result confirmed that the increase in salivary -amylase was caused by physical activity exceeding a certain workload (i.e., the at). chatterton. also investigated the correlation between salivary -amylase and plasma catecholamines and showed significant correlations between them (r = 0.64 for norepinephrine and r = 0.49 for epinephrine)24. this result directly indicates that salivary -amylase reflects the activity of sympathetic nervous system. since salivary -amylase is an index that sensitively reflects the activity of the sympathetic nervous system and the activity of the sympathetic nervous system is caused by workloads above the at, it is suggested that tsa, which is the point of inflection of salivary -amylase, is a good index by which to estimate at. the results of the present study also indicated that tsa was at least as good as dpbp as a method for estimating at quickly because the correlation between the at and tsa was 0.951 (p < this result suggests that salivary -amylase reflects the activity of the sympathetic nervous system more sensitively than dp, the components of which are controlled by the sympathetic nervous system. spence. compared the systolic and diastolic blood pressure responses to exercise before the at with those after the at by using a cycle ergometry33. the authors concluded that the rate of increase of systolic blood pressure significantly increased after the at. tanaka. also demonstrated that the slope of a regression line of systolic blood pressure and heart rate increased after the at by comparing the slope of a regression line of systolic blood pressure and heart rate before and after the at13. however, other researchers reported that the slope of a regression line of systolic blood pressure and heart rate may not increase after the at. riley. investigated the relationship between dpbp and at, as determined from analysis of expiration gas, by measuring systolic blood pressure and heart rate during an exercise test14. their results indicated that, although dpbp and at are strongly correlated, the rates of increase of systolic blood pressure and heart rate do not always increase after the at. in the study of riley., the rate of increase of systolic blood pressure increased after the at for some subjects but that for heart rate did not ; other subjects indicated the opposite. furthermore, conconi. researched the relationship between hrt and lt by obtaining the hrt, which was calculated from running speed and heart rate18. the results indicated that there was a strong correlation between hrt and lt and that increased running speed involved an increase in heart rate. however, the rate of increase of heart rate after the hrt decreased, which is in contrast to the results of tanaka13. both riley.14 and conconi.18 demonstrated that changes in systolic blood pressure and heart rate before and after the at differed among individuals. dp is highly correlated with at because it consists of 2 variables, so that even if only 1 variable shows unique changes, the other variable is able to change in a complementary manner. however, using the product of variables that vary among individuals may lead inaccurate estimation of at. salivary -amylase is therefore superior to dp in the degree of correlation to at because salivary -amylase can measure the activity of the sympathetic nervous system using only 1 variable. there are other ways in which tsa is superior to dpbp for estimation of at. to calculate dp, blood pressure and heart rate must be measured, but blood pressure measurement temporarily restricts subjects physical activity. additionally, measurement of heart rate requires equipment such as an electrocardiograph monitor, thus restricting the measurement environment. in contrast, measurement of salivary -amylase requires only insertion of the reagent test strip directly into the subject s oral cavity to collect saliva samples without the need to interrupt activity, and it can be performed in various environments. thus, salivary -amylase estimates at simply and has the advantages of easier measurement and more sensitive reflection of sympathetic nervous system activity than can be achieved with dp. interestingly, the results of the present study suggest that a more correct estimation of at can be achieved by combining tsa and dpbp. in this research, although at was identified in all subjects using expired - gas analysis, tsa and dpbp were unable to be determined in 2 subjects. however, because data allowing determination of dpbp were always collected, combining tsa and dpbp allowed identification of at in all subjects. this finding suggests that the variables salivary -amylase, systolic blood pressure, and heart rate are controlled by the sympathetic nervous system but that the expression of these variables in vivo differs among individuals. therefore, inclusion of all the variables in the estimation of at may allow a more precise estimation. at estimation using salivary -amylase is useful for subjects in whom tsa can be identified even if this is the only method used because tsa and at are strongly correlated. however, the combination of dpbp and tsa might be desirable for some subjects, since tsa was unable to be identified in about 18% of our study subjects. while it is better to measure salivary -amylase to estimate at because of its superiority to dpbp in the degree of correlation with at, if tsa is not able to be determined, dp can be calculated to complement the detection of at. as a result, at can be determined with a detection rate as high as that for expired - gas analysis. this result is in line with the study of calvo.25, who mentioned that differences among subjects in their hydration state before reporting to the laboratory or in the contribution of each salivary gland to the production of total saliva explained the inability to detect tsa in 20% of their subjects. in this study, although we encouraged subjects to drink water before measurement and allocated a point under the tongue for saliva collection in order to minimize these influences, the detection rate was not improved. further studies are needed to improve the precision of measurement of salivary -amylase itself. the results of this study clarified that tsa is a useful index for estimating at during an incrementally increasing workload. tsa was not able to be detected in 2 subjects, but in such cases, the rate of determination of at can be improved by combining tsa with dpbp. determination of at using salivary -amylase is superior to other methods because the measurement device is easily transportable, making measurement possible in various environments, and because of the noninvasiveness of the technique. we believe that the ability to adjust exercise to the optimal intensity in various environments using salivary -amylase can make exercise safer and more effective. | [purpose ] the purpose of this study was to clarify the validity of salivary -amylase as a method of quickly estimating anaerobic threshold and to establish the relationship between salivary -amylase and double - product breakpoint in order to create a way to adjust exercise intensity to a safe and effective range. [subjects and methods ] eleven healthy young adults performed an incremental exercise test using a cycle ergometer. during the incremental exercise test, oxygen consumption, carbon dioxide production, and ventilatory equivalent were measured using a breath - by - breath gas analyzer. systolic blood pressure and heart rate were measured to calculate the double product, from which double - product breakpoint was determined. salivary -amylase was measured to calculate the salivary threshold. [results ] one - way anova revealed no significant differences among workloads at the anaerobic threshold, double - product breakpoint, and salivary threshold. significant correlations were found between anaerobic threshold and salivary threshold and between anaerobic threshold and double - product breakpoint. [conclusion ] as a method for estimating anaerobic threshold, salivary threshold was as good as or better than determination of double - product breakpoint because the correlation between anaerobic threshold and salivary threshold was higher than the correlation between anaerobic threshold and double - product breakpoint. therefore, salivary threshold is a useful index of anaerobic threshold during an incremental workload. |
although the reconstruction of anterior cruciate ligament (acl) in patient with acl injury has been considered a standard treatment, the ideal time of reconstruction has been debated among various studies.1 the earlier reconstruction surgery after acl injury can facilitate early return to sports activity and decrease the incidence of meniscal injury. however, loss of range of motion (rom) and wound complications are reported to occur more frequently in patients treated with an early acl reconstruction.2 hence, shelbourne.2 suggested that delaying acl reconstructions for more than 3 weeks till maximum of 2 months after an acute injury may decrease the incidence of arthrofibrosis, and also result in superior recovery of rom and lower extremity power including quadriceps muscle. early acl reconstruction, before retuning to activity eliminates recurrent episodes of instability and thereby decreases chances of meniscal and cartilage injury. many studies have reported that the delay in acl reconstruction is associated with an increased incidence of meniscus and cartilage lesions.134567891011121314 therefore, early rather than delayed acl reconstruction has been recommended. however, there are no clear and uniform guidelines regarding the timing of acl reconstruction or clarity in the definition of early and delayed reconstruction to reduce the complications after reconstruction in the acl injured knee. moreover, only a few studies compared the results of earlier acl reconstruction regarding proprioception or recovery of muscle power compared with delayed acl reconstruction.615 we hypothesized that (1) the early acl reconstruction can reduce the incidence of meniscal or cartilage injuries compared with delayed acl reconstruction and (2) the early acl reconstruction has more benefits regarding recovery of muscle power and proprioception than delayed acl reconstruction would offer. the objectives of this study were to compare the incidence of meniscal or cartilage injuries based on the arthroscopic finding during the reconstruction between early and delayed acl reconstruction. we also compared clinical outcomes, quadriceps and hamstring muscle power and proprioception of the early acl reconstruction with those of the delayed acl reconstruction. 101 patients who underwent acl reconstruction from september 2008 to march 2012 were included in this prospective study. during the study period, all patients who received primary acl reconstruction with a quadruple hamstring tendon within 3 weeks or more than 3 months after injury and followed up to minimum of 2 years were included in this study. within this cohort, five patients with prior knee surgery, two patients with a multi - ligament knee injury, and three patients who lacked information regarding the date of injury were excluded leaving a total of 91 patients for our study group. we excluded patients that had aclr between 3 weeks and 3 months (intermediate period) because we want to compare acute and chronic cases. the early (within 3 weeks) reconstruction group had 48 knees and delayed (more than 3 months) group was 43 knees. the average period from injury to surgery in the early reconstruction group was 2 weeks (range 13 weeks). patients in this group included 35 men and 13 women with an average age of 30.1 years at the time of surgery. in the delayed group, the average period from injury to surgery was 35.6 weeks (range 1274 weeks), and it included 36 men and seven women with an average age of 30.0 years at the time of surgery. the sex, age at surgery, and followup duration, as well as preinjury activity score, were similar in both groups [table 1 ]. comparison of the two groups with respect to meniscal injury the diagnosis of an acl tear was made by the surgeon based on history, physical examination or magnetic resonance imaging finding. the acl reconstruction was usually performed within 1 week after the patient presented at outpatient clinic if knee showed grade ii or more than grade ii instability according to lachman or pivot - shift test. in acl reconstruction, we used single bundle transtibial technique, and used endobutton in femur and bio - absorbable interferential screw along with spiked washer and cortical screw in tibia fixation. meniscus repair or resection was performed based on the surgeon 's intra - operative discretion. location and grade of cartilage injury were recorded for any lesions noted intra - operatively according to international cartilage repair society (icrs) grade system.16 the number of patients with cartilage or meniscal lesions found during the reconstruction surgery was compared between two groups. in patients with meniscal tear, we performed meniscus repair. the indications for meniscal repair include the following : (1) a complete vertical longitudinal tear more than 10 mm in length, (2) a tear within the peripheral 1030% of the meniscus or within 3 or 4 mm of the meniscocapsular junction, (3) a peripheral tear that can be displaced toward the center of the plateau by probing, thus demonstrating instability. we also compared clinical outcomes with regards to international knee document committee (ikdc) score,17 rom including flexion contracture, and tegner activity scale.18 the stability with regard to lachman and pivot - shift test, and instrumented anterior laxity using telos (metax, hungen, germany) device were also evaluated and compared between two groups preoperatively under anesthesia before reconstruction and at the final followup. for the comparison of proprioception, we performed a sensory organization test (sot) by using a computerized dynamic posturography using smart balance master (neurocom international, clackamas, or, usa). after documenting medical history and daily activities, subjects were instructed to stand on the footplate of the smart balance master unit and to face the visual surround. the 2 footplates were supported by 5 force transducers (strain gauges) mounted symmetrically on a supporting center plate. the computer receives force measurements from the dual footplates, analyzes the information, and generates a screen display or printed report. the sot was designed to assess static and dynamic postural balancing ability under 6 different conditions that involve altered visual inputs and different support surfaces (sot-1, eyes open ; sot-2, eyes closed ; sot-3, sway vision ; sot-4, eyes open with a swaying support ; sot-5, eyes closed with a swaying support ; and sot-6, sway vision with a swaying support). among them, we evaluated the sot-5 in the dynamic conditions. each of the sot condition was conducted 3 times, and the average value of the 3 trials was used for data analysis. the highest possible score was 100%, which indicated that the subject did not sway at all, and a score of 0% indicated a fall from the footplate. quadriceps and hamstring isokinetic strength was assessed at the velocities of 180/s with a dynamometer (biodex system 2 ; biodex medical system, shirley, new york, usa) at the final followup visit after the acl reconstruction. a standardized application of equipment, data collection and procedure for warm - up were applied. before data collection the uninjured extremity was tested first and then the same procedure was performed for the injured one. quadriceps and hamstring isokinetic peak torques of the injured extremity were expressed as a percentage compared with those of the uninjured extremity. the same postoperative rehabilitation protocol was used in both groups. from the 1 day after surgery, a brace without angle limitation was worn, and rom training and quadriceps strengthening exercise were initiated. partial weight - bearing was allowed at 2 weeks after operation, and full weight - bearing was allowed at 6 weeks postoperatively. descriptive statistics were calculated as follows : continuous variables are presented as mean standard deviation and categorical variables are described as frequencies and percentages. we used an independent sample 's t - tests for continuous values and chi - square test for categorical values for the comparisons of two groups. the analysis was performed using spss software (spss for windows release 16.0, chicago, il, usa). descriptive statistics were calculated as follows : continuous variables are presented as mean standard deviation and categorical variables are described as frequencies and percentages. we used an independent sample 's t - tests for continuous values and chi - square test for categorical values for the comparisons of two groups. the analysis was performed using spss software (spss for windows release 16.0, chicago, il, usa). arthroscopic finding revealed, 25 (52%) of 48 patients in early group and 27 (62.8%) of 43 patients in delayed group had meniscal injury, which was not statistically significant (p = 0.06) [table 1 ]. among them, we performed repair in 40% (10 out of 25 cases) in early group and 11% (3 out of 27 cases) in delayed group (p = 0.04). regarding cartilage injuries more than icrs grade i, there was no significant difference between two groups (15 and 20 cases in early and delayed group, respectively ; p = 0.14) [table 2 ]. comparison of the two groups with respect to cartilage injury based on international cartilage repair society grading lysholm knee (lk) score at the final followup were similar in two groups ; the early reconstruction group showed 94.5 8.9 and the delayed reconstruction group showed 96.3 3.7 (p = 0.28) [table 3 ]. preinjury and postoperative tegner activity scales also were similar in two groups. at the final followup, the tegner activity scores were slightly reduced in both groups compared with those of preinjury activity. the tegner activity scores was 6.0 1.6 in early reconstruction group and 5.6 1.5 in delayed reconstruction group (p = 0.27) [table 3 ]. comparison of the two groups with respect to clinical outcomes the maximal flexion averaged 138.6 4.1 and 138.8 5.6 in the early and the delayed reconstruction groups at the final followups (p = 0.89) [table 3 ]. the flexion contracture were 0.3 1.7 in the earlier reconstruction and 0.4 1.3 in the delayed reconstruction groups (p = 0.93) [table 3 ]. in both groups, all patients showed, at least, grade ii instability preoperatively and improved to normal or grade i except for four cases of grade ii in early group and three cases of grade ii in delayed group. these results indicate that there were no significant differences regarding preoperative and postoperative grade in both groups (p = 0.93 and 0.58, respectively) [table 4 ]. in the pivot - shift test at preoperatively and final followup, we could not find any inter - group differences (p = 0.96, preoperatively ; p = 0.71, followup) [table 4 ]. comparison of the two groups with respect to knee stability according to lachman and pivot shift test the muscle power results were not significantly different regarding extension and flexion power at the final followup between two groups (p = 0.07, p = 0.06). the mean extension and flexion power at the final followup were 83.3 7.6% and 86.0 7.4% in the early reconstruction group and 80.7 5.9% and 83.2 6.9% in the delayed reconstruction. in posture control, the equilibrium scores on stage 5 at the final followup also were similar in the two groups (p = 0.66). the equilibrium scores on stage 5 were 82.8 8.7% in early group and 82.0 8.3% in delayed group. this study compared before 3 weeks and after 3 months acl reconstruction groups regarding the incidence of meniscal or chondral injury and clinical outcomes including proprioception and muscle power with a minimum of 2 years followup. our data demonstrated that the early reconstruction group showed the higher possibility of repair for meniscal injury. however, we were unable to identify any differences regarding functional scores, muscle power and posture control, rom and incidences of meniscal or cartilage injuries. therefore, most of our hypothesis were not proved positively by the data obtained in this study. some studies have cited that increased time to surgery is a risk factor for increased medial meniscus injury.57111920 papastergiou.11 reported that the prevalence of medial meniscus tear was increased with time, especially after 3 months from injury. they recommend acl reconstruction within 3 months after injury for reducing the risk of meniscal injury. our results were different from previous findings that delay in surgery by 3 months has increased chances of meniscal injury. similarly to our results, smith.21 could not find any significant increase in meniscal injury with time after acl injury in meta - analysis study. michalitsis.14 also reported no significant increase of meniscal lesion after 3 or 12 months from injury. however, this study showed a higher probability of meniscal repair in early than delayed reconstruction. while the prevalence of articular cartilage damage has been variably reported as about 20% in acute acl tears, chronic acl - deficient knees showed cartilage damage about 40% after 12 months.4212223 michalitsis.14 also reported that there was a significant increase of chondral lesion after 12 months from injury, but not after 3 months. however, smith.21 injury in meta - analysis study reported no significant increase of chondral injury with time after acl injury. similar to their findings, time to surgery > 3 months from injury did not have a strong increase in cartilage injury in this study. however, we could not say that our results were different from other 's study because 3 months after injury might be too short period to result in cartilage damage. in this study, we could not find any statistical significance between the early and delayed acl reconstruction groups for the lk score and tegner activity score, which were similar to others study.421 in addition, the postoperative tegner activity scores slightly reduced in both groups when compared with those of preinjury activity. we assume that this finding is probably due to the patient 's unwillingness to participate in stressful activities. recently, early acl reconstruction is preferred thanks to aggressive rehabilitation after acl reconstruction.34 however, the limited rom is still major problem in early acl reconstruction and the ideal time of early acl reconstruction is debatable., we could not observe any significantly limited rom even after early reconstruction (<3 weeks). however, there has been an increasing trend toward earlier reconstruction because early and aggressive rehabilitation after acl reconstruction can prevent the loss of rom after reconstruction. recovery of muscle hamstring and quadriceps power is important for the returning to sports and occupational work. in this study, we hypothesized that the early acl reconstruction can prevent the decrease in muscle power after acl injury. however, in this study, early reconstruction had no benefit regarding proprioception and muscle deficit in comparison to late reconstruction group after minimum of 2 years followup. the limitations of the study are that rom was measured using goniometer instead of x - ray, not a prospective randomized study. the patients were allocated into one of study group based on the time from injury date when they were seen at the outpatient clinic. the last limitation is that we could not evaluate the success of meniscal repair in both groups. however, no patients showed symptoms related to meniscal tear at the followup in both groups. early acl reconstruction (within 3 weeks) had good clinical results and stability as good as delayed reconstruction (more than 3 months) without the limitation of knee motion, muscle power, and postural control. moreover, early acl reconstruction should be recommended for the increase of possibility of repair of the torn meniscus. this study was supported by a grant of the national research foundation of korea funded by the ministry of education, science and technology (2014r1a1a2059147 and 2011 - 0030034). this study was supported by a grant of the national research foundation of korea funded by the ministry of education, science and technology (2014r1a1a2059147 and 2011 - 0030034). | background : early acl reconstruction, before retuning to activity eliminates recurrent episodes of instability and thereby decreases chances of meniscal and cartilage injury. however, there are no clear and uniform guidelines regarding the timing of acl reconstruction or clarity in the definition of early and delayed reconstruction to reduce the complications after reconstruction in the acl injured knee. the purpose of this study was to compare the clinical outcome, stability, muscle power, and postural control after early and delayed anterior cruciate ligament (acl) reconstruction.materials and methods : patients who had acl reconstruction with a quadruple hamstring tendon with a minimum 2-year followup were evaluated. early (within 3 weeks) reconstruction group was 48 knees and delayed (more than 3 months) group was 43 knees. we compared the two groups with regard to lysholm knee score, range of motion (rom), lachman test, tegner activity scale, associated meniscal or chondral injuries, and anterior laxity. we also compared muscle strength with an isokinetic dynamometer and postural control with computed dynamic posturography at the final followup.results:while 50% of early and 70% of delayed group had meniscal injuries (p = 0.06), of which were reparable in 42% of early group and 17% of delayed group (p = 0.04). however, there was no significant difference in cartilage injury (p = 0.14). at the final followup, no significant differences were found between two groups for lysholm score (p = 0.28), tegner activity scale (p = 0.27), and rom. the stabilities regarding lachman and pivot - shift tests, and anterior laxity also showed no significant differences between two groups. the mean extension and flexion muscles power, and postural control showed no significant inter - group differences (p > 0.05).conclusions : early acl reconstruction had excellent clinical results and stability as good as delayed reconstruction without the problem of knee motion, muscle power, and postural control. moreover, early reconstruction showed the high possibility of meniscal repair. therefore, early acl reconstruction should be recommended. |
there is increasing interest globally in equity in health and the pathways by which inequities arise and are perpetuated or exacerbated. concerns over persistent inequalities in health outcomes across individuals and social groups have been voiced across both the developed and developing worlds. the crucial role of health systems in maintaining and improving population health makes equity in the inputs towards managing and delivering health services also a subject of considerable interest. a recent review of the scientific literature suggested that the majority of publications on equity in health focus on access, utilization and financing of health services. receiving relatively less attention is the role of human resources in the health system, and this despite the argument by the world health organization that human resources are " the most important of the health system 's inputs ". imbalance in the supply, deployment and composition of human resources for health (hrh), leading to inequities in the effective provision of health services, is an issue of social and political concern in many countries. inequitable distribution of health workers remains a major problem even in countries with strong central planning structures and apparently rigorous regulations and staffing norms (such as india, pakistan and nepal). imbalances, especially shortages of skilled health personnel in rural or poor areas, are reported to have a number of adverse consequences for health system performance. the potential negative consequences include lower quality and productivity of health services, closure of hospital wards, increased waiting times, reduced numbers of available beds for inpatients, diversion of emergency department patients and under - utilization of remaining personnel or substitution with persons lacking the required skills for performing critical interventions. common underlying problems that may contribute to imbalances include inadequate pay, benefits or other incentives, as well as poor management of health personnel. in some cases, they may include the active recruitment of health personnel from poorer to richer areas within a country, or from poorer to richer countries. in the north american context, for example, cross - border traffic along the mexico - united states border is intense, involving migrants of all social and occupational groups. but most movement of health personnel takes place within countries, and internal disparity in the geographical distribution of skilled workers is an important issue in many developing countries. for instance, the nicaraguan capital of managua contains around half the country 's available health personnel, but only one - fifth of the population. similarly, in bangladesh the health sector is characterized with a strong concentration of physicians (35%) and nurses (30%) in four metropolitan districts where less than 15% of the population live. there is a wide diversity in the approaches to how imbalances are monitored, reflecting the diversity of definitions and measures for addressing the topic. imbalances can be considered in terms of the various health occupations, such as shortages or surpluses of physicians or nurses, as well as within a given occupation, such as medical specialists of one type or another. they can be addressed in terms of geography, such as by rural / urban typology or by administrative or epidemiological regions. for instance in mexico, whereas gender imbalance in terms of enrolment in medical schools diminished over the period 19701998, unemployment rates for women physicians remained much higher than those for their male counterparts. in this study, we focus on geographical imbalance in the health workforce, which can be defined as observed differences in the supply of health personnel across spatial units. geographical variations of hrh can have a critical impact in terms of equity of access to health services. it has been suggested that virtually all countries suffer to some extent from an unequal distribution of hrh (except, perhaps, certain countries that are geographically small). it is widely speculated that health workers, especially highly skilled professionals, are more likely to prefer settling in selected (usually more urban and affluent) areas, which may offer greater opportunities for professional development, education and other amenities for their families, and attractive employment possibilities. despite the undoubted importance of monitoring the distribution of hrh and the impacts on health system performance, many sources that can potentially produce statistics relevant to this issue remain underused in health research, especially among developing countries. although a range of standard data sources can be exploited for conducting hrh assessments including population censuses, routine administrative records and sample surveys their potential for monitoring health human resources including questions of distribution and equity has generally not been met. household - based censuses can be a key source for statistics describing human resources for health, providing precise information on the total stock and composition of the health workforce as well as distribution by spatial units and socio - demographic characteristics. censuses offer an advantage over surveys in that they do not suffer from problems of sample sizes too small to allow estimates at the sub - national level. moreover, routine administrative records, which are commonly used in countries to estimate their hrh stock, tend to be subject to differences in the underlying regulations and administrative procedures of government ministries and professional associations, rendering comparisons difficult across countries and over time. the main objective of this study is to investigate the uses of census data for assessing the extent of geographical imbalance (if any) that characterizes the health workforce, as a step towards monitoring and evaluation of health policy planning and management. for illustrative purposes, we draw on data from the national censuses for three developing countries from three different regions : kenya, mexico and viet nam. at the same time, many studies on geographical imbalance have considered only physicians [9,12 - 14 ], or sometimes physicians and nurses. but health human resources encompass a wide range of personnel with different occupations and skill levels working across different domains of health systems : public and private sectors ; clinical, research and public health interventions ; preventive and curative personal care ; and health services planning and management. who broadly defines hrh as " the stock of all individuals engaged in the promotion, protection or improvement of population health ". from this perspective, we include in the analysis a grouping of occupations with health - related specializations. since health care professions can have different national histories and cultures, and the roles of health care workers can vary from country to country, census findings are processed against internationally standardized classifications to enhance comparability. data used in this research are drawn from the integrated public use microdata series (ipums), a collaborative project dedicated to collecting and distributing census data and documentation from around the world. its goals are to collect, preserve, harmonize and disseminate microdata for scientific and policy research. while census microdata samples exist for many countries, access to these data has generally been limited, and comparisons across countries or time periods are difficult because of inconsistencies in both data and documentation. the ipums - international project addresses these issues by converting census microdata for multiple countries into a consistent format. machine - readable census microdata samples, once processed to respect privacy and confidentiality standards, are made available to registered users through a world wide web - based data dissemination system. among the data currently available from the ipums - international archives are samples from the 1989 census of kenya (population census of 24/25 august 1989) as well as the latest censuses of mexico (xii censo de poblacin y vivienda 2000) and viet nam (population and housing census 1999). while ipums has data from the 1999 kenya census, this later round did not include questions on occupation that would have enabled distinguishing the health workforce, and so was not used here. health human resources were identified through questions on main occupation among the economically active population, and matched against the international standard classification of occupations (isco) to enhance cross - national comparability. under isco, occupations are essentially organized according to two dimensions : skill level and skill specialization. occupational titles are pooled into a hierarchical four - digit system, which can be aggregated to progressively broader groups, representing a value set describing the different tasks and duties of jobs. the last revision of isco, in 1988, aimed to produce an international classification that would better reflect the labour markets of developing countries as well as of industrialized countries, covering the " informal " as well as the " formal " sectors, and also better represent women 's positions in the labour market. in particular, we were interested in two categories of health care practitioners classified at the three - digit or minor group level : " health professionals " (generally well - trained workers in jobs that normally require a university or advanced - level degree for recruitment) and " health associate professionals " (generally requiring skills at a tertiary non - university educational qualification level). the former includes physicians and other health care professionals such as dentists and pharmacists (isco-88 code 222), as well as nursing and midwifery professionals (code 223) (see table 1). classified as associate professionals are modern health associate professionals including medical assistants, dental assistants, physiotherapists and others (code 322), nursing and midwifery associate professionals (code 323), and traditional medicine practitioners (code 324). selected health occupations in the international standard classification of occupations (1988 revision) source : international labour office while many national occupational classifications for countries participating in the ipums project were mapped to the isco-88 standard for data dissemination, the precision of mapping would have depended on the level of detail in the national system. in particular, it was not always possible to differentiate traditional health practitioners, so these occupations were merged with modern health associate professionals in the final analysis. moreover, while certain related occupations aside from medical and nursing practitioners are identifiable at the four - digit isco classification level such as medical equipment operators (code 3133), health and safety inspectors (code 3152) and institution - based personal care workers (code 5132) they were excluded from the present analysis to maintain comparability with data where the selection of occupations was possible only at the three - digit level or equivalent. the primary area of concern in the scientific literature on geographical imbalance is usually the physician workforce, offering only a limited view of hrh issues. but in addition to health care practitioners, health systems employ a large number of workers with non - health occupational backgrounds, such as administrators, accountants, drivers and other support staff. capturing this wider range of workers entails expansion of the information requirements across all of the major isco groups, so further details may be needed, notably industry of employment. however, data on industry were not collected in the kenya census, and so this variable was not used here to maintain cross - national comparability of findings. depending on the data availability and quality, indicators that can be used to estimate the incidence and extent of workforce imbalances include employment indicators (such as vacancy rate, occupational unemployment rate and turnover rate), activity indicators (overtime), monetary indicators (real wage rate, rate of return on education) and population - based indicators (ratio of health workers to the population). the health worker - to - population ratio is among the most commonly used indicators in health services research and planning, because it is simple to construct from standard sources, such as the census, but still offers a basis upon which more sophisticated indices of spatial distribution can be built. following a preliminary assessment of health workforce statistics that were derived from the census, we conducted spatial analyses of hrh in each of the three countries. a range of summary measures, which may reflect different normative positions, can be used to monitor the magnitude of inequality in a distribution. we opted to use the gini coefficient to summarize relative inequality in the distribution of health workers according to geopolitically aggregated units. using this index allows us to examine the extent of inequality across the specified groups independently of the countries ' population or workforce sizes. the gini coefficient ranges from 0 to 1, with 0 representing perfect equality (that is, all spatial units of the country having the same health worker - to - population ratio) and 1 denoting total inequality (all health workers located in only one unit). results are interpreted in comparative terms, by contrasting the calculated value across occupational groups. previous research using gini indices to evaluate spatial patterns of health practitioners suggested that this measure is most useful for fairly aggregated forms of economic analysis, involving relatively few and large geographical divisions. we therefore focused our analysis on the spatial distribution of hrh across the largest - scale geographical identifier provided in the census data for each country (states, provinces or regions). one aspect of spatial distribution that is often considered in the scientific literature but which was not assessed here was comparisons between urban and rural areas. the project 's selection of variables for inclusion in the public - use datasets is based largely on the ease of conducting comparative analyses. on the other hand, widespread differences exist in national definitions of what settlements are regarded as urban or rural. the definition may be based on population size, economic activities, administrative region or other (sometimes not officially documented) criteria. a lack of common or precise classification hinders comparative analyses of urban - rural differences. for instance, one review of the literature on rural mental health and sociology reported that 43% of the 178 articles examined did not even include a formal definition of " rural ". stocks can be expressed in terms of the ratio of hrh with regard to the total population, as an indicator of the capacity of the health system. data from the three countries included in this study reveal that the total stock was considerably higher in mexico (621 health workers per 100,000 inhabitants) compared to viet nam (266 per 100,000) and to kenya (255 per 100,000), where the lowest density was found. this result is not surprising : a previous analysis of internationally reported estimates of health personnel suggested that countries in the african region most often have the lowest supplies of hrh. large cross - national variations are also found in the occupational mix of the health workforce (see figure 1). differences in occupational distribution may be partially linked to differences in health care delivery and financing systems, but may also possibly be related to inconsistencies in the classification mapping procedures of the different types of health workers during data collection and processing. for example, only a handful of nursing and midwifery professionals were found in the vietnamese census sample, while the number of nursing and midwifery associate professionals was very low in kenya. occupational mix of the health workforce in three developing countries, according to national population census results table 2 offers a portrait of the health workforce by educational attainment. it is expected that, given the isco hierarchical nature, professional - level occupations should be universally characterized with a tertiary educational attainment. high levels of education were generally found among health professionals (except in nursing) in mexico and viet nam. but this pattern did not hold for kenya, where fewer than half had a tertiary - level education. kenya was also burdened with lower education levels among the overall health workforce and the total population, compared to the two other countries. percentage of health workers with tertiary - level educational attainment, according to occupation source : ipums census microdata samples among nursing and midwifery professionals, high education levels were still found in mexico, but less predominantly so in viet nam. moreover, the proportion with tertiary attainment was not only extremely low in kenya, but even lower than that observed for health associate professionals. cross - national differences in educational attainment by occupational grouping might be explained in part by differences in education systems. but discrepancies between mappings of national occupation classifications with the international standard have been suggested in other household - based data sources even in statistically developed countries, particularly with regard to the nursing and midwifery specializations. the distinction in isco-88 between nursing and midwifery professionals (code 223) and their associate counterparts (code 323) was designed to reflect differences in the tasks and duties that may be a consequence of differences in work organization as well as in education and training. in some countries, this distinction may be of limited relevance ; inadequacies in the reporting system or incomparability of the education systems and measures of technical capacity may mean that some nursing and midwifery jobs do not fit easily into these two categories. as such, to minimize possible errors related to occupational classification, for the following analyses on spatial distribution the categories of nursing and midwifery personnel are merged into one combined group. as seen in figures 2, 3 and 4, each of the three countries were characterized with large geographical imbalances in the overall health workforce. in particular, there was a fivefold discrepancy between the density of hrh across provinces of kenya : from some 138 health workers per 100,000 inhabitants in the north - eastern province, to 688 in nairobi. in mexico the stock ranged from fewer than 350 per 100,000 inhabitants in the states of oaxaca and chiapas to nearly 1200 in the distrito federal, or more than three times higher. the distribution of the vietnamese health workforce was likewise distorted albeit to a lesser magnitude, varying twofold, between 169 per 100,000 in central highlands and 330 in the northwest region. distribution by province of the stock of health workers (per 100,000 inhabitants), kenya, 1989 census microdata sample distribution by state of the stock of health workers (per 100,000 inhabitants), mexico, 2000 census microdata sample distribution by region of the stock of health workers (per 100,000 inhabitants), viet nam, 1999 census microdata sample quantitative summary measures of inequality in the geographical distribution of hrh are presented in terms of gini coefficients in table 3. reflecting the patterns described above, the gini coefficient was found to be highest in kenya (0.287) and lowest in viet nam (0.150). at the same time, the extent of inequality among all three of these developing countries appears to be greater than that reported for many countries with developed market economies. in an examination of regional disparities in hrh for selected industrialized countries, the most recent estimates of gini coefficients summary measures of inequality in the spatial distribution of health workers, according to occupation source : ipums census microdata samples note : kenya was divided into 9 provinces at the time of the 1989 census ; north and south rift valley provinces were combined to represent the country 's current situation. part of the cross - national differences in magnitude of distributional inequality might be related to differences in the skill mix of hrh. health professionals (except in nursing) were most susceptible to inequitable geographical distribution in kenya and viet nam, followed by nursing and midwifery personnel (table 3). within the health labour market, highly skilled professionals are presumed to have greater mobility and be more sensitive to push - and - pull factors, and thus be characterized with greater inequality in their spatial distribution. however the nature of the imbalance was found to vary across countries. in mexico, it was health associate professionals (except in nursing) who were least equitably distributed. one possible confounding factor for differential cross - national patterns could be related to the geographical delimitation. we relied on the largest - scale geographical identifier provided in the public - use census data ; this may have had varying distinctions across countries in terms of patterns in human settlement, economic activities, epidemiological patterns, health system organization, political administration, or other factors. as such, gini coefficients were also calculated at lower levels of geographical aggregation for two of the countries for which a second identifier was provided : kenya (40 spatial units at the district level) and viet nam (61 units at the provincial level). similar trends in geographical imbalance were found according to occupational group for both countries compared to results for the higher - level aggregation, although even greater distributional inequality was suggested in viet nam, with a strong concentration of health professionals and nursing and midwifery personnel in hanoi (results not shown). stocks can be expressed in terms of the ratio of hrh with regard to the total population, as an indicator of the capacity of the health system. data from the three countries included in this study reveal that the total stock was considerably higher in mexico (621 health workers per 100,000 inhabitants) compared to viet nam (266 per 100,000) and to kenya (255 per 100,000), where the lowest density was found. this result is not surprising : a previous analysis of internationally reported estimates of health personnel suggested that countries in the african region most often have the lowest supplies of hrh. large cross - national variations are also found in the occupational mix of the health workforce (see figure 1). differences in occupational distribution may be partially linked to differences in health care delivery and financing systems, but may also possibly be related to inconsistencies in the classification mapping procedures of the different types of health workers during data collection and processing. for example, only a handful of nursing and midwifery professionals were found in the vietnamese census sample, while the number of nursing and midwifery associate professionals was very low in kenya. occupational mix of the health workforce in three developing countries, according to national population census results table 2 offers a portrait of the health workforce by educational attainment. it is expected that, given the isco hierarchical nature, professional - level occupations should be universally characterized with a tertiary educational attainment. high levels of education were generally found among health professionals (except in nursing) in mexico and viet nam. but this pattern did not hold for kenya, where fewer than half had a tertiary - level education. kenya was also burdened with lower education levels among the overall health workforce and the total population, compared to the two other countries. percentage of health workers with tertiary - level educational attainment, according to occupation source : ipums census microdata samples among nursing and midwifery professionals, high education levels were still found in mexico, but less predominantly so in viet nam. moreover, the proportion with tertiary attainment was not only extremely low in kenya, but even lower than that observed for health associate professionals. cross - national differences in educational attainment by occupational grouping might be explained in part by differences in education systems. but discrepancies between mappings of national occupation classifications with the international standard have been suggested in other household - based data sources even in statistically developed countries, particularly with regard to the nursing and midwifery specializations. the distinction in isco-88 between nursing and midwifery professionals (code 223) and their associate counterparts (code 323) was designed to reflect differences in the tasks and duties that may be a consequence of differences in work organization as well as in education and training. in some countries, this distinction may be of limited relevance ; inadequacies in the reporting system or incomparability of the education systems and measures of technical capacity may mean that some nursing and midwifery jobs do not fit easily into these two categories. as such, to minimize possible errors related to occupational classification, for the following analyses on spatial distribution the categories of nursing and midwifery personnel are merged into one combined group. as seen in figures 2, 3 and 4, each of the three countries were characterized with large geographical imbalances in the overall health workforce. in particular, there was a fivefold discrepancy between the density of hrh across provinces of kenya : from some 138 health workers per 100,000 inhabitants in the north - eastern province, to 688 in nairobi. in mexico the stock ranged from fewer than 350 per 100,000 inhabitants in the states of oaxaca and chiapas to nearly 1200 in the distrito federal, or more than three times higher. the distribution of the vietnamese health workforce was likewise distorted albeit to a lesser magnitude, varying twofold, between 169 per 100,000 in central highlands and 330 in the northwest region. distribution by province of the stock of health workers (per 100,000 inhabitants), kenya, 1989 census microdata sample distribution by state of the stock of health workers (per 100,000 inhabitants), mexico, 2000 census microdata sample distribution by region of the stock of health workers (per 100,000 inhabitants), viet nam, 1999 census microdata sample quantitative summary measures of inequality in the geographical distribution of hrh are presented in terms of gini coefficients in table 3. reflecting the patterns described above, the gini coefficient was found to be highest in kenya (0.287) and lowest in viet nam (0.150). at the same time, the extent of inequality among all three of these developing countries appears to be greater than that reported for many countries with developed market economies. in an examination of regional disparities in hrh for selected industrialized countries, the most recent estimates of gini coefficients were found to be at most 0.124 for physicians summary measures of inequality in the spatial distribution of health workers, according to occupation source : ipums census microdata samples note : kenya was divided into 9 provinces at the time of the 1989 census ; north and south rift valley provinces were combined to represent the country 's current situation. part of the cross - national differences in magnitude of distributional inequality might be related to differences in the skill mix of hrh. health professionals (except in nursing) were most susceptible to inequitable geographical distribution in kenya and viet nam, followed by nursing and midwifery personnel (table 3). within the health labour market, highly skilled professionals are presumed to have greater mobility and be more sensitive to push - and - pull factors, and thus be characterized with greater inequality in their spatial distribution. however the nature of the imbalance was found to vary across countries. in mexico, it was health associate professionals (except in nursing) who were least equitably distributed. one possible confounding factor for differential cross - national patterns could be related to the geographical delimitation. we relied on the largest - scale geographical identifier provided in the public - use census data ; this may have had varying distinctions across countries in terms of patterns in human settlement, economic activities, epidemiological patterns, health system organization, political administration, or other factors. as such, gini coefficients were also calculated at lower levels of geographical aggregation for two of the countries for which a second identifier was provided : kenya (40 spatial units at the district level) and viet nam (61 units at the provincial level). similar trends in geographical imbalance were found according to occupational group for both countries compared to results for the higher - level aggregation, although even greater distributional inequality was suggested in viet nam, with a strong concentration of health professionals and nursing and midwifery personnel in hanoi (results not shown). census results pointed to large inequalities in the geographical distribution of the health workforce in all three countries, with the highest densities of hrh tending to be found in the capital areas. moreover, strong variations were consistently found across countries, and this despite their selection for inclusion in this analysis having been based on the data source (availability of microdata from the ipums - international archives) rather than necessarily any a priori assumption of a problem of inequality. health professionals and nursing and midwifery personnel were most susceptible to imbalance in kenya and viet nam, but it was health associate professionals in this rank in mexico. one potential explanation of differential cross - national patterns could be related to the classification of health occupations. we mapped occupations against isco-88, which assigns items into categories according to presumed shared characteristics, as our framework for harmonizing health workforce statistics. in practice, however, the precision of coding to standardized classifications depends largely on the level of specification in the raw data. for instance, problems were already suggested in mapping national definitions of nursing and midwifery specializations to the international standard. it is further possible that in mexico, where the occupational mix was observed to be skewed towards health professionals (except in nursing), the eventual coding may have excessively favoured this category over the associate professional counterparts independently of the actual job tasks and duties. several factors play a role in determining the numbers and distributions of health care workers in a particular health system, including resource availability, accessibility to training in the health field, location of health facilities and employment opportunities, regulatory environment, culture and customs. an assessment of the extent to which any one of these factors influences the equitable distribution of hrh across countries remains beyond the scope of the census alone as information source. census findings offer little information on the demand for health services and hrh, other than in relation to population size under an assumption of uniform needs across spatial units. moreover, the available data do not enable distinguishing the specific activities of health practitioners. it may be that the health system 's requirements for skilled workers are greater in capital areas, for instance, for carrying out nationwide public health planning and management activities. while many governments have adopted strong policies such as combinations of compulsory service and incentives to address workforce imbalance, especially rural / urban imbalance, it is acknowledged that none so far has solved the problem. for instance, simply expanding the supply of medical personnel does not appear to be an effective solution for improving geographical distribution, as suggested by findings from mexico of the coexistence of high unemployment and underemployment among physicians in major cities while many rural areas go underserved. empirical evidence elsewhere has further suggested that spatial distributions tend to be highly stable for most categories of health personnel, making it problematic whether these distributions can be changed through normal public policy interventions. even in developed countries, an analysis of geographical inequalities in the united kingdom reported that policies intended to improve the distribution of general practitioners did not lead to a reduction in inequality over time, either in relative or absolute terms. rather, unintended interactions among different policies were believed to have contributed to a reduction in their overall effectiveness. this does not necessarily mean that policies and programmes designed to reduce imbalance have no effect. for example, thailand offers a success story of having begun to stem the migration of health professionals from rural to urban areas and from public to private facilities through a range of financial incentives. the main goal of this study was to investigate uses of national population censuses for monitoring geographical imbalances among human resources for health, as a basis for formulation of evidence - based health policy options. census data were used to offer a breakdown of the health workforce by spatial units (states / provinces / regions) in three developing countries : kenya, mexico and viet nam. while at first glance it might appear easy to determine whether imbalances exist or not, operationalization is hampered by the lack of a single empirical measure of workforce imbalance. furthermore, many previous studies of health personnel issues have been based on data of limited validity and with highly inconsistent categories of occupations and skills. we used population - based indicators for monitoring hrh distribution, in part because they made it easy to quantify imbalances, in part because of the nature of the data available. the findings from the census were useful for illuminating the magnitude of geographical imbalance, especially as in developing countries where many standard data sources tend to be underused in health research and the evidence often fragmented. at the same time, the types of indicators used here are prone to a certain degree of subjectivity ; the interpretation of findings should be based on a broad discussion of how communities and decision - makers view the implied value judgements. the consistency of the problem of imbalance across wide - ranging countries holds important implications at the local and national levels but also at the international level, in terms of constraints for the effective deployment, management and retention of hrh and ultimately for the equitable delivery of health services. in particular, inequitable distribution of hrh in less - developed countries can be exacerbated by emigration of health workers to countries promising higher salaries and other incentives, putting greater pressure on the health systems and workers remaining. who advocates better cooperation between the many agencies supporting processes for strengthening national health systems, with equitable geographical distribution as one of the core policy areas. somewhat unexpected from our findings was the cross - national diversity in the magnitude of distributional inequality according to occupational group. while speculation about settlement processes according to skill level implies a rational sequence, the reality of individuals ' decision - making does not always appear to follow such presumed logic. qualitative evidence may be needed to better understand why health workers seem to prefer remaining in certain areas rather than others where their services may be more needed, and why this phenomenon affects some categories of health workers more than others. the comparative analysis was facilitated by the availability of public - use microdata samples with harmonized variable structures through the ipums collaborative research project. in the present study, we used data from three developing countries with occupational information that enabled differentiation of health occupations. despite certain problems apparent from the mapping of occupational titles to the internationally standardized classification, the census can be an important source for health workforce statistics, particularly at the sub - national level. many countries have a long history of collecting information on labour force participation and occupation in their censuses. but the content and quality of census data can vary across countries and over time, and microdata have generally not been made available in a timely manner, if at all. increasing access to microdata, and the mapping of relevant variables to international standards (notably on occupation, but also industry and education), will enable even greater future research opportunities. for one, it is anticipated that data from the latest censuses for two more developing countries (brazil and ghana) will be accessible through ipums very soon. possibilities are also emerging through other international databank projects. among these are the african census analysis project, which has archived census microdata from at least six countries with occupation data detailed minimally at the three - digit isco level or equivalent (gambia, kenya, mali, nigeria, south africa and uganda), with plans to eventually facilitate distribution of the data with the aid of tools being developed for guided statistical application. other potential sources include the centro latinoamericano y caribeo de demografa for access to census data and specialized tables from selected countries in the latin american and caribbean region, and the economic commission for europe for data from a number of developed market and transitional countries. moreover, comparative analyses are expected to be increasingly facilitated as use of or mapping to isco-88 becomes more widespread across countries. the uses of census data for hrh assessment are numerous, including profiling of the health workforce and its organization in relation to the rest of the labour force and the population at large. for instance in viet nam, monitoring of health professionals in private practice is becoming increasingly imperative, as the country moves from a centrally planned economy to a more open economic model. but relatively long periodicity between censuses (usually once every ten years) remains a drawback to conducting analyses for short - term hrh planning and policy monitoring purposes. even in the medium term, analyses can be hampered in the absence of occupational data from successive census rounds ; the case of kenya is a prime example, given the lack of adequate information on occupation in the most recent enumeration. moreover, the shorter length of census questionnaires tends to preclude possibilities for conducting refined analysis of workforce activities compared to labour force and other sample surveys. nevertheless, the census remains one valuable source that, in combination with other complementary information, can help provide the evidence base required for better understanding hrh as an input to health systems performance. we recommend that special attention be paid to the availability of data relevant to health workforce issues in such periodic data collection activities, as part of the strengthening of national health information systems, including the processing of occupation and other variables through the use of internationally standardized classifications at the greatest level of detail possible. all authors participated in interpreting the results, and read and approved the final manuscript. the authors would like to thank the minnesota population centre for having lent permission to use the ipums census microdatasets for the empirical analysis. we are grateful to christopher murray and robert mccaa for much support and expert advice offered during the course of our research. the boundaries and names shown and the designations used on the maps presented here do not imply the expression of any opinion whatsoever on the part of the world health organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. | backgroundimbalance in the distribution of human resources for health (hrh), eventually leading to inequities in health services delivery and population health outcomes, is an issue of social and political concern in many countries. however, the empirical evidence to support decision - making is often fragmented, and many standard data sources that can potentially produce statistics relevant to the issue remain underused, especially in developing countries. this study investigated the uses of demographic census data for monitoring geographical imbalance in the health workforce for three developing countries, as a basis for formulation of evidence - based health policy options.methodspopulation-based indicators of geographical variations among hrh were extracted from census microdata samples for kenya, mexico and viet nam. health workforce statistics were matched against international standards of occupational classification to control for cross - national comparability. summary measures of inequality were calculated to monitor the distribution of health workers across spatial units and by occupational group.resultsstrong inequalities were found in the geographical distribution of the health workforce in all three countries, with the highest densities of hrh tending to be found in the capital areas. cross - national differences were found in the magnitude of distributional inequality according to occupational group, with health professionals most susceptible to inequitable distribution in kenya and viet nam but less so in mexico compared to their associate professional counterparts. some discrepancies were suggested between mappings of occupational information from the raw data with the international system, especially for nursing and midwifery specializations.conclusionsthe problem of geographical imbalance among hrh across countries in the developing world holds important implications at the local, national and international levels, in terms of constraints for the effective deployment, management and retention of hrh, and ultimately for the equitable delivery of health services. a number of advantages were revealed of using census data in health research, notably the potential for producing detailed statistics on health workforce characteristics at the sub - national level. however, lack of consistency in the compilation and processing of occupational information over time and across countries continues to hamper comparative analyses for hrh policy monitoring and evaluation. |
an elevation in brain temperature is common in acute ischemic stroke and is associated with a worse outcome [13 ]. one potential explanation is that pyrexia increases the brain temperature and the brain metabolic rate and could result in a more rapid exhaustion of limited energy and oxygen supplies and the increased production of free radicals and other toxic substances in ischemic tissue [4, 5 ]. for different blood flow and metabolic states, brain temperature could be different in different regions of ischemic tissue, that is, the infarct core, ischemic penumbra (ip), and the oligemic region. conventional methods of measuring brain temperature by invasive probes may monitor only one or a few locations simultaneously and cursorily, and they are difficult to be accepted clinically [6, 7 ]. several magnetic resonance parameters can be used for the noninvasive measurement of regional temperatures, including the diffusion coefficient, the longitudinal relaxation time constant (t1), and the proton resonance frequency (prf), and the latter is more popular [8, 9 ]. magnetic resonance (mr) therefore, it is possible to more accurately measure brain temperature in different regions of ischemic lesions, especially in the ip. many experimental and clinical studies have focused on brain temperature in the fields of cerebral infarction measured by magnetic resonance spectroscopy (mrs) [6, 10, 11 ]. the brain temperature is elevated in the ischemic brain soon after stroke, prior to any increase in body temperature, and is significantly higher in probable penumbral tissue than in the infarct core or normal brain [2, 12 ]. a previous study found that there was a significant difference in brain temperature between ischemic tissue and the contralateral normal hemisphere in patients with middle cerebral artery occlusion. additionally, the brain temperature of ischemic tissue could increase with the enlargement of lesion volume. however, there have been no reports on the detailed evolution of brain temperature in different regions of ischemic lesion at the early stage of onset. in this work, we modified equations to measure brain temperature based on mrs prf techniques in a physiological solution and used the final equation to calculate the brain temperature of different regions in ischemic lesion before and after reperfusion, especially the ip, in monkey middle cerebral artery occlusion (mcao) models. the experiment was approved by the animal care and use committee of tianjin medical university. six mature male monkeys (cynomolgus macaque) with a mean weight of 8.6 kg (range from 8 to 10 kg) were supplied by experimental animal center of beijing military medical academy. anesthesia was induced by ketamine (0.25 mg intramuscular) and atropine (810 mg / kg intramuscular) followed by 100 mg ketamine and 50 mg diazepam in a 500 ml ns intravenous drip. physiological parameters, such as respiration, heart rate, body temperature, and blood pressure, were monitored. a one - inch incision was made in the right femoral groove, and the femoral artery and vein were isolated. the right femoral artery was punctured, and a 5 french catheter sheath was inserted. the 5 french catheter was introduced over a guide wire and navigated under fluoroscopy in a common carotid artery in close proximity to the internal carotid artery. a 1.7 french microcatheter (prowler 10 with two markers, cordis corporation, 0.55 mm outside diameter) was introduced over a transcend microwire (cordis). the microcatheter and the microguide wire were navigated into a selected branch of the middle cerebral artery (mca) and advanced into a wedged position. the animals were maintained on a slow, continuous, heparinized drip infusion (100 u / hour) during the ischemia period. after one hour of ischemia, the microguide wire was removed, and the animal was analyzed with a magnetic resonance imaging (mri) scanner. if cerebral ischemia was confirmed by diffusion - weighted imaging (dwi), the microcatheter was removed, and 250,000 units of urokinase in 50 ml normal saline (ns) was infused with a high - pressure injector (2 ml / min). mri and mrs were performed with a ge 1.5 t twin speed infinity with excite i magnetic resonance system 1 h after occlusion and 1 h, 3 h, 6 h, 12 h, and 24 h after recanalization. we performed dwi, perfusion - weighted imaging (pwi), and t2-weighted imaging (t2wi) with a head coil. the t2wi parameters were the following : repetition time (tr) = 4000 ms ; echo time (te) = 106.4 ms ; averages = 2 ; slice thickness = 2.5 mm ; gap = 0 mm ; field of view (fov) = 18 cm 18 cm ; and matrix = 288 256. dwi was obtained using a single - shot spin - echo echo planar sequence with tr = 6000 ms ; te = 96.8 ms ; averages = 2 ; b = 1000 s / mm ; fov = 18 cm 18 cm ; and matrix = 128 128. pwi was obtained using a single - shot gradient - recalled echo planar imaging t2wi sequence, that is, dynamic susceptibility contrast (dsc) mri, with tr = 2000 ms ; te = 80 ms ; average = 1 ; fov = 18 cm 18 cm ; and matrix = 128 128. a gd - dtpa bolus (0.1 mmol / kg) was administered with a power injector at a rate of 2 ml / s, and 10 ml of isotonic saline was injected to wash the pipe. we used multivoxel point - resolved - spectroscopy- (press-) localized proton mrs with the voxel grid centered on the slice showing the maximum ischemic lesion on dwi. the imaging parameters were the following : tr = 1500 ms, te = 135 ms, fov = 18 cm 18 cm, matrix = 24 24, slice thickness = 10 mm, and total acquired time = 96. the voxel grid (1 ml) was carefully placed within the brain to include as much of the visible ischemic lesion ipsilateral and contralateral normal brain as possible and to avoid contamination of the spectra with the lipid signal from bone marrow or subcutaneous fat. we collected standard three - pulse chemical shift - selective (chess) water suppression and nonsuppression imaging. spectroscopic data were analyzed with the java - based magnetic resonance user interface (jmrui, http://www.mrui.uab.es/mrui/) package, spectroscopic time domain analysis software. initially, the zero - order phase correction of the water proton peak (effectively bringing water to a chemical shift of 4.70 ppm) was performed, and the residual water signal was then removed using the hanckel - lanczos singular value decomposition (hlsvd) method. spectroscopic data were fourier transformed for display and visual quality control purposes using the advanced method for accurate robust and efficient spectral fitting (amares) algorithm within the jmrui package. gaussian components were modeled in the time domain, including choline (cho), creatine (cr), n - acetyl aspartate - containing compounds (naa), and lactate (lac). the chemical shifts (i.e., frequency) of the fitted metabolite peaks were reported to a precision of 0.001 ppm to confirm the chemical shifts of naa. spectra were automatically discarded if the fitted line widths were less than 1 hz or greater than 10 hz. we inspected the spectroscopic data visually and discarded voxels laying on the edges of the press excitation region, voxels containing cerebrospinal fluid (csf) and those with poor quality, for example, having a badly elevated baseline or containing spurious peaks. temperature t was derived from the chemical shift of water (csh2o) using the following equation : (1)t = tref+k(csh2ocsref). csref is the (temperature - independent) chemical shift of a reference compound, k is the coefficient of proportionality, and tref is the reference temperature. temperature - dependent changes in hydrogen bonding cause the water chemical shift to vary linearly with temperature at 0.01 ppm per c. in our scanner, with 4.7 ppm as the chemical shift of water and 37c as the reference temperature, (1) csnaa is the apparent chemical shift of naa, and csnaaref is the reference chemical shift of naa. we measured the temperature of a physiological solution (naa 12.5 mmol, cr 10.0 mmol and cho 3.0 mmol) with constant temperature equipment to acquire the csnaaref, which could be fit to the mr scan. there was a linear correlation between the prf and the temperature of the solution ranging from 34c to 44c, and the value of csnaaref was 2.039 ppm by this method. therefore, the following was used to calculate the brain temperature of each voxel grid in our study : (3)t=37 + 100(csnaa2.039). the mean transit time (mtt) map of pwi was processed by perfusion software using a ge sun aw4.2 workstation, and the final infarct lesion was defined as abnormal signal intensity on t2wi at 24 h of the reperfusion stage. we classified the ischemic lesion (abnormal signal intensity on mtt) into three types of tissue by dwi and mtt at the artery occlusion stage and t2wi at 24 h of the reperfusion stage : the infarct core had high signal intensity on both dwi and t2wi, the ip had high signal intensity on dwi but normal on t2wi, and the oligemic region had normal signal intensity on both dwi and t2wi but abnormal signal intensity on mtt (figure 1). if one type of tissue was greater than 75% within one voxel grid of mrs, we named the voxel grid as this type of tissue. the brain temperature of each voxel grid and the mean and standard deviation of brain temperature for each tissue voxel category were calculated. the paired t - test was used to analyze the differences in brain temperature between normal bilateral hemispheres and between different ischemic regions and the contralateral brain. one - way anova was performed between the brain temperature of three types of tissues. the normal (baseline) brain temperature of the six monkeys was measured before the operation (table 1). the mean baseline brain temperature was 37.16c in left hemispheres, 37.17c in right hemispheres, and 37.16c in bilateral hemispheres. there was no significant difference in the baseline brain temperature between the bilateral hemispheres by paired t - test (t = 1.659, p > 0.05). mcao models were successfully established in four out of the six monkeys, including one right and three left sides. two monkeys were dead at 2 and 14 hours after reperfusion, respectively, probably because of large area of hemorrhagic infarction. and table 2 shows the brain temperature of different ischemic tissues and the contralateral hemisphere at the artery occlusion stage (0 h) and different time points of the reperfusion stage. at the artery occlusion stage, the mean brain temperature of ischemic tissue was 1.16 0.15c higher than the baseline brain temperature. the increase in brain temperature was region dependent, with 1.72 0.08c in the ip, 1.08 0.19c in the infarct core, and 0.62 0.14c in the oligemic region compared to the basal brain temperature. in the reperfusion stage, the evolution of brain temperature differed in the infarct core, ip and oligemic region (figure 2). after recanalization, the brain temperature of the infarct core showed a pattern of an initial decrease accompanied by a subsequent increase. however, the brain temperature of the ip and oligemic region showed a monotonously and slowly decreased pattern. the brain temperature of the contralateral hemisphere increased slightly (0.23c) compared to the baseline brain temperature. table 3 shows the differences in brain temperature between different ischemic tissues and the contralateral hemisphere. there were significant differences in brain temperature between the infarct core and contralateral hemisphere at 0 h, 1 h, 12 h, and 24 h, between the ip and contralateral hemisphere within 6 h, and between the oligemic region and the contralateral hemisphere within 3 h (p 0.05). one - way anova was performed on the brain temperature of different ischemic tissues at different time points (table 4). there were significant differences in the brain temperature among different ischemic tissues at each time point (f values were 27.64, 8.32, 26.46, 21.55, 27.64, and 47.90 at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, resp., p 0.05). previous studies of brain temperature were limited by the reliance on invasive measurement techniques, such as probes inserted in brain tissue [1517 ]. in this situation, the brain temperature could be lower than the actual temperature if the brain surface was exposed to cooler environmental air. invasive techniques may inevitably induce local microlesions and inflammatory responses adjacent to probes, which might affect brain temperature. infrared thermometry is a noninvasive method, but it fails to measure the deep brain temperature due to the limited depth penetration and can not make an accurate localization because of the limited spatial resolution of this method. mrs is a validated method for noninvasively measuring brain temperature using the principle that the water frequency shift relative to n - acetyl aspartate is temperature dependent [8, 10, 18 ]. moreover, mrs thermometry could avoid most of the above - mentioned weaknesses of other methods. because equations for calculating brain temperature (especially csnaaref) are dependent on the mr scanner and sequence parameters, the temperature equation can be modified based on in vivo models or healthy volunteers. in our study the signal intensity from naa as a chemical shift reference should be high enough for calculating the temperature. however, naa decreases gradually after a stroke ; as a consequence, studies of the temperature of ischemic brain tissue by mrs should be limited in acute stoke. a previous study found that there was a statistically significant reduction in temperature of 0.09c per scan (p = 0.0001) across four sequential mrs scans on 4 normal volunteers, presumably due to cooling by the air current in the bore of the magnet (or relative cerebral inactivity during the scan). in our study, the mr examination was arranged as dwi, t2wi, pwi, and mrs for 15 min for all of the time points, which can at least partly reduce the effect of environmental temperature on the longitudinal changes of brain temperature. the perfusion state is critical for the temperature of the brain tissue ; for example, reduced blood flow could impair heat exchange and result in a higher temperature in ischemic tissue. according to the perfusion state theory, the brain temperature in the infarct core should increase the most because it has the lowest blood flow. however, we found that the highest increase in brain temperature was in the ip, followed by the infarct core and the oligemic region at the artery occlusion stage in the monkey mcao model. this finding suggests that the perfusion state was not the only factor for brain temperature elevation in ischemic tissue, and other factors might play a role. the brain tissue temperature is mainly determined by two processes, heat production and heat radiation. in the infarct core, reduced blood flow induces the reduction of heat radiation and increased anaerobic metabolism induces the increase in heat production, which may both result in the elevated brain temperature. in ip tissue, reduced blood flow may induce the reduction of heat radiation, while an increase in heat production is induced by the subsequent events, including both aerobic and anaerobic metabolism [1922 ], inflammation and the release of excitatory amino acids [23, 24 ]. these complex processes may account for the higher elevated brain temperature in the ip than in the infarct core or oligemic regions at the artery occlusion stage. in addition, uncoupling protein 2 (ucp-2) is a natural neuroprotective factor in the human ischemic brain [2528 ]. ucp-2 upregulation may regulate atp synthesis by uncoupling oxidation from phosphorylation, thus dissipating energy as local heat, and may simultaneously be responsible for the early rise in lesion brain temperature after stroke. after recanalization, the brain temperature of the ischemic tissue was decreased with time, which can be mainly explained by the changes of the perfusion states in these brain regions (figure 3). in the infarct core, a temporary and rapid increase in rcbf and shortening of the rmtt, that is, over - perfusion, result in the clearance of accumulated heat, which can account for the sharply decreased brain temperature. however, after 6 h, both decreased rcbf and delayed mtt lead to the reincrease in the temperature. in particular, at 12 h after recanalization, the brain temperature of the infarct core was even higher than the ip and oligemic region and was accompanied by the lowest rcbf and highest rmtt in the infarct core. however, the brain temperature of the ip and oligemic region recovered to normal levels as in the contralateral hemisphere without repeated elevation, suggesting that the cells in these regions were viable or salvaged. the brain temperature in the contralateral hemisphere (which may be more closely related to body temperature) is slightly elevated after a stroke, reflecting increased neuronal activity in response to the ischemia. therefore, in our study, the brain temperature in the ischemic region was evaluated by comparison with the baseline brain temperature, which is more reasonable than the previously used method that regarded the brain temperature of the contralateral hemisphere as a control state. in the present study, we focused on detailed changes in the brain temperature of different ischemic tissues before and after reperfusion by h mrs. we found that the ip and infarct core showed different evolution patterns of brain temperature, which may provide us with a method to discriminate the ip from the infarct core. the first limitation is that the classification of different ischemic tissues in our study was based on dwi and pwi at the artery occlusion stage and t2wi at 24 h of the reperfusion stage. ip was defined as low - perfusion tissue with high signal intensity on dwi but normal intensity on t2wi, but this definition may miss part of the ip tissue peripheral to the abnormal dwi regions [2931 ]. the last limitation is that we do not know whether the results obtained from the monkey mcao model can be generalized to human stroke patients. further studies are needed to overcome the above limitations and to clarify the mechanism underlying the evolution of the brain temperature of ischemic tissue. after ischemic stroke, the infarct core and ip have different evolution patterns of brain temperature, which suggests that in vivo measurement of brain temperature could help to identify whether ischemic tissue survives. | brain temperature is elevated in acute ischemic stroke, especially in the ischemic penumbra (ip). we attempted to investigate the dynamic evolution of brain temperature in different ischemic regions in a monkey model of middle cerebral artery occlusion. the brain temperature of different ischemic regions was measured with proton magnetic resonance spectroscopy (1h mrs), and the evolution processes of brain temperature were compared among different ischemic regions. we found that the normal (baseline) brain temperature of the monkey brain was 37.16c. in the artery occlusion stage, the mean brain temperature of ischemic tissue was 1.16c higher than the baseline ; however, this increase was region dependent, with 1.72c in the ip, 1.08c in the infarct core, and 0.62c in the oligemic region. after recanalization, the brain temperature of the infarct core showed a pattern of an initial decrease accompanied by a subsequent increase. however, the brain temperature of the ip and oligemic region showed a monotonously and slowly decreased pattern. our study suggests that in vivo measurement of brain temperature could help to identify whether ischemic tissue survives. |
62.4 million indians were reported to have type 2 diabetes mellitus (t2 dm) putting india on the forefront of diabetic epidemic across globe. fear of hypoglycaemia and gain in body weight are barriers for initiation of insulin therapy. modern insulin analogues are a convenient new approach or tool to glycaemic control, associated with low number of hypoglycaemia and favourable weight change. a1chieve, a multinational, 24-week, non - interventional study, assessed the safety and effectiveness of insulin analogues in people with t2 dm (n = 66,726) in routine clinical care. please refer to editorial titled : the a1chieve study : mapping the ibn battuta trail. the patient characteristics for the entire cohort divided as insulin - nave and insulin users is shown in table 1. the majority of patients (71.5%) started on or switched to biphasic insulin aspart. other groups were insulin detemir (n = 158), insulin aspart (n = 124), basal insulin plus insulin aspart (n = 19) and other insulin combinations (n = 54). overall demographic data after 24 weeks of treatment, overall hypoglycaemic events reduced from 0.2 events / patient - year to 0.0 events / patient - year in insulin nave group, whereas overall hypoglycaemia was nil in insulin user group similar to that of baseline. body weight and blood pressure decreased from baseline, while overall lipid profile and quality of life improved at week 24 in the total cohort [tables 2 and 3 ]. all parameters of glycaemic control improved from baseline to study end in the total cohort [table 4 ]. overall efficacy data of the total cohort, 893 patients started on biphasic insulin aspart ogld, of which 259 (29.0%) were insulin nave and 634 (71.0%) were insulin users. for both insulin nave and insulin user groups, hypoglycaemic events reduced from 0.1 events / patient - year to 0.0 events / patient - year after 24 weeks of treatment. a decrease in body weight and improvement in quality of life biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [table 7 ]. biphasic insulin aspartoral glucose - lowering drug efficacy data of the total cohort, 19 patients started on basal + insulin aspart ogld, of which 10 (52.6%) were insulin nave and 9 (47.4%) were insulin users. body weight decreased and quality of life improved after 24 weeks of treatment [tables 8 and 9 ]. basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for both insulin nave and insulin user groups [table 10 ]. basal+insulin aspartoral glucose - lowering drug efficacy data of the total cohort, 158 patients started on insulin detemir ogld, of which 124 (78.5%) were insulin nave and 34 (21.5%) were insulin users. after 24 weeks of starting or switching to insulin detemir, hypoglycaemic events reduced from 0.1 events / patient - year to 0.0 events / patient - year in insulin naive group, while, hypoglycaemia was nil similar to that of baseline in insulin users. body weight decreased and quality of life improved at the end of 24 weeks [tables 11 and 12 ]. insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for both insulin - nave and insulin user groups [table 13 ]. insulin detemiroral glucose - lowering drug efficacy data of the total cohort, 124 patients started on insulin aspart ogld, of which 71 (57.3%) were insulin nave and 53 (42.7%) were insulin users. after 24 weeks of starting or switching to insulin aspart, hypoglycaemic events reduced from 0.2 events / patient - year to 0.0 events / patient - year in insulin naive group, while, hypoglycaemia was nil similar to that of baseline in insulin users. body weight decreased and quality of life improved after 24 weeks of treatment [tables 14 and 15 ]. insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart oglds for both insulin nave and insulin user groups [table 16 ]. of the total cohort, 893 patients started on biphasic insulin aspart ogld, of which 259 (29.0%) were insulin nave and 634 (71.0%) were insulin users. for both insulin nave and insulin user groups, hypoglycaemic events reduced from 0.1 events / patient - year to 0.0 events / patient - year after 24 weeks of treatment. a decrease in body weight and improvement in quality of life biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [table 7 ]. of the total cohort, 19 patients started on basal + insulin aspart ogld, of which 10 (52.6%) were insulin nave and 9 (47.4%) were insulin users. body weight decreased and quality of life improved after 24 weeks of treatment [tables 8 and 9 ]. basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for both insulin nave and insulin user groups [table 10 ]. of the total cohort, 158 patients started on insulin detemir ogld, of which 124 (78.5%) were insulin nave and 34 (21.5%) were insulin users. after 24 weeks of starting or switching to insulin detemir, hypoglycaemic events reduced from 0.1 events / patient - year to 0.0 events / patient - year in insulin naive group, while, hypoglycaemia was nil similar to that of baseline in insulin users. body weight decreased and quality of life improved at the end of 24 weeks [tables 11 and 12 ]. insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for both insulin - nave and insulin user groups [table 13 ]. of the total cohort, 124 patients started on insulin aspart ogld, of which 71 (57.3%) were insulin nave and 53 (42.7%) were insulin users. after 24 weeks of starting or switching to insulin aspart, hypoglycaemic events reduced from 0.2 events / patient - year to 0.0 events / patient - year in insulin naive group, while, hypoglycaemia was nil similar to that of baseline in insulin users. body weight decreased and quality of life improved after 24 weeks of treatment [tables 14 and 15 ]. insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart oglds for both insulin nave and insulin user groups [table 16 ]. our study reports improved glycaemic control and quality of life following 24 weeks of treatment with any of the insulin analogues (biphasic insulin aspart ; basal + insulin aspart ; insulin detemir ; insulin aspart) with or without ogld. though the findings are limited by number of patients, still the trend indicates that insulin analogues can be considered effective and possess a safe profile for treating type 2 diabetes in hyderabad, india. | background : the a1chieve, a multicentric (28 countries), 24-week, non - interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with t2 dm (n = 66,726) in routine clinical care across four continents.materials and methods : data was collected at baseline, at 12 weeks and at 24 weeks. this short communication presents the results for patients enrolled from hyderabad, india.results:a total of 1249 patients were enrolled in the study. four different insulin analogue regimens were used in the study. patients had started on or were switched to biphasic insulin aspart (n = 893), insulin detemir (n = 158), insulin aspart (n = 124), basal insulin plus insulin aspart (n = 19) and other insulin combinations (n = 54). at baseline glycaemic control was poor for both insulin nave (mean hba1c : 9.0%) and insulin user (mean hba1c : 9.5%) groups. after 24 weeks of treatment, both the groups showed improvement in hba1c (insulin nave : 0.9%, insulin users : 1.1%). sadrs including major hypoglycaemic events or episodes did not occur in any of the study patients.conclusion:starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia. |
to illustrate the potential role for heidelberg edge perimetry (hep) in the assessment of early functional loss in glaucoma. a 46-year - old male presented with raised intraocular pressure but healthy optic discs and normal standard automated perimetry (sap). he was diagnosed with ocular hypertension, but after 16 years, he developed glaucomatous changes to the right optic disc and inferior retinal nerve fiber layer thinning. despite the evidence of progressive structural damage, hep, on the other hand, revealed an extensive superior visual field damage in agreement with the structural changes. additionally, and in contrast to sap, the hep indices of glaucoma hemifield test, mean deviation and pattern standard deviation were all outside normal limits. this case illustrates that hep may have an important role in the early detection of functional damage in glaucoma, with potential advantages over sap. glaucoma is a heterogeneous group of potentially blinding disorders characterized by progressive optic neuropathy. given the irreversible nature of this disease, early diagnosis and detection of progression is important, particularly as appropriate treatment can slow or halt visual loss. the assessment of visual function is an essential component of glaucoma diagnosis and monitoring, with the current gold standard for visual function testing being standard automated perimetry (sap). however, qualitative and quantitative analyses of the optic nerve and retinal nerve fiber layer (rnfl) have shown that significant structural changes are present in many patients before detectable changes in sap. as such, sole reliance on sap, especially in early stages of the disease, has the potential for underestimating both neural loss and risk of functional impairment. based on these observations, new perimetric techniques have been developed with the goal of detecting glaucomatous functional damage at an earlier stage. one recently developed device is the heidelberg edge perimeter, which uses a flicker - defined form (fdf) stimulus. fdf perimetry employs a temporally driven illusion within which an array of randomly positioned black and white dots flicker at high temporal frequency in counterphase. the spots reverse polarity without changing their position, which means that white dots are replaced with black dots and black dots are replaced with white dots. at high temporal frequency, subjects perceive an illusory circular edge contour, which appears as a gray patch against the mean luminance background. this stimulus is thought to stimulate the magnocellular pathway, which is considered to be damaged in early stages of glaucoma. although the exact mechanism underlying the fdf stimulus and its effect on the magnocellular pathway have not yet been fully elucidated, there is some evidence to suggest that fdf perimetry could be more sensitive at detecting early functional loss than sap and other types of perimetry [5, 6 ]. the following report illustrates the potential role of heidelberg edge perimetry (hep) in the assessment of early functional loss in a case of confirmed glaucomatous structural damage in the absence of a significant sap defect. this is a case of an asymptomatic male, who originally presented in 1993, at age 46, due to an incidental finding of raised intraocular pressure (iop). there was no family history of glaucoma and no significant past medical or ocular problem aside from low hyperopia (od : + 1.00/+0.25 155, os : + 1.25/+0.50 30). on goldmann applanation tonometry, iops were 27 mm hg in the right eye (od) and 24 mm hg in the left eye (os). gonioscopy revealed open angles in both eyes. on dilated fundoscopic examination, both optic discs appeared healthy and there were no rnfl defects visible. the patient was diagnosed with ocular hypertension and the decision was made to monitor him without treatment. for the next 16 years, iops were consistently in the mid - teens and optic disc and sap parameters remained normal ; however, in 2009, changes to the right optic disc were noted (fig. 1). there was an increase in the excavation of the right disc with a thinning of the inferior neuroretinal rim, and an optic disc hemorrhage was present inferiorly. iops had increased to 40 mm hg od and 30 mm hg os. a structural abnormality of the right eye the patient was commenced on topical ocular antihypertensive treatment in both eyes ; however, reduction in iop was insufficient. he had a stepwise increase in treatment and, by 2012, was using 3 antiglaucoma medications (brinzolamide t.i.d. he also underwent 360 degree selective laser trabeculoplasty in the right eye. at his most recent visit, iops were 19 mm hg od and 18 mm hg os. despite the glaucomatous optic disc changes in the right eye, sap global indices were within statistically normal limits (fig. 3b). due to the apparent mismatch between structural and functional damage, the patient underwent hep to determine whether functional damage might in fact be present. hep revealed an extensive abnormality, with a marked nasal and superior paracentral scotoma (fig. in contrast to the sap indices, the hep indices of the glaucoma hemifield test (ght), mean deviation (md) and pattern standard deviation (psd) were all outside normal limits. the hep md was 9.55 db (p < 0.05) and the hep psd was 5.25 db (p < 0.05). figure 3 compares sap, frequency doubling technology (fdt) and hep, which were completed on the same day. both fdt and hep detected a statistically significant defect while sap did not ; however, the hep defect was more extensive than the defect observed on fdt. there was also a good spatial correlation between the defect on the heidelberg edge perimeter and the defect observed on optic disc photographs and sd - oct (fig. this was a case of a patient with ocular hypertension who developed glaucoma as documented by structural changes on stereoscopic optic disc photographs and sd - oct imaging. despite evidence of progressive structural damage (fig. 1), the sap global indices ght, md and psd remained within statistically normal limits (fig. 1, fig., a marked visual field defect was detected on the heidelberg edge perimeter and the hep indices ght, md and psd were all abnormal (fig. the difference between sap and hep was of particular significance as hep indicated that the visual field defect involved an area close to fixation. sap uses a differential contrast stimulus consisting of a white light presented on a white background. most subtypes of retinal ganglion cells (rgc) can respond to a white - on - white stimulus and, therefore, due to redundancy in the visual system, loss of a large number of rgc can occur before a sap defect is detected. histological studies in humans and monkeys have investigated the relationship between sap and rgc counts. these studies also indicated that a large reduction in rgc density could occur before significant sap defects develop. the result is that in early glaucoma, reliance on sap may lead to an underestimation of the amount of glaucomatous damage. functional tests targeting specific subtypes of rgc, with lower levels of redundancy, may be able to detect damage at an earlier stage. such tests would be particularly effective if they were to target groups of rgc susceptible to early glaucomatous damage [5, 10, 11, 12 ]. although it is unlikely that any stimulus can be specific for a single visual pathway, it is possible to design stimuli to minimize the potential input from other pathways. hep uses an fdf stimulus to create a high temporal frequency, contrast detection task. previous histopathologic studies have reported that the magnocellular pathway is damaged early in the glaucomatous process, and this might explain why hep was able to detect a functional abnormality before sap in our patient [4, 5, 12 ]. other perimetric methods have also been introduced with the goal of earlier detection of glaucomatous visual field loss [13, 14 ] ; however, a recent study comparing hep with these techniques reported hep to be a more sensitive test with a stronger correlation to structural measurements. the aim of this report was to explore the potential use of fdf perimetry in the management of a glaucoma patient with statistically normal sap results. as demonstrated, hep detected a clinically and statistically significant perimetric defect and documented the area of damage that other perimetric technologies were not able to detect. there was also a good structure - function correlation with the superior nasal defect detected by hep, corresponding to the area of the inferior thinning of the neuroretinal rim and rnfl. hep also indicated a small inferior visual field defect, which was consistent with the thinning of the superior rnfl ; the sd - oct indicated a superior quadrant rnfl thickness of 107 m, which although was still within statistically normal limits, was significantly lower than the 132 m thickness in the same sector in the left eye. this case illustrates that hep may have an important role in the early detection of functional damage in glaucoma, with potential advantages over sap. hep may have the potential to become an important component of our armamentarium for the diagnosis and monitoring of glaucoma ; however, prospective longitudinal studies are required to fully validate this technology. aerie : financial support ; alcon : consultant ; allergan : consultant ; altheos : consultant ; amakem : consultant ; bausch & lomb : consultant ; carl zeiss - meditec : consultant ; genentech : financial support ; haag - streit : financial support ; heidelberg engineering : financial support ; konan : financial support ; lumenis : financial support ; national eye institute : financial support ; nidek : financial support ; optovue : consultant ; quark : consultant ; solx : consultant ; topcon : consultant. carl - zeiss : financial support ; heidelberg engineering : financial support ; topcon : financial support ; alcon : financial support ; allergan : financial support ; sensimed : financial support ; reichert : financial support. | purposeto illustrate the potential role for heidelberg edge perimetry (hep) in the assessment of early functional loss in glaucoma.case reporta 46-year - old male presented with raised intraocular pressure but healthy optic discs and normal standard automated perimetry (sap). he was diagnosed with ocular hypertension, but after 16 years, he developed glaucomatous changes to the right optic disc and inferior retinal nerve fiber layer thinning. despite the evidence of progressive structural damage, functional testing using sap was repeatedly normal, based on conventional criteria. hep, on the other hand, revealed an extensive superior visual field damage in agreement with the structural changes. additionally, and in contrast to sap, the hep indices of glaucoma hemifield test, mean deviation and pattern standard deviation were all outside normal limits.conclusionthis case illustrates that hep may have an important role in the early detection of functional damage in glaucoma, with potential advantages over sap. |
the pulp and periodontal tissues share close embryologic, anatomic and functional interrelationship. although communication pathways of the developmental origin, pathological origin and iatrogenic origin have been suggested, the most primitive and demonstrable relationship between the two tissues is through vascular system in the presence of the apical foramen and aberrant, accessory communications. there is general consensus that pulpal disease can initiate or perpetuate periodontal disease through the apical foramen. it has been suggested in the past that since inflammation follows venous drainage and venous blood flows outward from pulp into periodontium, periodontal disease can not affect pulp. however, later studies demonstrated that teeth affected with chronic periodontitis showed pathologic changes in pulp in the form of inflammatory alterations, localized necrosis, calcification, root resorption and deposition of secondary dentin. these changes occurred because of spread of noxious inflammatory substances in a reverse direction through the lateral and accessory canals. czarnecki and schilder and torabinejad and kiger in their study disapproved this correlation between periodontal pathology and changes in pulp. the variation of opinions is due to differences in periodontal diagnostic criteria, difficulties in pulpal tissue fixation or lack of clear histologic criteria for definition of observations. with this regard, the study was undertaken to address the controversy surrounding the relationship between periodontal disease and pulpal tissue changes. to examine the degree to which pulpal pathosis is associated with periodontal disease, we evaluated the pulp of teeth affected by moderate to severe chronic periodontitis. the aim of this study was to evaluate the histopathological changes in pulp due to moderate to severe chronic periodontitis. the aim of this study was to evaluate the histopathological changes in pulp due to moderate to severe chronic periodontitis. the present study was carried out in the department of periodontics, v. s. dental college and hospital, bengaluru, and was approved by the institutional ethics committee. forty human teeth affected with moderate to severe chronic periodontitis were extracted from patients with age range of 1855 years. chronic periodontitis was diagnosed based on the criteria of the american academy of periodontology (1999). teeth were extracted in the department of oral and maxillofacial surgery of the same institution. before extraction, clinical examination was carried out and radiographs were taken as needed. patients providing teeth for the study were selected based on the following criteria : patients age (whose teeth were extracted) between 18 and 55 yearsgrade iii mobile teethnoncarious and nonattrited teethsingle or multirooted teethpocket probing depth of more than 6 mm. patients age (whose teeth were extracted) between 18 and 55 years grade iii mobile teeth noncarious and nonattrited teeth single or multirooted teeth pocket probing depth of more than 6 mm. adolescents (1115 years) and older patients (> 55 years) were excludedimmunocompromised patientsrestored teethteeth with developmental disorders (amelogenesis imperfecta, dentinogenesis imperfecta, dentin dysplasia, regional odontodysplasia, etc.)teeth with a history of trauma, bruxism or clenchingorthodontically treated teethteeth having any history of periodontal treatment. adolescents (1115 years) and older patients (> 55 years) were excluded immunocompromised patients teeth with developmental disorders (amelogenesis imperfecta, dentinogenesis imperfecta, dentin dysplasia, regional odontodysplasia, etc.) teeth with a history of trauma, bruxism or clenching orthodontically treated teeth teeth having any history of periodontal treatment. using local anesthesia, the teeth were extracted as atraumatically as possible to prevent the histological sequel of traumatic extraction. immediately following extraction, the apical 23 mm of the roots were sectioned with a straight fissure bur and kept in 10% neutral buffered formalin solution for a week. the sectioning of apical 23 mm of roots provided a clear accessibility for the solution to fix the pulp. after 7 days, teeth were decalcified with 6%8% nitric acid in a microwave oven (lg, 700 watts, model 1911he) placed in an aluminum enclosure fitted with an exhaust fan and vent. a beaker containing 6%8% nitric acid with teeth was placed in oven and irradiated for 5 cycles of 30 s each (at 1 h intervals) per day. the decalcified specimens were embedded in paraffin wax and sectioned longitudinally mesiodistally using a microtome (leica rm2245, germany) set at 5 m thickness. two specimens were excluded as pulpal tissue was lost during processing and only 38 teeth were available for histological evaluation. all specimens were examined under compound light microscope (model magnus mlx) using magnification of 10, 40 and 100 for inflammatory and degenerative changes (fibrosis, calcification and necrosis). edema was defined as accumulation of interstitial fluids in pulp and its presence or absence was recorded. patients age (whose teeth were extracted) between 18 and 55 yearsgrade iii mobile teethnoncarious and nonattrited teethsingle or multirooted teethpocket probing depth of more than 6 mm. patients age (whose teeth were extracted) between 18 and 55 years grade iii mobile teeth noncarious and nonattrited teeth single or multirooted teeth pocket probing depth of more than 6 mm. adolescents (1115 years) and older patients (> 55 years) were excludedimmunocompromised patientsrestored teethteeth with developmental disorders (amelogenesis imperfecta, dentinogenesis imperfecta, dentin dysplasia, regional odontodysplasia, etc.)teeth with a history of trauma, bruxism or clenchingorthodontically treated teethteeth having any history of periodontal treatment. adolescents (1115 years) and older patients (> 55 years) were excluded immunocompromised patients teeth with developmental disorders (amelogenesis imperfecta, dentinogenesis imperfecta, dentin dysplasia, regional odontodysplasia, etc.) teeth with a history of trauma, bruxism or clenching orthodontically treated teeth teeth having any history of periodontal treatment. using local anesthesia, the teeth were extracted as atraumatically as possible to prevent the histological sequel of traumatic extraction. immediately following extraction, the apical 23 mm of the roots were sectioned with a straight fissure bur and kept in 10% neutral buffered formalin solution for a week. the sectioning of apical 23 mm of roots provided a clear accessibility for the solution to fix the pulp. after 7 days, teeth were decalcified with 6%8% nitric acid in a microwave oven (lg, 700 watts, model 1911he) placed in an aluminum enclosure fitted with an exhaust fan and vent. a beaker containing 6%8% nitric acid with teeth was placed in oven and irradiated for 5 cycles of 30 s each (at 1 h intervals) per day. the decalcified specimens were embedded in paraffin wax and sectioned longitudinally mesiodistally using a microtome (leica rm2245, germany) set at 5 m thickness. two specimens were excluded as pulpal tissue was lost during processing and only 38 teeth were available for histological evaluation. all specimens were examined under compound light microscope (model magnus mlx) using magnification of 10, 40 and 100 for inflammatory and degenerative changes (fibrosis, calcification and necrosis). edema was defined as accumulation of interstitial fluids in pulp and its presence or absence was recorded. immediately following extraction, the apical 23 mm of the roots were sectioned with a straight fissure bur and kept in 10% neutral buffered formalin solution for a week. the sectioning of apical 23 mm of roots provided a clear accessibility for the solution to fix the pulp. after 7 days, teeth were decalcified with 6%8% nitric acid in a microwave oven (lg, 700 watts, model 1911he) placed in an aluminum enclosure fitted with an exhaust fan and vent. a beaker containing 6%8% nitric acid with teeth was placed in oven and irradiated for 5 cycles of 30 s each (at 1 h intervals) per day. the decalcified specimens were embedded in paraffin wax and sectioned longitudinally mesiodistally using a microtome (leica rm2245, germany) set at 5 m thickness. two specimens were excluded as pulpal tissue was lost during processing and only 38 teeth were available for histological evaluation. all specimens were examined under compound light microscope (model magnus mlx) using magnification of 10, 40 and 100 for inflammatory and degenerative changes (fibrosis, calcification and necrosis). edema was defined as accumulation of interstitial fluids in pulp and its presence or absence was recorded. several microscopic sections indicated that the pulp could range from intact to necrotic in different sections of the same tooth [table 1 and graph 1 ]. percentage distribution of various pathologic changes in pulp percentage distribution of various pathological changes in pulp pulpal calcification (52.62%) and partial necrosis of pulp (52.62%) were found to be the most common finding [figures 1 and 2 ] inflammation, which was found in 47.38% of the cases, ranged from mild to severe in most sections and was always chronic [figure 3 ] pulp with complete necrosis was seen in 26.32% of cases [figure 4 ] fibrosis [figure 5 ] and pulpal edema was seen in 36.84% of cases. pulpal calcification (52.62%) and partial necrosis of pulp (52.62%) were found to be the most common finding [figures 1 and 2 ] inflammation, which was found in 47.38% of the cases, ranged from mild to severe in most sections and was always chronic [figure 3 ] pulp with complete necrosis was seen in 26.32% of cases [figure 4 ] fibrosis [figure 5 ] and pulpal edema was seen in 36.84% of cases. (a) histopathological image showing pulpal calcification (h & e stain) under 10. (b) histopathological image showing pulpal calcification (h & e stain) under 40 histopathological image showing partial necrosis of pulp (h&e stain) under 10 (a) histopathological image showing mild inflammation of pulp (h&e stain) under 40. (b) histopathological image showing moderate inflammation of pulp with edema (h&e stain) under 40. (c) histopathological image showing severe inflammation of pulp (h&e stain) under 40 histopathological image showing complete necrosis of pulp (h&e stain) under 40 histopathological image showing pulpal fibrosis (h&e stain) under 40 table 2 shows samples of pulpal inflammation, which exhibited calcification and partial necrosis : pulpal inflammation samples showing calcification and partial necrosis of 18 samples of inflammation, nine had calcification (50%) and 13 had partial necrosis (72.22%). of 18 samples of inflammation, nine had calcification (50%) and 13 had partial necrosis (72.22%). a lot of speculation seems to exist with regard to potential or actual relationship between pulp and periodontal tissues. since the effect of periodontal disease on pulp is unclear and controversial, this study examined the changes in dental pulp in teeth with moderate to advanced periodontitis. seltzer. stressed the importance of accessory and lateral canals in spreading the noxious substance from periodontal tissue to pulp and concluded that periodontal disease could lead to a greater incidence of inflammatory and degenerative changes pulpal changes. on the other hand, mazur and massler criticized this study because of the lack of controlled specimens and contradicted the relationship between the severity of periodontal disease and pulpal changes. later, other authors who conducted human and animal studies suggested that degenerative pulpal changes develop independent of periodontal disease. the most common pulpal changes reported in literature are inflammatory and degenerative changes in the form of fibrosis and calcifications and the results of our study are in agreement with earlier studies. these studies, however, employed cross - sectional specimens whereas we employed longitudinal sections. although the chances of developing an artifact are more in longitudinal section, it allows to view the pulp entirely and also its relations to accessory and lateral canals. instead of merely dropping the tooth in a formalin jar, the apical 12 mm of root was sectioned which provided a more prominent entry for formalin to fix the pulpal tissues as inadequate pulp fixation can result in artifacts. the microwave oven method of demineralization is more efficient as it reduces the time required for decalcification and is devoid of drawbacks related to the specimen preparation. table 2 shows that of 18 samples of inflammation, nine showed calcification and 13 showed partial necrosis. inflammation probably occurred in response to toxic products from plaque bacteria colonizing the denuded root surface. the dynamics of pulp inflammation is not different to that of inflammation in the periapical and other tissues. depending on the severity and duration of the irritants, the pulp response ranges from reversible to irreversible pulpitis, then to partial necrosis which leads to complete necrosis. the dental pulp may also respond to irritation with a range of degenerative changes including fibrosis and calcification. a wide range of nonspecific mediators of inflammation such as histamine, bradykinin, serotonin, interleukins and arachidonic acid metabolites (pge2) the tissue becomes edematous as a result of filtration of serum proteins and fluid from the vessels. the flow stasis causes an aggregation of red blood cells and an elevation of blood viscosity. it also produces tissue hypoxia or ischemia, which suppress cellular metabolism in the affected area of the pulp. in addition, neutrophils in the area degenerate and release intracellular lysosomal enzymes to digest the surrounding tissue, forming necrotic tissue. as time progresses, necrotic pulp tissue becomes infected by oral microorganisms penetrating into the root canal system through exposed dentinal tubules and lateral / accessory canals. however, as long as the accessory canals are protected by sound cementum, necrosis usually does not occur. in addition, if the microvasculature of the apical foramen remains intact, the pulp will maintain its vitality. thrombi in pulp blood vessels and collagen sheaths around vessel walls may become nidi for mineralization, resulting in pulp calcification. it may also be a normal physiologic response to aging and genetic predisposition may play a role. this study is limited by the fact that no attempt was made to correlate the extent and severity of periodontal disease with pulpal changes. in the absence of age - matched controls, it is difficult to discuss whether changes observed are attributable to age alone. degenerative changes, increased calcification and reduction in cellularity have been described with aging pulp. over many decades, the enigma of periodontal disease affecting pulp has prevailed. more case in the presence of moderate to severe chronic periodontitis, degenerative changes such as inflammation, fibrosis, edema, calcification and pulpal necrosis were observed to a variable degree. it is not important what kind of degenerative changes develop in pulp, as the treatment modality remains the same for all of them. | introduction : over the past century, the dental literature has consistently reflected a controversy related to the effect of periodontal disease on the dental pulp. nonetheless, practitioners are of the opinion that teeth having deep periodontal pockets show variable pulpal response, which may necessitate root canal treatment. thus, this study aimed to evaluate the changes in pulp due to advanced periodontal disease.materials and methods : forty caries - free teeth affected with severe periodontitis were collected from patients aged between 18 and 55 years. the collected teeth were stored in formalin for 24 h and were then decalcified and examined histologically after staining with hematoxylin and eosin to note the changes that occurred in pulp.results:pulpal calcification (52.62%) and partial necrosis of pulp (52.62%) were found to be the most common findings. inflammation, which was found in 47.38% of the cases, ranged from mild to severe in most sections and was always chronic. pulp with complete necrosis was seen in 26.32% of cases. fibrosis and pulpal edema were seen in 36.84% of cases.conclusion:in the presence of moderate to severe chronic periodontitis, degenerative changes such as inflammation, fibrosis, edema, calcification and necrosis were observed to variable degree. |
post - transcriptional gene silencing (ptgs) is a general term for a variety of phenomena that repress gene expression by causing degradation of mrna. ptgs was discovered ' by accident ' in organisms that carried a transgene, were virally infected, or were treated with exogenous rna. a form of ptgs triggered by transgenic dna, called co - suppression, was initially described in plants, and a related phenomenon, termed quelling, was later observed in the filamentous fungus neurospora crassa. co - suppression was first noticed when a transgenic petunia, expected to express a transgene involved in pigment formation at a high level, instead expressed neither the transgene nor related, endogenous genes. subsequent work indicated that viral infection can also trigger co - suppression in plants, leading to the hypothesis that the biological role of ptgs is as an anti - viral defense mechanism. meanwhile, other experiments with the soil nematode caenorhabditis elegans uncovered a phenomenon triggered by double stranded (ds) rna, called rna interference (rnai), as well as transgene - induced co - suppression. rnai is an extremely valuable tool for ' reverse ' genetic studies because it gives researchers a quick means to determine the loss - of - function phenotype for a gene. a wide variety of organisms have now been shown to respond to rnai, including drosophila and mouse (see, for example,). because of the utility of ptgs for reverse genetic studies and its possible clinical uses, there is a great deal of interest in the underlying molecular mechanism(s). it has become apparent that different ptgs phenomena have common characteristics, and this realization has led to the speculation that ptgs in different organisms may be mediated by similar molecular mechanisms. genes involved in ptgs have been identified in n. crassa (qde genes), c. elegans (rde, mut, and ego-1 genes), and the mustard plant arabidopsis thaliana (sde, sgs genes). intriguingly, related proteins function in ptgs in these organisms (table 1), as would be expected if different forms of ptgs occur by similar mechanisms. among proteins associated with ptgs to date, the most widely conserved are those with homology to tomato rna - directed rna polymerase (rdrp) : neurospora qde-1, c. elegans ego-1, and arabidopsis sgs-2/sde-1 (; figure 1). the recent addition of arabidopsis genes to this collection suggests that ptgs is a widely conserved, evolutionarily ancient means of gene regulation. two additional protein families have been linked to ptgs in nematodes and neurospora (table 1) but such a link has yet to be made in plants. piwi / sting proteins are related to eif2c, a proposed translation factor (see) ; several members of this family are known to have important developmental functions. the mut-7 and qde-3 genes encode members of the wrn (werner 's syndrome) protein family, whose members include several recq dna helicases ; werner 's syndrome is associated with premature aging. qde-3 and mut-7 are predicted to have nucleic acid binding activity : qde-3 has strong homology to the dna helicase domains and mut-7 has homology to the rnase catalytic domains. independent screens by the vaucheret and baulcombe laboratories for co - suppression - defective mutants in arabidopsis have recovered mutations in a rdrp - related gene, named sgs2 (suppressor of gene silencing) or sde1 (silencing defective). for the purposes of this review, the gene is referred to as sgs2/sde1. several other rdrp - related genes exist in arabidopsis, but no mutations were recovered in those genes. because co - suppression in plants can be triggered by viral infection and, in fact, is hypothesized to be a viral defense mechanism, both groups investigated the effects of viral infection on sgs2/sde1 mutants. was virally induced ptgs defective ? were the mutants super - susceptible to viral infection ? dalmay and colleagues found that ptgs triggered by tobacco mosaic virus and tobacco rattle virus is not defective in sde1 mutants. they interpret their data as meaning that sde1 is not required for virally induced ptgs, but only for transgene - induced ptgs. they suggest that virally encoded rdrp activity can substitute for the putative rdrp activity of sde1. in contrast, mourrain and colleagues found that sgs2 mutants had increased susceptibility to infection by certain viruses, such as a cucumovirus, but not others, such as a tobamovirus or a potyvirus. they interpret these results as indicating that ptgs indeed acts as a defense against at least some viruses. interpretation of infection data is complicated by the fact that many viruses can inhibit ptgs : tobamovirus and potyvirus strongly inhibit ptgs, whereas cucumovirus inhibits it only weakly. logically, the loss of a plant component of ptgs, such as sgs2/sde1, will have no effect on a virus that fully blocks ptgs by itself, but would allow a higher level of infection by a virus that disables ptgs only partially or not at all. at any rate, these results suggest that the dalmay. model is unlikely to be correct for all viruses ; rdrp activity has been known in plants for decades, although its physiological function has been unclear. many models for ptgs in plants postulate an rna amplification step that could be accomplished by rdrp activity (for example in), but it has yet to be shown definitively that rdrp is in fact involved in ptgs. in light of these models, it is intriguing that proteins related to the purified tomato rdrp have now been linked to ptgs in diverse organisms. it should be noted, however, that these proteins have not yet been shown to have rdrp activity. they contain large regions of sequence conservation with tomato rdrp, which may encode rdrp activity, but they also contain extensive regions of divergent sequences (see figure 1). analysis of genome sequence data has shown that c. elegans and arabidopsis contain several rdrp - related genes. in c. elegans, for example, there are three rrf (rdrp family) genes in addition to ego-1. thus far, six other rdrp - related genes have been found in the arabidopsis genome in addition to sgs2/sde1. genome sequencing has uncovered rdrp - related genes in many other organisms, including several plant species, such as wheat and petunia, and the fission yeast schizosaccharomyces pombe. it is not yet clear though how many of these rdrp - related proteins actually function in ptgs. indeed, ego-1 was identified originally as being important in development of the c. elegans germ line, and was only later shown to function in rnai ; the connection between development and rnai is not yet clear, but one possibility is that ego-1 protein has different functions in the two processes. since alleles of only one rdrp - related gene, sgs2/sde1, were recovered as suppressors of ptgs, perhaps the related genes in arabidopsis do not function in ptgs. alternatively, some or all of these genes may be redundant, so that simultaneous mutations in two or more of them would have to be induced in order to see a phenotype. in addition, if any rdrp - related gene is essential, it would not have been identified because the screens for silencing - defective mutants did not allow for recovery of lethal mutations. rdrp - related genes are apparently absent from at least one organism that is susceptible to rnai : drosophila melanogaster. genome sequence data are nearly complete for drosophila, yet no gene with homology to either tomato or viral rdrp has been found. since viral and cellular (for example, tomato) rdrp have little amino acid homology to each other, perhaps rdrp function in drosophila is accomplished by an enzyme with yet a different amino acid sequence. molecular characterization of another arabidopsis sgs gene, sgs3, by mourrain. revealed that it encodes a novel protein. in general, sgs3 mutants behave like sgs2 mutants, including having an increased susceptibility to cucomovirus infection. no sgs3 relative has been found yet in any other organism, including those with fully sequenced genomes such as c. elegans and drosophila and this result is consistent with sgs3 functioning in a plant - specific aspect of ptgs. the extensive and growing genome sequence database is an important resource for studying the mechanics of ptgs and the role of rdrp - related genes in this and other cellular processes. genome sequence information has allowed researchers to identify paralogs and orthologs very quickly, and provides a focus for future experiments. for example, reverse genetic approaches can be used to generate deletion mutations in these related genes for use in studying their possible role in ptgs. we can expect a more complete molecular portrait of ptgs to be revealed in the near future. schematic alignment of tomato rdrp with proteins known to be involved in ptgs in fungus (qde-1), nematodes (ego-1), and plants (sgs2/sde1). the total number of amino acids in each protein is listed to the right ; amino acid positions delimiting the conserved regions are indicated. within the shaded region, the percentage of rdrp amino - acid sequence identical to each related gene sequence is as follows : qde-1, 17% ; ego-1, 25% ; sds2/sde1, 41%. substantial sequence similarity between rdrp and sgs2/sde1 is present in the non - shaded region as well ; qde-1 is relatively divergent from the other three proteins. conserved proteins involved in ptgs gene names in full : ego, enhancer of glp-1 ; mut, mutator ; qde, quelling defective ; rde, rnai defective ; rdrp, rna - directed rna polymerase ; sde, silencing defective ; sgs, suppressor of gene silencing ; wrn, werner 's syndrome. the author thanks john belote and m kathryn barton for helpful comments on the manuscript. | proteins with homology to rna - directed rna polymerases function in post - transcriptional gene silencing : in quelling in the fungus neurospora crassa, rnai in the nematode caenorhabditis elegans, and co - suppression in the mustard plant arabidopsis thaliana. these findings are consistent with a conserved mechanism operating in these diverse species. |
the two prairie dogs from whom data are presented here came from a group of approximately 200 prairie dogs that were housed at a wholesale pet store with multiple species of exotic african rodents. of the 15 ill prairie dogs, 10 died rapidly, and 5 exhibited anorexia, wasting, sneezing, coughing, swollen eyelids, and ocular discharge. initially, tularemia was suspected clinically, and two of the ill prairie dogs were euthanized for pathologic confirmation. fresh lung tissue specimens were evaluated for the presence of viable infectious virus by injecting them into bsc-40 tissue culture and observing them daily for typical cytopathic effect. fresh, unfixed tissues from lung were examined for specific signatures of monkeypox virus by using pcr. samples were initially evaluated by single - gene pcr, followed by restriction - endonuclease fragment length polymorphism (rflp) identification of monkeypox - specific fragment patterns (79). an additional novel multiplex standard pcr assay (10) discriminated monkeypox from vaccinia and variola orthopoxvirus species on the basis of specific dna polymerase gene amplicons. real time (rt)-pcr assays included a specific monkeypox virus nucleic acid signature encoded in the envelope gene (monkeypox - b6r) and orthopoxvirus nucleic acid signatures in the dna polymerase gene (e9l non var.). controls included dna from monkeypox virus, other orthopoxviruses, and no - template controls. tissues were examined grossly and microscopically. hematoxylin- and eosin - stained slides were prepared from formalin - fixed, paraffin - embedded samples of the central nervous system, conjunctivae, tongue, salivary glands, lungs, heart, liver, gastrointestinal tract, spleen, adrenal glands, kidneys, and lymph nodes. ihc assays were performed as previously described for other infectious agents in the dako autostainer (dako corp., carpinteria, ca) (1114). tissue sections were then digested with 0.1 mg / ml proteinase k (roche diagnostics, indianapolis, in) in 0.6 m tris (ph 7.5)/ 0.1% cacl2 (proteinase k buffer) for 15 min and later blocked with 20% normal sheep serum in tris - saline - tween-20. primary antibodies included three polyclonal antiorthopoxvirus antibodies (rabbit antivariola virus, mouse antivaccinia virus, and rabbit antimonkeypox virus [centers for disease control and prevention (cdc), atlanta, ga ]) ; in addition, a monoclonal anti - francisella tularensis antibody (naval biodefense program, bethesda, md), and a polyclonal anti yersinia pestis antibody (cdc, fort collins, co). this was followed by sequential application of swine antimouse or swine antirabbit link antibody, avidin - alkaline phosphatase, and naphthol / fast red substrate (dako corp). sections were then counterstained in meyer s hematoxylin (fisher scientific, pittsburgh, pa). positive controls included formalin - fixed, paraffin - embedded cells infected with variola virus, vaccinia virus, and monkeypox virus. negative controls included similar cells infected with influenza a virus and human herpesvirus 1 (herpes simplex) and 3 (varicella - zoster) ; animal tissue samples infected with ebola virus, y. pestis, and f. tularensis ; human skin lesions known to have human herpesvirus 1 and 3 ; and human skin samples with noninfectious dermatitis caused by poison ivy or drug eruptions. negative controls for the prairie dogs specimens consisted of sequential tissue sections incubated with normal rabbit or mouse serum. specimens for em were excised from paraffin - embedded blocks of lung and tongue in areas that corresponded to positive ihc results. tissues were deparaffinized for 1 h in xylene warmed to 60c, rehydrated through a graded series of alcohols, postfixed in phosphate - buffered 2.5% glutaraldehyde and 1% osmium tetroxide, stained with 4% uranyl acetate, dehydrated through a graded series of alcohols and propylene oxide, and embedded in a mixture of epon - substitute and araldite. fresh lung tissue specimens were evaluated for the presence of viable infectious virus by injecting them into bsc-40 tissue culture and observing them daily for typical cytopathic effect. fresh, unfixed tissues from lung were examined for specific signatures of monkeypox virus by using pcr. samples were initially evaluated by single - gene pcr, followed by restriction - endonuclease fragment length polymorphism (rflp) identification of monkeypox - specific fragment patterns (79). an additional novel multiplex standard pcr assay (10) discriminated monkeypox from vaccinia and variola orthopoxvirus species on the basis of specific dna polymerase gene amplicons. real time (rt)-pcr assays included a specific monkeypox virus nucleic acid signature encoded in the envelope gene (monkeypox - b6r) and orthopoxvirus nucleic acid signatures in the dna polymerase gene (e9l non var.). controls included dna from monkeypox virus, other orthopoxviruses, and no - template controls. eosin - stained slides were prepared from formalin - fixed, paraffin - embedded samples of the central nervous system, conjunctivae, tongue, salivary glands, lungs, heart, liver, gastrointestinal tract, spleen, adrenal glands, kidneys, and lymph nodes. ihc assays were performed as previously described for other infectious agents in the dako autostainer (dako corp., tissue sections were then digested with 0.1 mg / ml proteinase k (roche diagnostics, indianapolis, in) in 0.6 m tris (ph 7.5)/ 0.1% cacl2 (proteinase k buffer) for 15 min and later blocked with 20% normal sheep serum in tris - saline - tween-20. primary antibodies included three polyclonal antiorthopoxvirus antibodies (rabbit antivariola virus, mouse antivaccinia virus, and rabbit antimonkeypox virus [centers for disease control and prevention (cdc), atlanta, ga ]) ; in addition, a monoclonal anti - francisella tularensis antibody (naval biodefense program, bethesda, md), and a polyclonal anti this was followed by sequential application of swine antimouse or swine antirabbit link antibody, avidin - alkaline phosphatase, and naphthol / fast red substrate (dako corp). sections were then counterstained in meyer s hematoxylin (fisher scientific, pittsburgh, pa). positive controls included formalin - fixed, paraffin - embedded cells infected with variola virus, vaccinia virus, and monkeypox virus. negative controls included similar cells infected with influenza a virus and human herpesvirus 1 (herpes simplex) and 3 (varicella - zoster) ; animal tissue samples infected with ebola virus, y. pestis, and f. tularensis ; human skin lesions known to have human herpesvirus 1 and 3 ; and human skin samples with noninfectious dermatitis caused by poison ivy or drug eruptions. negative controls for the prairie dogs specimens consisted of sequential tissue sections incubated with normal rabbit or mouse serum. specimens for em were excised from paraffin - embedded blocks of lung and tongue in areas that corresponded to positive ihc results. tissues were deparaffinized for 1 h in xylene warmed to 60c, rehydrated through a graded series of alcohols, postfixed in phosphate - buffered 2.5% glutaraldehyde and 1% osmium tetroxide, stained with 4% uranyl acetate, dehydrated through a graded series of alcohols and propylene oxide, and embedded in a mixture of epon - substitute and araldite. the lungs showed patchy areas of red - brown consolidations involving about 50% of the pulmonary parenchyma. typical orthopoxvirus sequences were revealed in lung tissue samples by use of a novel multiplex pcr assay, which detected the essential dna polymerase gene ; however, the standard single - gene pcr and rflp analysis did not show the presence of monkeypox virus. viral cytopathic effects were observed at days 4 and 5 in cultures inoculated with fresh lung samples. histopathologic examination of the eyelids showed a necrotic, ulcerated lesion of the palpebral conjunctiva. the ulcer bed consisted of necrotic debris and pyknotic epithelial cells. columnar epithelial cells surrounding the ulcer were swollen and contained dense, eosinophilic, cytoplasmic granules of various sizes that suggested guarnieri - like inclusions. other areas of the palpebral conjunctiva and skin showed inflammatory foci in the epithelium without ulcer formation but with ballooning degeneration of epithelial cells, acantholysis, and occasional cell necrosis. abundant orthopoxvirus antigens were detected in areas with grossly and microscopically identified lesions (figure 1a, b) by using orthopoxvirus ihc assays. viral antigens were present prominently in the squamous and columnar epithelium (figure 1b) and in lesser amounts in fibroblasts and histiocytes in the ulcer bed or underlying the lesions. in epithelial cells, antigens were observed in the cytoplasm intensely staining the cytoplasmic guarnieri - like inclusions. immunohistochemical staining of a prairie dog eyelid infected with monkeypox virus, showing orthopox virus antigen staining of the cytoplasm of the epithelium of the palpebral conjunctivae (assay using anti variola virus antibody ; original magnifications : a, 12.5x ; b, 25x). histopathologically, the tongue ulcer demonstrated necrosis and mixed inflammation at the ulcer bed (figure 2a). nonulcerated mucosa showed focal areas of lichenoid interface, mixed inflammatory infiltrate with necrosis, ballooning degeneration, and dense eosinophilic cytoplasmic granules (guarnieri - like inclusions) in the squamous epithelium. ihc assays showed viral antigens only in lesions (figure 2b) ; the antigens had a pattern similar to that described for the necrotic, ulcerated lesion of the conjunctivae. em examination revealed abundant mature and immature poxvirus particles in the cytoplasm of epithelial cells (figure 2c, d). ulcer on tongue of a prairie dog infected with monkeypox virus (a : hematoxylin and eosin stain, 12.5x original magnification). orthopox viral antigens are abundant in the squamous epithelium, with lesser amounts in the ulcer bed (b : immunohistochemical stain using the anti - smallpox antibody, 12.5x original magnification). tongue epithelial cell adjacent to epidermal basement membrane (small arrows) with guarnieri - like inclusion (large arrow) (c : transmission electron microscopy, 2,400x original magnification). higher magnification of the guarnieri - like inclusion shows intracellular immature (arrowhead) and mature (arrows) orthopox virions. the mature virions consist of a dense core surrounded by several laminated zones and enclosed within an outer membrane. airways showed necrosis and mixed infiltrate of neutrophils and histiocytes in the lumen and epithelium, and the bronchial epithelium showed a reactive proliferative response (figure 3a). inflammation extended through the bronchilolar walls into surrounding alveoli, which demonstrated fibrinous edema, necrosis, and marked infiltrate of macrophages, some having intranuclear cytoplasmic inclusions, while others showed multinucleation. ihc assays demonstrated abundant viral antigens in the areas with bronchioalveolar inflammation (figure 3d). viral antigens were observed in the cytoplasm of macrophages, bronchial epithelial cells, and fibroblasts ; viral antigens were also present in the necrotic debris and interstitial connective tissue (figure 3c). immature and mature poxvirus particles were demonstrated inside bronchial epithelial cells by using em (figure 3b). lung of prairie dog infected with monkeypox virus, showing abundant intraalveolar mixed inflammatory infiltrate and necrosis (a : hematoxylin and eosin stain, 50x original magnification). orthopox viral antigens are abundant in the cytoplasm of the bronchiolar epithelium (d : immunohistochemical assay anti variola virus antibody, 100x original magnification). macrophages, fibroblasts, and alveolar epithelial cells, as well as necrotic debris demonstrate orthopox viral antigens in pneumonic areas of the lung (c : immunohistochemical stain anti variola virus antibody, 100x original magnification). accumulation of intracellular mature virions (arrow) in bronchial epithelial cell (arrowhead pointing to cilia) (b : transmission electron microscopy, 2,400x original magnification). except for mild portal inflammation in the liver and reactive hyperplasia in the spleen viral antigens were not observed in other tissues, with the exception of occasional medullary and subcapsular sinusoidal histiocytes in a submandibular lymph node. during the 2003 outbreak of human monkeypox in the united states, a shipment of african rodents that contained gambian rats and dormice is thought to have resulted in secondary infection of prairie dogs (14). exposure to infected prairie dogs resulted in 37 human infections involving exotic pet dealers, pet owners, and veterinary care workers in the united states (13). the mode of transmission of the monkeypox virus between infected animals and humans is not clearly defined, partly because histopathologic and immunohistochemical studies of animals with naturally acquired infection have not been published. the prairie dogs in this study demonstrated abundant viral antigens and mature poxvirus particles in the tongue and conjunctival lesions ; hence, direct contact with saliva or exudates from these lesions could have inoculated monkeypox virus to skin or mucous membranes of other hosts. in addition, the lungs demonstrated abundant replicating monkeypox virus in the bronchi and lung parenchyma ; thus, transmission to other rodents and humans may have occurred when the infected animal coughed and dispersed infective droplets. furthermore, the pneumonic process in these prairie dogs suggests a respiratory route of infection between rodents. thus, the pathologic study of severely ill prairie dogs in this outbreak provided evidence that direct mucocutaneous contact and respiratory routes played a role in transmission, as has been suggested in african outbreaks of human disease (1519). prairie dogs may be an excellent animal model for the further study of monkeypox infections because they are small, plentiful, and susceptible to severe monkeypox virus disease. in naturally or experimentally infected animals, a spectrum of clinical illness will develop ; for example, of the nonhuman species that naturally acquire monkeypox virus infections, skin lesions have only been observed in some african primate species and rope squirrels (funiscuirus spp.) the pathologic features observed in prairie dogs, including a necrotizing bronchopneumonia, have been described in cynomolgus monkeys infected experimentally by inhalation of monkeypox virus (21). in these animals, monocytes carried the virus to lymphoid tissues, where a secondary viral replication occurred and resulted in the seeding of other tissues, including skin, oral mucosa, gastrointestinal tract, and the tissues of the reproductive system. in this monkey model, secondary viral replication sites had necrotizing lesions. necrotizing lesions with viral antigens in lymphoid tissues have been seen in other animals, including prairie dogs that fell ill and died during the u.s. the prairie dogs in this study did not have necrotizing lymphadenitis or splenitis, which may indicate that these rodents were euthanized relatively early in the disease course. histopathologically, infected epithelial cells showed prominent ballooning degeneration and dense, eosinophilic, cytoplasmic granules that were difficult to distinguish from keratohyalin bodies. epithelial cells occasionally coalesced, forming syncytia, and their nuclei showed eosinophilic, ground - glass staining that must be differentiated from herpetic inclusions for diagnostic purposes. by use of ihc, the eosinophilic cytoplasmic granules seen in infected epithelial cells were proven to be viral inclusions (guarnieri - like inclusions), and em examination corroborated these findings. in the prairie dogs studied, orthopoxvirus antigens were also demonstrated in other cells, including macrophages and fibroblasts in areas adjacent to infected epithelial cells. histopathologic studies of human monkeypox skin vesicular lesions showed an ihc staining pattern similar to that found in the tongue and conjunctiva of the infected prairie dogs (14,22,23). monkeypox virus outbreak demonstrated how new diseases can emerge due to facile movement of species from one location to another (including the illegal transporting of species). the investigation of human cases at the wholesale pet store that housed a variety of african rodents and the two prairie dogs studied revealed a spectrum of monkeypox - associated disease ranging from only serologic evidence of monkeypox infection to febrile vesicular rash illness (3). differences in disease severity may relate to the source of exposure, transmission route (i.e., inhalational versus direct mucocutaneous contact), amounts of virus inoculated, virus strain, or host susceptibility. epidemiologic studies of human monkeypox infections have shown that younger children and persons not vaccinated against smallpox can have severe disease and complications, which supports the importance of host susceptibility, including previous immunity (18,24,25). this outbreak of monkeypox virus infection in humans and nonhuman animals is an important reminder to monitor surveillance programs for febrile rash illnesses designed to detect potential bioterrorism attacks with smallpox virus, which may be beneficial for detecting emerging infections (26). a variety of methods were used to diagnose and study monkeypox virus infection in these prairie dogs. em and culture demonstrated viral replication, while molecular studies were essential for determining the specific signatures of monkeypox virus. in however, rt - pcr detected monkeypox viral dna since rt - pcr is more sensitive and can be used to accurately titrate up to 410 dna copies (27). research into the natural biology of monkeypox has been limited because the disease is rare in humans and no descriptions exist of naturally acquired animal infections. the pathologic findings in this study of prairie dogs can be used to better define possible transmission routes and pathogenesis of human and animal monkeypox, and such a model may help develop new vaccine and treatment strategies for orthopoxvirus infections. | during may and june 2003, the first cluster of human monkeypox cases in the united states was reported. most patients with this febrile vesicular rash illness presumably acquired the infection from prairie dogs. monkeypox virus was demonstrated by using polymerase chain reaction in two prairie dogs in which pathologic studies showed necrotizing bronchopneumonia, conjunctivitis, and tongue ulceration. immunohistochemical assays for orthopoxviruses demonstrated abundant viral antigens in surface epithelial cells of lesions in conjunctiva and tongue, with less amounts in adjacent macrophages, fibroblasts, and connective tissues. viral antigens in the lung were abundant in bronchial epithelial cells, macrophages, and fibroblasts. virus isolation and electron microscopy demonstrated active viral replication in lungs and tongue. these findings indicate that both respiratory and direct mucocutaneous exposures are potentially important routes of transmission of monkeypox virus between rodents and to humans. prairie dogs offer insights into transmission, pathogenesis, and new vaccine and treatment trials because they are susceptible to severe monkeypox infection. |
oral health is considered as one of the most important issues that affect on the general health of the intensive care units (icus) admitted patients. omitting tooth brushing from dental hygiene program will cause the formation of dental plaque and consequently gingivitis. in fact, poor oral health has been incremented as the primary cause of gingivitis. dental plaques alone can be the major cause of initiation and progression of periodontal diseases. the direct correlation between dental plaques and severity of gingival inflammation or gingivitis was demonstrated. untreated periodontitis is also considered as a risk factor for systemic disorders specifically bacterial pneumonia and cardiovascular diseases. however, gingivitis and its related complications are readily preventable through controlling dental plaques and provision of oral and dental hygiene procedures. intubated patients in icu are not able to take care of their mouth health, so they are at risk of bacterial colonization and dental plaques formation that can lead to systemic diseases such as pneumonia and gingivitis. interventions for removing the dental plaques in intubated patients in icus include mechanical approaches and pharmaceutical approaches. one of the most effective methods of controlling dental plaque is the use of mouth rinses. chlorhexidine is considered as the gold treatment for controlling the dental plaque and gingivitis due to its efficacy against different kinds of bacteria, fungi, and viruses and so far, no microbial resistance has been reported for it. although chlorhexidine has been introduced as the most effective anti - plaque agent, but there is no sufficient evidence in regard to its efficacy and safety. some adverse effects include changing in color teeth and mucosa, mucosal desquamation, salivary stones creation, irritation, dryness of mouth, and side systemic effects as the result of swallowing were reported. therefore, finding the new alternative mouthwash with undesired side effects is mandatory. the world health organization (who) has recommended on finding the new natural sources such as the herbal extracts for overcoming on side effects of chemical agents. salvadora persica is traditionally used for oral and dental health, and it was recommended by who for oral hygienic procedures. the antibacterial activity of s. persica on cariogenic oral bacteria was confirmed. other pharmacological effects such as anti - inflammatory, antioxidant, antiulcer, and sedative effects were confirmed. aloe vera gel is the clear, jelly - like substance found in the inner part of the aloe leaf with anti - inflammatory, antimicrobial, antiulcer effects. therefore, the aim of this investigation was to evaluate the efficacy of oral botanical extract mouth rinse containing s. persica ethanol extract (miswak) and a. vera (aloe) gel on gingival index (gi) of intubated patients of icu and its efficacy was compared to chlorhexidine as current treatment. a randomized, double - blind controlled clinical trial was conducted on 76 patients under mechanical ventilation of three icus of teaching hospital, yazd, iran, from may to september 2014. the dentist measured the gingival health index of the patients before and after the intervention. the clinical design was approved by the university committee of ethics, and all private data of patients remained confidential. this clinical study was also registered in the iranian clinical trial center under the code number 2014062418212n1. participants with the following criteria were included all intubated patients (1865 years old with mean age 40.35 13.2) with normal teeth that had hospitalization time in icu 0.05). the results of the present study demonstrated that the use of chlorhexidine rinse and herbal extract mouthwash along with mechanical methods both reduced the gi in intubated patients, but this reduction in gi was more considerable in the herbal mouthwash group, and their differences were statistically significant (p < 0.05) [table 2 ]. gingival index for intubated patients before and after rinsing of mouthwashes there was no significant between the means of gi of intubated patients in herbal mouthwash (3.3 0.63) and chlorhexidine (3.2 0.71) groups at the baseline of the study (p = 0.4). administration of both mouthwashes decreased the gi to 2.31 0.73 and 1.5 0.6 in the patients that were treated with chlorhexidine and herbal extract mouthwash, respectively. the results were shown ; the herbal extract mouthwash was superior to chlorhexidine as the current treatment in reducing the gi of intubated patients in icus. the results of our study exhibited the high gi for patients that were admitted to icus, and high gis were not suitable for intubated hospitalized patients due to causing the systemic disorders specifically bacterial pneumonia and cardiovascular diseases. therefore, gingival disorders as silent epidemic are most commonly chronic bacterial infection that affects on 6090% of school children and nearly 100% of adults. furthermore, hospitalization has negative effects on oral health conditions of hospitalized patients and accelerates the accumulation of dental plaque that is associated with the destruction of mucosal membranes and increases the rate of gingivitis. in addition, because the oral cares of hospitalized patients are not the right priority of nurses, however, in these patients, oral problems are rapidly intensified and increased the nosocomial infections. therefore, oral health surveillance is one of the prominent topics of the icu which should be performed by nurses to prevent the onset of infection. the results of our study exhibited the prominent potency of mouthwash containing s. persica ethanol extract and a. vera gel than that of chlorhexidine in intubated hospitalized patients of icus. chlorhexidine (0.2%) as the gold standard for oral health care has been accepted. although, there are some studies that confirmed the beneficial effects of s. persica extracts and a. vera gel on oral cariogenic bacteria in vitro and in vivo conditions, separately, but our literatures survey did not show any research on the efficacy of a combination of s. persica and a. vera on gi of patients hospitalized in icus. in other study showed that mouthwash containing s. persica alone improves the gi as same as chlorhexidine. the effectiveness of a. vera gel mouthwashes and chlorhexidine on improving gi have been demonstrated the same. indeed, the higher efficacy of this natural mouthwash than that of chlorhexidine is related to the synergistic effects of s. persica and a. vera gel extracts against oral cariogenic bacteria that finally improve the gi of hospitalized patients. other biological activities of extracts including the anti - inflammatory effects, antiulcer potency, and sedative activities of s. persica and a. vera and their synergic effects improve the status of oral health. a. vera gel administration due to its efficacy in improvement of periodontal condition, nature 's soothing healer property on gingiva has been recommended as a local drug delivery system in periodontal pockets. therefore, the combination of s. persica ethanol extract and a. vera gel extract in mouthwash formula acts better than chlorhexidine in reducing the gi of intubated hospitalized patients in icus. improvement in gi of hospitalized patients is one of the most important factors which can protect the patients from pathogenic microorganisms and therefore, prevent the spread of pulmonary and nosocomial pathogens in hospital and in the lungs and blood of patients. other larger clinical studies are required for evaluating the efficacy and safety of s. persica and a. vera mouthwash. | background : intubated patients in intensive care unit (icu) are not able to take care of their mouth health, so they are at risk of bacterial colonization and dental plaques formation that can lead to systemic diseases such as pneumonia and gingivitis.aims:in randomized, double - blind clinical study, the efficacy of natural herbal mouthwash containing salvadora persica ethanol extract and aloe vera gel was compared with chlorhexidine on gingival index (gi) of intubated patients in icu.materials and methods : seventy - six intubated patients (1864 years old with mean age 40.35 13.2) in icu were admitted to this study. the patients were randomly divided into two groups : (1) herbal mouthwash and (2) chlorhexidine solution. before the intervention, the gis was measured by modified gi device into two groups. the mouth was rinsed by mouthwashes every 23 h for 4 days. 2 h after the last intervention, gis were determined.results:along with mechanical methods, herbal mouthwash in reducing gi was statistically significant than that of chlorhexidine (p < 0.05).conclusion : the results of this study introduce a new botanical extract mouthwash with dominant healing effects on gi (1.5 0.6) higher than that of synthetic mouthwash, chlorhexidine (2.31 0.73). |
the world health organization (who) estimates that globally ~293 million young children and 468 million non - pregnant women suffer from anemia, among which ~50% are estimated to be attributable to iron deficiency (i d). in addition to inadequate intake of iron, infection and blood loss are common causes of i d, and there is growing evidence suggesting that impairment of mucosal absorption of iron may contribute to the high rates of i d in developing countries. i d impairs physical activity and cognitive performance and may also cause irreversible intergenerational effects when it occurs in women of reproductive age. i d anemia (ida) during pregnancy increases the risk of adverse pregnancy and perinatal outcomes including maternal and neonatal deaths, particularly in severe anemia. because of limited resources, i d is usually diagnosed based on the estimation of hemoglobin levels despite the gross lack of sensitivity and specificity of this method. however, there is no feasible and universally accepted single indicator that can diagnose i d precisely. low plasma ferritin indicates depletion of iron stores, but values between 20 and 100 g / l are difficult to interpret because of its nature as a positive acute - phase protein. transferrin receptor (tfr) is a relatively recent addition to the iron markers and is less affected by inflammation. few studies have assessed determinants for poor iron status by using multiple iron parameters and at the same time measured the association between maternal iron status and that of their breastfed infants. the limited knowledge base is drawn primarily from studies conducted before the universal recommendation of iron and folic acid supplementation to pregnant women. widespread use of iron and folic acid supplementation during pregnancy and lactation, secular changes in dietary patterns and rapid urbanization have likely changed the contributions of i d to anemia in low- and middle - income countries such as nepal. in this paper, we aimed to describe prevalence and predictors of anemia and i d in breastfed infants and explore associations between maternal and infant iron status in a periurban nepali population. bhaktapur municipality is located in the bhaktapur district in the kathmandu valley at an altitude of 1400 m above sea level. the population according to census report of 2011 is 81 748 people, and the majority have agriculture as their main occupation. the study site includes a periurban area with a large proportion of low - income inhabitants. bhaktapur is the one of the most densely populated districts of nepal, with a population density of 11 058 people per square km. the selection criteria and details on field procedures have been described in detail elsewhere. from january 2008 to february 2009, we enrolled 500 lactating women between 15 and 44 years of age and 474 of their infants below 1 year of age from bhaktapur municipality in nepal. we used a two - stage cluster sampling procedure whereby 66 neighborhood streets called ' toles ' were randomly selected as the primary sampling unit from a list of 160. we listed all women living in these toles, and women and infant pairs were again randomly selected from this list. the inclusion criteria for the study were that both mothers and children had no ongoing infection (clinically assessed), resided in selected clusters, were willing to provide the household information and consented to participate. because of acute blood loss and many physiological and hormonal changes during delivery that usually last up to 2 months, we enrolled lactating mothers 2 months after delivery. infant pairs were recorded during the 1 year of longitudinal follow - up, and 582 were approached for enrollment. of these, the study had ethical clearance from the institutional review boards at the institute of medicine, kathmandu, nepal. four hundred and fifty mothers were required to detect this prevalence with an absolute precision of 4%, that is, with a 95% confidence interval from 21 to 29%. assuming 10% loss - to - follow - up, we enrolled 500 women and infant pairs. blood samples were collected from one of the cubital veins using micronutrient - free heparinized polypropylene tubes (eppendorf, sarstedt, germany). hemoglobin was tested on hemocue immediately after blood collection (hemocue, vedbk, denmark) with regular calibration of the instrument as instructed by the manufacturer. the samples were then centrifuged (760 g, for 10 min, room temperature), and plasma was allocated onto micronutrient - free polypropylene vials (eppendorf, hinz, germany) and stored at 20 c in a freezer in the field site laboratory. every day these samples were transported to the central laboratory in kathmandu with an ice pack where they were then stored at 70 c until they were transported on dry ice to norway where they were temporarily stored at 80c before analysis at the laboratory of clinical biochemistry, haukeland university hospital (bergen, norway). the biochemical iron parameters were analyzed on a modular analytics system by roche diagnostics (roche diagnostics gmbh, mannheim, germany) with a 5% coefficient of analytical variation for each test. the plasma ferritin was analyzed by an electrochemiluminescence immunoassay, whereas the soluble tfr and transferrin were analyzed by immunoturbidimetry. total iron - binding capacity (tibc) was calculated from transferrin concentrations. according to the who, anemia among infants between 6 and 12 months of age is defined as hemoglobin 83 mol / l, which is the upper limit value of our laboratory, to indicate an alteration in iron status. we defined ida as the concurrent presence of anemia and low plasma ferritin or a high tfr concentration. anemia was considered to have other causes when it was accompanied with high ferritin or low tfr concentration. the tfr - f ratio was calculated by dividing tfr with the log transformation of plasma ferritin, and the tfr - f index was calculated by dividing tfr and ferritin concentrations after converting milligrams of tfr to microgram units. maternal body mass index (bmi) was calculated as kg / m, and bmi 83 mol / l, which is the upper limit value of our laboratory, to indicate an alteration in iron status. we defined ida as the concurrent presence of anemia and low plasma ferritin or a high tfr concentration. anemia was considered to have other causes when it was accompanied with high ferritin or low tfr concentration. the tfr - f ratio was calculated by dividing tfr with the log transformation of plasma ferritin, and the tfr - f index was calculated by dividing tfr and ferritin concentrations after converting milligrams of tfr to microgram units. maternal body mass index (bmi) was calculated as kg / m, and bmi 6 months of age (72%) were anemic (hb 6 months of age (30%) were iron deficient (plasma ferritin 6 months of age, respectively. using combined indicators of hemoglobin and plasma ferritin, the prevalence of ida was 4% in mothers and 26% among infants > 6 months of age. however, enrolling apparently healthy subjects and screened clinically, 5% women and 21% infants had a crp concentration > 5 mg / l, suggesting a possible subclinical acute infection. none of the iron - deficient women were having high crp concentration (> 5 mg / l), but 12% of iron - deficient infants of > 6 months of age were having high crp concentration. after restricting analyses to infants with normal crp concentration (crp 6 months, respectively. slightly more than one - third (36%) of infants 6 months with normal crp concentrations had plasma ferritin concentrations 6 months of age. the prevalence of ida in infants 6 months (ferritin 6 months (ferritin 83 mol / l in 15% (n=77) of the women and 18% (n=79) of the infants. younger women (6 months of age (72%) were anemic (hb 6 months of age (30%) were iron deficient (plasma ferritin 6 months of age, respectively. using combined indicators of hemoglobin and plasma ferritin, the prevalence of ida was 4% in mothers and 26% among infants > 6 months of age. however, enrolling apparently healthy subjects and screened clinically, 5% women and 21% infants had a crp concentration > 5 mg / l, suggesting a possible subclinical acute infection. none of the iron - deficient women were having high crp concentration (> 5 mg / l), but 12% of iron - deficient infants of > 6 months of age were having high crp concentration. after restricting analyses to infants with normal crp concentration (crp 6 months, respectively. slightly more than one - third (36%) of infants 6 months with normal crp concentrations had plasma ferritin concentrations 6 months of age. the prevalence of ida in infants 6 months (ferritin 6 months (ferritin 83 mol / l in 15% (n=77) of the women and 18% (n=79) of the infants. younger women (6 months) infants. however, the average concentrations of plasma ferritin were more than threefold higher among the young infants (figure 1) probably due to temporary redistribution of iron from the red blood cells to the iron stores during the first 6 months of life when fetal hemoglobin is replaced by adult hemoglobin. in addition to this physiological process, the impact of inflammation and infectious diseases on plasma ferritin complicates selection of an adequate cutoff level to estimate the prevalence of i d and ida. this is evident from the finding that ida (17%), which did not explain more than 25% of the total prevalence of anemia (69%) in infants (table 2a), was significantly increased when we used a ferritin cutoff of 30 g / l (table 2b). most likely, an important reason for the discrepancy between prevalence of anemia and ida is the use of erroneous ferritin cutoffs, which do not relate adequately to true i d. this is particularly relevant for infants in the first months of life where 30 g / l may be is too low with respect to the enlarged iron stores with median ferritin levels above 80100 g / l. on the other hand, despite enrolling clinically healthy children, 21% had increased crp. therefore, we can not rule out that subclinical, mild infection may have biased the true ferritin cutoff limit with the greatest impact in the older infants where the frequency of higher crp concentration was higher compared with that in the younger. plasma ferritin is very sensitive to infection and may even in mild virus infection raise 2060 g the plasma concentration of tfr is increased by i d, but in contrast to ferritin, it is not seriously affected by infection. among the infants, there is good consistency between ida diagnosed by combining anemia with either increased tfr (i.e., ide) or with ferritin 17%. this point is strongly supported by the large difference between ide (76%) and depleted iron stores (20%). increased tibc, which is a measure of long - standing reduced iron absorption, was increased in 18% of infants, which corresponded well with our ida results. because of lower iron status and faster growth rate, low birth weight infants are susceptible for developing i d, which is also supported by our data as they had substantially lower hb concentration compared with normal weight babies. our finding of better iron status (reflecting higher hb and ferritin and lower tfr and tibc) among female infants is also consistent with previous findings. this is perhaps due to differences in iron requirements and metabolism or different sized iron stores in early infancy. our findings of lower hb and ferritin concentrations among infants who practiced more than 3 months of exclusive breastfeeding are also in accordance with findings from an earlier study and are a reflection of the low iron content of breast milk, but in discordance with others. we used a cutoff of 3 months for exclusive breastfeeding instead of the standard who recommendation of 6 months. this is because about 50% of our enrolled infants were < 6 months of age, and early introduction of complementary foods or drinks is common in this population. caution is required while interpreting these finding as we used a strict definition of exclusive breastfeeding based on any introduction of food / drink since birth and because feeding patterns are constantly changing, particularly among young infants. our study clearly illustrates the well - known problem of using a common, single cutoff limit to get a true estimate of the prevalence of ida and distinguish it from other causes that include deficiencies in other nutrients, inflammation, hematological abnormalities and parasitic infestation. since the finding of an alarmingly high anemia prevalence, particularly among pregnant women and infants in a micronutrient survey conducted in 1998, the government of nepal, in collaboration with different agencies including the who, has endorsed several strategies to reduce anemia. the strategy includes the following : daily supplementation with 60 mg elemental iron sulfate for 180 days during pregnancy and the postpartum period irrespective of baseline iron status ; single deworming drugs administered during the second trimester of pregnancy ; insecticide - treated bed nets in areas where malaria is a common problem ; and delayed cord clamping (after 23 min of birth). although we lack data on trends, our findings of a relatively low contribution of iron to overall anemia burden among women, high adherence to iron supplementation during pregnancy and the positive association between maternal ferritin with infant plasma ferritin suggest that these strategies have been effective, at least in the periurban area of bhaktapur. nevertheless, as shown in our study, combining tests associated with different stages of negative iron balance makes it possible to discriminate between i d, ide and ida. this may be a valuable approach to target more precisely subgroups with different need of iron in a population at risk. the prevalence of i d and anemia among infants was high, particularly among male infants born to mothers with low iron stores. the contribution of i d to the total anemia prevalence, particularly among mothers, was low, suggesting that anemia may have other causes in this community, which require further exploration. the coverage of iron supplementation among pregnant and lactating women in this population was high, and the observation that high maternal plasma ferritin was associated with lower risk of anemia among infants suggests that increased coverage of this program in rural nepal and other parts of south asia could help reduce anemia burden. | background / objectives : iron deficiency anemia is a widespread public health problem, particularly in low- and middle - income countries. maternal iron status around and during pregnancy may influence infant iron status. we examined multiple biomarkers to determine the prevalence of iron deficiency and anemia among breastfed infants and explored its relationship with maternal and infant characteristics in bhaktapur, nepal.subjects/methods:in a cross - sectional survey, we randomly selected 500 mother infant pairs from bhaktapur municipality. blood was analyzed for hemoglobin, ferritin, total iron - binding capacity, transferrin receptors and c - reactive protein.results:the altitude - adjusted prevalence of anemia was 49% among infants 26-month - old (hemaglobin (hb) < 10.8 g / dl) and 72% among infants 712-month - old (hb < 11.3 g / dl). iron deficiency anemia, defined as anemia and serum ferritin < 20 or < 12 g / l, affected 9 and 26% of infants of these same age groups. twenty percent of mothers had anemia (hb < 12.3 g / dl), but only one - fifth was explained by depletion of iron stores. significant predictors of infant iron status and anemia were infant age, sex and duration of exclusive breastfeeding and maternal ferritin concentrations.conclusions:our findings suggest that iron supplementation in pregnancy is likely to have resulted in a low prevalence of postpartum anemia. the higher prevalence of anemia and iron deficiency among breastfed infants compared with their mothers suggests calls for intervention targeting newborns and infants. |
cockayne syndrome (cs) is a rare autosomal recessive genetic disease characterized by growth failure and progressive neurological degeneration. here we report a mild form of cs patient who was homozygous for the c526 t transition resulting in a new nonsense mutation, which converts arg176 to a stop codon. it is characterized by severe growth retardation, progressive neurological degeneration, and accelerated aging. associated clinical features of cs patients are gait defects, structural eye abnormalities such as cataracts and progressive pigmentary retinopathy, sensorineural hearing loss, dental caries, and cutaneous photosensitivity. the disease is clinically heterogeneous, with a wide range in type and severity of symptoms. the phenotypic spectrum of cs can be divided into three general clinical groups : cs type i (moderate cs) is characterized by normal prenatal growth with the normal length, weight, and head circumference. however, the disease would become fully manifest, and the onset of growth and developmental would fall below normal in the first 2 years. progressive impairment of central and peripheral nervous system function, vision, hearing, dental caries, and photosensitivity leads to severe disability, with death occurring in the first or second decade. cs type ii (severe cs or early - onset cs) is evident by growth failure at birth, with little or no postnatal neurological development. affected individuals have early postnatal contractures of the spine (kyphosis, scoliosis) and joints. cs type ii overlaps clinically with the cerebro - oculofacioskeletal syndrome (cofs), which is also referred to as pena - shokeir syndrome type ii. cellular defects in the response to ultraviolet (uv) irradiation appear to be similar for type i patients and type ii patients. cs type iii (mild cs or late - onset cs) is assigned to individuals with some late - onset features of cs, with mostly normal growth and cognitive development. cs belongs to the family of nucleotide excision repair (ner) deficient disorders, together with xeroderma pigmentosum (xp), trichothiodystrophy (ttd), and uv - sensitive syndrome (uvss). the process of ner protects cells from a wide range of dna lesions including uv - induced lesions. cs cells are specifically defective in transcription - coupled dna repair (tcr), a subpathway of ner, which is closely linked to the basal transcription machinery and preferentially targets helix - distorting dna lesions in the transcribed dna strand of active genes. this correlated with earlier findings that rna synthesis recovered rapidly after uv irradiation of normal cells, whereas this recovery did not occur in cs cells. the other subpathway of ner (global genome repair or ggr) is responsible for the removal of helix - distorting dna lesions throughout the genome including those from the nontranscribed strand in the active gene. classic cs is diagnosed by clinical findings, comprising postnatal growth failure and progressive neurological dysfunction along with other minor criteria. this rare disease is linked to mutations in the csb / ercc6 and csa / ercc8 genes encoding proteins involved in the tcr pathway. molecular genetic testing or a specific dna repair assay on fibroblasts (available only on a research basis) can confirm the diagnosis. mutations in csb are distributed along the whole genomic sequence and almost all types of mutations are found, such as short insertions and deletions, nonsense mutations, splice mutations, missense mutations, promoter mutation, and polymorphisms. here, we report a chinese patient with a clinical phenotype of mild cs who has a homozygous nonsense mutation in the csb gene resulting in a new stop codon from the mutation. the patient was referred at 16 years of age, with tremor of hands, global developmental delay, and abnormal walking gait. a physical examination showed him to be 149 cm (< 2 sd) in height and 30 kg in weight (fig.1). an examination by the ophthalmologist established that the patient had retinal pigment degeneration. an examination by the otolaryngologist showed that he had bilateral nerve deafness and hearing loss 3050 db. he had early old face, long limbs, and his bone age was 19. otherwise, growth hormone, adrenal hormone, thyroid hormone, and sex hormone appeared normal. a head computed tomography scan showed calcifications of the subcortical white matter and bilateral basal ganglions, leukoencephalopathy and brain atrophy. brain magnetic resonance imaging revealed demyelination of generalized white matter and dentate nucleus, abnormal signal stove of bilateral basal ganglions. the patient at 16 years of age, 149 cm tall and 30 kg in weight. he had early old face and long limbs, with tremor of hands, and abnormal walking gait. sequence analysis of the ercc6 gene using genomic dna from the patient was applied to all 20 exons and the flanking splice junction consensus sequences of the ercc6 (csb) gene (by bgi, china). the patient was referred at 16 years of age, with tremor of hands, global developmental delay, and abnormal walking gait. a physical examination showed him to be 149 cm (< 2 sd) in height and 30 kg in weight (fig.1). an examination by the ophthalmologist established that the patient had retinal pigment degeneration. an examination by the otolaryngologist showed that he had bilateral nerve deafness and hearing loss 3050 db. he had early old face, long limbs, and his bone age was 19. otherwise, growth hormone, adrenal hormone, thyroid hormone, and sex hormone appeared normal. a head computed tomography scan showed calcifications of the subcortical white matter and bilateral basal ganglions, leukoencephalopathy and brain atrophy. brain magnetic resonance imaging revealed demyelination of generalized white matter and dentate nucleus, abnormal signal stove of bilateral basal ganglions. the patient at 16 years of age, 149 cm tall and 30 kg in weight. he had early old face and long limbs, with tremor of hands, and abnormal walking gait. sequence analysis of the ercc6 gene using genomic dna from the patient was applied to all 20 exons and the flanking splice junction consensus sequences of the ercc6 (csb) gene (by bgi, china). the patient was homozygous for three mutations : a nonsense mutation c526 t, which converts arg176 to a stop codon (cga 526 : arg176 to tga 526 : stop, located at exon3), and two missense mutations g1196a and c3962 g, respectively, resulting in the amino acid changes gly399asp and ser1321cys (fig.2). these missense mutations are not relevant for the pathological phenotype since they are located downstream of the nonsense mutation. this c to t transition generates the nonsense codon tga at amino acid position 176. this study demonstrated for the first time that a mild cs patient resulted from a homozygous nonsense mutation (cga 526 : arg176 to tga 526 : stop), located at csb gene exon3. our findings are consistent with the prior reports that patients with no detectable csb fared better than patients with slightly truncated csb. it has been hypothesized that the mutated csb protein might have some additional inhibitory functions in transcription or in oxidative damage repair, which could specifically lead to the severe cs phenotype. this hypothesis would probably imply that mutated csb polypeptides had a dominant negative effect, but direct experimental evidence for this inhibitory effect has not been provided yet. the clinical features of cs, as a rare disorder of dna repair system, were first described by cockayne in 1936. venema. first pointed out that cs cells had a defect in tcr of uv - induced dna damage. the csb gene, located on chromosome 10q11, encoded a 168-kda protein of 1493 amino acids. the csb protein included seven helicase - like atpase motifs and pertained to the swi2/snf2 family of proteins generally involved in chromatin remodeling, transcription, and dna repair. after irradiation rna synthesis was depressed in both normal and cs cells, but it recovered rapidly in normal cells and failed in cells from patients with cs. when the ercc6 cdna was introduced into different human cell lines, it was able to correct the uv sensitivity and post uv irradiation rna synthesis defect in a cs - b cell line, cs1an, and this cell strain was found to contain mutations in the ercc6 gene. csb protein has been related to various repair and transcription processes, but the details of these mechanisms remained unclear. the actual mechanism of cs remained poorly unknown and the great differences between severity and age of onset could still not be observed at the molecular level. the study by horibata. indicated that the very mild clinical presentation of uvss was due to a homozygous stop mutation at csb gene position 308 (cga 308 : arg77 to tga 308 : stop) inducing a severely truncated csb protein, which contained only the n - terminal 76aa generated in uvs1ko cells. newman. have raised the hypothesis that the fusion protein comprising the first five exons of csb gene and the open reading frame of a pgbd3 transposon nested in intron 5 could be responsible for the pathogenesis of cs. according to this hypothesis, mutations downstream of csb exon 5/6 boundary in the atpase and c - terminal domains would cause cs by impairing expression of the full - length csb protein without affecting expression of the csb - pgbd3 fusion protein. however, we found out that a nonsense mutation upstream of exon 6 caused a mild cs phenotype, this molecular research was in sharp contrast with the mild clinical feature presented by the patient, who had slight growth failure and progressive neurological degeneration. comparatively, the much more severe clinical picture of csb patients was usually due to apparently milder csb mutations, such as missense mutations or truncating mutations downstream to position 77 described previously. phenotype correlation researches have attempted to state why certain mutations in the same gene are expressed separately, but no such simple, obvious correlations have been proved. in conclusion, it is proposed that carrying a truncated or mutated version of csb may be highly harmful, whereas the complete absence of the product may give rise to mild phenotypes. it is tempting to suppose that an abnormal csb protein not only completely lost its functional activity but probably also its ability to interact with other cellular proteins, which a normal csb protein interacts with. it is reasonable to speculate on a direct relation between the severity of symptoms and the length of truncated csb proteins. thus, the mildest cases of cs without any detectable protein would stand for one end of the spectrum, and the other end would contain the severest phenotype of cs type ii, with mutations impacting import ant functions. in summary, we report a mild form of cs patient who was homozygous for the c526 t transition resulting in a new nonsense mutation, which converts arg176 to a stop codon. all of these observations are brand new and provide a deeper level of understanding about mechanisms of cs. therefore, our results will likely provide more specific targets to prevent or improve cs. | key clinical messagecockayne syndrome (cs) is a rare autosomal recessive genetic disease characterized by growth failure and progressive neurological degeneration. here we report a mild form of cs patient who was homozygous for the c526 t transition resulting in a new nonsense mutation, which converts arg176 to a stop codon. |
abdominal swelling is an uncommon presentation in newborn babies. a combination of huge abdominal swelling, obstructive uropathy and imperforate hymen in newborns has not been reported in the medical literature. we report a 4 days old newborn with a rare presentation of hydrometrocolpos which posed a diagnostic challenge and consequently resulted in delays in diagnosis and treatment. hydrometrocolpos should be considered as a differential diagnosis in neonates who present with huge abdominal swelling. a 23 days old female neonate born to a 35 years old para iv lady at gestational age of 38 + 6weeks was referred to the neonatal intensive care unit (nicu) of hawassa university referral hospital from a nearby district hospital. delivery was done by spontaneous vertex at a district hospital after 20hrs of labor ; membrane was ruptured during labor. her birth weight was 2500 grams ; apgar score was unknown but the baby cried immediately after delivery. few minutes after delivery, the baby failed to suck and was admitted to newborn unitof the hospital. blood workup revealed a white cell count (wbc) of 22,400/mm and an absolute neutrophil count (anc) of 13,216/mm. based on these clinical clues, infection was suspected, and the newborn was started on ampicillin (100mg / kg bid) and gentamicin (5 mg / kg once daily). on the 4 postnatal day, a supra - pubic swelling and failure to pass urine were noticed. pelvic ultrasound revealed an intra abdominal cystic mass with bilateral moderate hydronephrosis. even though the neonate was referred on the 4 postnatal day to hawassa university hospital (huh) nicu, the parents could not come immediately for social reasons and brought her on the 23 postnatal day because of a progressively increasing abdominal swelling, failure to pass urine and difficulty of breathing. by then, her vitals were : pulse rate, 152/minute ; respiratory rate, 82/minute ; temperature, 37.2 celsius. weight, 2590 grams ; length, 51 cm ; and head circumference, 35 cm. she was noted to have emaciation, failure to thrive, tachypnia and labored breathing.a huge cystic abdominal mass with difficulty to delineate the lower border was appreciated. the genitalia looked grossly normal, but the hymeneal membrane was imperforate (noticed after the operation). upon catheterization, 30cc of urine was evacuated.repeat laboratory tests showed : wbc, 30,100/mm ; anc, 21,390/mm ; hemoglobin, 15gm / dl ; platelets, 412,000/mm ; blood group / rh, o/+ ; creatinine, 10.9 mg /dl ; sodium, potassium and calcium within normal limits. repeated ultrasound showed a distended fluid containing mass with echo - debris arising from pelvis with a conclusion of ascites, bilateral moderate hydronephrosis, and possibility of endometrial cavity abscess or adnexal cyst (figure c). since the abdominal swelling was noted to be huge and the imperforate hymen was noticed late and with the controversial ultrasound findings, decision was made to do exploratory laparatomy. intra - operative findings were a 2010 cm distended uterus with the bladder adherent to the abdominal wall which caused inadvertent injury upon incising the abdominal wall. around 1250cc of whitish fluid with debris was drainedfrom the uterine cavity using a wide bore needle. a : photo of the abdominal swelling on day, 21 b : photo of abdominal swelling on day 23, c : ultrasound examination on day 21. following the procedure, the baby was persistently hypothermic with irregular breathing. since infection was initially considered, antibiotics were continued ; she was put on radiant warmer and was on oxygen. however, after 3 hours of the procedure, the baby started to gasp and was resuscitated which was not successful ultimately. the possible the common differential diagnoses to be considered in such babies include neuroblastoma, intra - peritoneal fluid collection following organ failure, ovarian cysts, intra - abdominal sacrococcygeal teratoma, mesoblastic nephroma, bowel duplication, genitourinary anomalies and anterior sacral meningocele (1). congenital hydrometrocolpos as a cause of abdominopelic mass in neonates has also been characterized in few case reports in the past 2 decades (16). in the current case, the presentation with huge abdominal swelling with distended veins and compromised breathing (figures a and b) prompted entertainment of various differential diagnosis and delayed the actual diagnosis of hydrometrocolpos which was made intra - operatively. hydrometrocolpos is a condition in which the uterus and vagina are distended by retained fluid other than blood in the presence of distal vaginal outlet obstruction. it is a very rare condition with incidence of 1 in 16,000 live births (7). the previously reported low incidence of hydrometrocolpos could be due to difficulty in diagnosis and high mortality rates as suggested by mittal (6). associated anomalies may include obstructive congenital malformations of the genital tract such as vaginal atresia, transverse vaginal septum and imperforate hymen. mckusick - kaufman syndrome, an autosomal recessive disorder characterized by vaginal atresia with hydrometrocolpos, polydactyly, congenital heart defects and non - immune mediated hydrops fetalis, has also been described (1,3). among the obstructive genitourinary anomalies, it is due to failure of partial resorption of the membrane during the embryonic development and also the hymen failing to rupture during the eighth week of gestation ; the incidence is 0.00140.01 % in full - term newborns (1, 8). previous authors described that hydrometrocolpos is usually diagnosed prenatally as the cause of abdominal cystic mass (4). however, in the present case, prenatal evaluations were negative, and abdominal swelling started to progressively enlarge after the 4 day when the swelling was first noticed. the fact that the presence of imperforate hymen was overlooked was the pitfall in the evaluation of this case. it resulted in major surgery under general anesthesia while it could have been enough to make incision on the imperforate hymen to drain the fluid. previous authors described the possibility of hydrometrocolpos causing urinary stasis and acute renal failure due to obstructive uropathy (9). because of the delay in diagnosis of hydrometrocolpos as a cause of the obstructive uropathy, the renal function was deteriorating progressively and contributed to the death of the baby. clinicians should consider the possibility of hydrometrocolpos as a cause of huge abdominal swelling in newborns. the genitals should be carefully examined to save patients from major surgeries and related complications. | backgroundabdominal swelling is an uncommon presentation in newborn babies. a combination of huge abdominal swelling, obstructive uropathy and imperforate hymen in newborns has not been reported in the medical literature.case detailswe report a 4 days old newborn with a rare presentation of hydrometrocolpos which posed a diagnostic challenge and consequently resulted in delays in diagnosis and treatment.conclusionhydrometrocolpos should be considered as a differential diagnosis in neonates who present with huge abdominal swelling. |
injuries are responsible for an increasing share of morbidity and mortality in low- and middle - income countries (lmics) where they currently account for 11% of the total disability - adjusted life years. injuries also have an enormous socioeconomic impact both at the household and national level. there are great disparities in the prevention and care of injuries worldwide, with 1 to 2 million preventable deaths in severely injured patients and 90% of deaths from road traffic injuries occurring in low- and middle - income countries [2, 3 ]. african children over 5, injuries account for more deaths than hiv, tuberculosis, and malaria combined. understanding the epidemiology of injury can help identify risk factors for injury and target interventions for prevention and treatment to decrease disability and mortality. despite the growing burden of injuries in lmics, however, there remain limited primary epidemiologic data to guide health policy and health system development [5, 6 ]. even if a health facility capable of providing quality emergency care exists, the absence of formal prehospital care in resource - constrained settings, along with multiple barriers to care such as social, educational, cultural, and financial factors, prevents access to these facilities. to improve epidemiologic surveillance of injuries in uganda, the injury control centre - uganda established a trauma registry in 1998, after piloting and validating an instrument for data collection. this registry has been used in five ugandan hospitals since 1998 [7, 8 ]. similar registries have also been established in other countries in the region. since then, a number of injury prevention interventions such as seat belt laws in motor vehicles, helmet laws for motorcyclists, speed bumps, improved street lighting, and school and household - based interventions for children have been implemented. efforts to improve trauma care have included trauma courses for hospital - based personnel and some first aid courses for police. in addition, a national injury policy has been drafted by the ministry of health. there has, however, been no formal epidemiologic analysis of the potential collective impact of these interventions since the trauma registry was first implemented. the goal of this study was to estimate the current epidemiology of injury in the capital city of kampala by using data at mulago national referral hospital, especially the most common causes of injury, and the prevalence of intentional and unintentional injury. the database for this study was constructed from prospectively collected data from the injury control centre - uganda, which is a private organization that is funded by both public and private sources ; trained nurses, clinical officers, or doctors in the casualty (or emergency) department of the mulago national referral hospital completed a one - page form (described previously in the literature) on each patient presenting to the casualty department, recording information on the patient condition, status, demographics (age, sex, residence, occupation), several clinical variables (blood pressure, pulse, respiratory rate, and neurological status), as well as causes and place of injury, severity of injury, and outcome. two weeks after initial presentation, the health care providers or records clerks completed the patient disposition from the casualty (treated and discharged from casualty, admitted, transferred to higher level facility, died in casualty, dead on arrival) as well as the disposition for those admitted (discharged, died, still in the hospital, transferred, or other). this registry was checked for accuracy by a hospital surgeon or senior doctor, and the data were entered into excel (microsoft, 2005), cleaned, and managed by the injury control centre - uganda. the most current full year of data available at this time is from july 2004 to august 2005. prospective injury data were no longer recorded after 2005 due to lack of funding, and at this time, these data represent the most recent injury epidemiology seen at mulago national referral hospital, a government hospital. all patients seen at mulago national referral hospital that were recorded in the database in the 12-month period from 1 july 20041 august 2005 were included in the data set, which was queried for descriptive statistics of all injuries to characterize patients by age, gender, type of injury, location of injury, intent, mode of arrival, distance, injury severity, and disposition. the kampala trauma score (kts) was chosen to categorize severity of injury. this score has been validated and was revised in 2004, where previous definitions of mild, moderate, and severe injuries (which were kts scores of 1416, 1113, and 894 0 - 893 1 - 492 undetectable1d. respiratory rate (breaths / min) 10 - 293 302 91e. neurological status (avpu system) alert4 responds to verbal stimuli3 responds to painful stimuli2 unresponsive1kts total = a+b+c+d+enotes : possible range for kts is 5165 = most severely injured16 = the least severely injuredthe avpu system is a simplified version of the glasgow coma scale kts total = a+b+c+d+e notes : possible range for kts is 516 5 = most severely injured 16 = the least severely injured the avpu system is a simplified version of the glasgow coma scale for patients that were eventually admitted, 9.8% (95% ci 8.2, 11.4) came by ambulance. of these admitted patients, more severely ill patients had 1.2 times the likelihood (95% ci 1.1, 1.4) of being taken to the hospital in an ambulance compared to those with lower severity (higher kts). sixty - five percent of injured people brought to mulago hospital were from the district of kampala. of those who were injured in kampala and brought to mulago hospital, 33% were admitted as compared to 46.7% of those who were injured outside of kampala (p 894 0 - 893 1 - 492 undetectable1d. respiratory rate (breaths / min) 10 - 293 302 91e. neurological status (avpu system) alert4 responds to verbal stimuli3 responds to painful stimuli2 unresponsive1kts total = a+b+c+d+enotes : possible range for kts is 5165 = most severely injured16 = the least severely injuredthe avpu system is a simplified version of the glasgow coma scale kts total = a+b+c+d+e notes : possible range for kts is 516 5 = most severely injured 16 = the least severely injured the avpu system is a simplified version of the glasgow coma scale for patients that were eventually admitted, 9.8% (95% ci 8.2, 11.4) came by ambulance. of these admitted patients, more severely ill patients had 1.2 times the likelihood (95% ci 1.1, 1.4) of being taken to the hospital in an ambulance compared to those with lower severity (higher kts). sixty - five percent of injured people brought to mulago hospital were from the district of kampala. of those who were injured in kampala and brought to mulago hospital, 33% were admitted as compared to 46.7% of those who were injured outside of kampala (p < 0.01 for difference). table 2 shows that 92% of the patients were mildly injured and only 1% of the sample severely injured. at 2 weeks, 67% of admitted patients had been discharged, 26% were still in the hospital, 6% had died, and 1% had left against medical advice. seventy - five percent of the patients who died of trauma in the hospital had head injury. a total of 72 patients died, which comprised 2% of the admitted patients. for those who died, the most common cause was road traffic injuries (n = 41) (table 3), which constituted over half of the fatalities. a multivariable regression (including age, sex, mechanism, occupation, place of injury, mode of arrival, and distance) looking at outcome of death in the hospital did not show any correlation with any variables except for kts, where each lower kts point was correlated with a decrease of 5% in the chance of survival (95% ci, 4.9%5.7%). table 2injury severity of patients seen in casualtycategorykts scorenumber% samplemild8 - 103,13592%moderate5 - 72357%severe<5331%3,403100%table 3fatal causes of injurycausenumberpercentroad traffic4156.9%burn1216.7%blunt / penetrating injury1115.2%fall79.7%sexual assault11.4%total72100.0% injury severity of patients seen in casualty fatal causes of injury the goal of this study was to analyze the injuries of patients that presented to the tertiary care hospital of the capital city of uganda. for a broader view of injury, it would be preferable to include data from all hospitals in kampala ; however, such data are not available since there is no systematic data collection across these facilities for trauma. although mulago hospital receives 75% of injured victims in kampala, most of the other hospitals in city are private and better resourced than the national hospital, although they do not as reliably have the 24-h coverage of the national hospital in addition, limitations in facility - based care that could have affected outcomes were not evaluated in this study. furthermore, we were unable to assess the number, characteristics, and types of trauma patients that did not present to this hospital because of the lack of a formal prehospital emergency system in kampala. earlier reports of trauma registries in uganda show that the young male population is most affected by trauma, and the findings of this more updated study confirm the impact on this age group. similar to previous studies, the data also show that road traffic injuries are the most common cause of injury overall, except for in children aged 5 and younger. worldwide, road traffic injuries account for approximately 30% of all childhood injury deaths, which mirrors our finding of overall injured people presenting to the hospital. while these results are no doubt partly a reflection of the underlying demographic patterns in sub - saharan africa, with a relatively younger population compared to more developed countries, these findings still have important potential implications for public health and for poverty eradication. first, children and the economically productive segments of the population are most affected by injury, but the precise impact on household poverty (e.g., income forgone, impact on family members, household wealth lost) is poorly characterized, even in other sub - saharan african nations with a similar burden of injury. though there was no specific variable to capture wealth or socioeconomic status, the preponderance of students and casual laborers that were injured suggests that poorer and more vulnerable groups are affected by injury, keeping with prior reports. this could be further analyzed either through closer evaluation of patients hospitalized with injuries or as part of community surveys or ongoing demographic surveillance programs. previous work has suggested that injury costs uganda 2% of the gdp per year ; however, the prevention and care of injuries has not been highlighted by the poverty eradication action plan of the ministry of finance or recognized as a key health - related aspect of uganda s progress toward the millennium development goals. in addition, the impact on school - age children suggests that more coherent policies for the prevention and care of injuries must be integrated into child health programs, as has been previously suggested from prospective studies in children and reviews of surgical conditions in the region [14, 1719 ]. in the under-5-years age group, the preponderance of burns and falls points to areas of further research for prevention and care. since the majority of child injuries occurred at home, one might consider how a household - based injury prevention program could be designed and implemented. in addition, though prior reviews suggest that school - based education programs for injury prevention have limited effectiveness, this may need to be reconsidered. multiple randomized controlled trials have shown that safety education can change pedestrian behavior in high - income countries, although the effects on injury rates are not known. however, there have been no large - scale studies on pedestrian education in low and middle - income countries. such interventions may need to be considered potentially in combination with other, more effective interventions at the household and primary care level to address the injury epidemic in kampala. second, our findings also show that, within road traffic injuries, more passengers are injured compared to drivers in both motorcycle and vehicular injuries in kampala. not only are road traffic injuries the most common cause of injury, they are also responsible for the majority of fatal injuries that occurred in this study. this is in contrast to previous findings showing that pedestrians were the most commonly injured in road traffic injuries in urban uganda. prior studies have also shown the high impact on vulnerable road users such as pedestrians and motorcycle drivers [16, 21 ]. the increased numbers of non - driver injuries reflect the pattern of transport in uganda, where few individuals own their own vehicles, and most use large vehicles that ferry numerous passengers at a time. this pattern provides a strong argument for interventions that increase the road safety of these large transportation vehicles. this is also in keeping with prior work documenting that more passengers per crash are injured in lmics compared to higher income countries. the authors have also noted that multiple casualty injuries are more the norm than the exception, and this has implications for the design of prehospital systems and the training and protocols in the casualty ward, which must be ready to receive several severely injured patients at a time. third, very few patients (less than 5%) were brought to the hospital by ambulance, with the majority of injured patients brought in private cars, by the police, and by bystanders. this rate has not changed significantly since earlier reports from the registry, suggesting that there has been limited progress in the development of a formal prehospital system. though ambulances do exist in kampala, they are privately run, and families must be able to afford the cost, which the majority can not. others have suggested that the majority of potentially avertable deaths in severely injured patients occur in the prehospital setting. in kampala, records of fatalities from the prehospital setting are captured by the police or by the public city mortuary. an analysis of trauma - related deaths from mortuary data may help to clarify the proportion of deaths and disabilities that may have been preventable by improved prehospital care. in the absence of an ambulance - based prehospital system, prior work has suggested that in regions of high injury incidence, initiatives to improve knowledge and skills of lay responders may be a feasible, cost - effective, and critical first step towards developing a formal emergency system. these initiatives deserve further study [23, 24 ]. finally, a primary finding of prior analysis is the preponderance of minor injuries presenting to the national hospital, congesting the hospital and perhaps impeding the care of more severely injured patients. our findings show a similar pattern and suggest that a more organized prehospital system might triage patients with less severe injuries to other health centers to decongest the tertiary care facility. this would require more thorough evaluation of the capacity of lower level health facilities to receive and care for injured patients. qualitative interviews with kampala residents during this study indicated that most patients prefer to attend mulago hospital because it is a government hospital with highly subsidized health care, unlike the only other options in the city, which are all private hospitals. nevertheless, many injured patients still do not seek care because of other barriers to access to care, whether these are cultural, social, economic, or geographic. prior work has suggested that up to 80% of patients with fractures present first to a traditional healer before coming to a hospital [25, 26 ]. hospital - based registries are limited because they do not capture injured patients who do not interact with the health system. a community survey in ghana showed that 30% of patients with severe injuries did not access formal care. prior community surveys in kampala have demonstrated that large proportion of injured patients die without reaching care, with a mortality rate of 2.2/1,000 per year, which is higher than that seen in this data. there were 72 deaths recorded in the trauma registry, which is likely to be a gross underestimate of total deaths from injuries since a large proportion of deaths are likely to have occurred in the prehospital setting. hospital - based death data should be analyzed with police and city mortuary data to calculate a better estimate of injury mortality in kampala. improving access to emergency neurosurgical care may have the potential to prevent a substantial proportion of these deaths. prior reports have suggested that the human resources and infrastructure for neurosurgical care are very limited in sub - saharan africa. a more detailed mortality audit at the hospital level may help to determine how to improve care for patients with head injuries who reach the hospital. other strategies to decrease trauma mortality in similar settings have included the introduction of a trauma education course for health personnel. this intervention is currently underway in uganda, and an understanding of its effectiveness will add greatly to the provision of injury care in kampala. road traffic injuries are by far the largest cause of both morbidity and mortality seen in the government hospital in kampala. they are the most common cause of injury for all ages except those less than 5, and school - aged children comprise a large proportion of victims from these incidents. the preponderance of minor injuries seen at the national hospital suggests that lower level facilities could be better equipped to handle these injuries to decongest the national hospital. the development of prehospital trauma care should be a high priority area given that less than 5% of patients arrived by ambulance. our data show that injuries of the head, face, and neck account for a large proportion of injured patients, and is the most common body part injured in both discharged and admitted (and those who died in the hospital) patients. while more research needs to be done on appropriate and effective interventions, our findings point to patterns of injury that should be targeted in future injury control programs. the integration of injury control programs with ongoing health initiatives is an urgent priority for health and development. | backgrounddespite the growing burden of injuries in lmics, there are still limited primary epidemiologic data to guide health policy and health system development. understanding the epidemiology of injury in developing countries can help identify risk factors for injury and target interventions for prevention and treatment to decrease disability and mortality.aimto estimate the epidemiology of the injury seen in patients presenting to the government hospital in kampala, the capital city of uganda.methodsa secondary analysis of a prospectively collected database collected by the injury control centre - uganda at the mulago national referral hospital, kampala, uganda, 2004-2005.resultsfrom 1 august 2004 to 12 august 2005, a total of 3,750 injury - related visits were recorded ; a final sample of 3,481 records were analyzed. the majority of patients (62%) were treated in the casualty department and then discharged ; 38% were admitted. road traffic injuries (rtis) were the most common causes of injury for all age groups in this sample, except for those under 5 years old, and accounted for 49% of total injuries. rtis were also the most common cause of mortality in trauma patients. within traffic injuries, more passengers (44%) and pedestrians (30%) were injured than drivers (27%). other causes of trauma included blunt / penetrating injuries (25% of injuries) and falls (10%). less than 5% of all patients arriving to the emergency department for injuries arrived by ambulance.conclusionsroad traffic injuries are by far the largest cause of both morbidity and mortality in kampala. they are the most common cause of injury for all ages, except those younger than 5, and school - aged children comprise a large proportion of victims from these incidents. the integration of injury control programs with ongoing health initiatives is an urgent priority for health and development. |
bipolar disorder (bd) is an episodic mood disorder and has a significant psychiatric comorbidity. attention deficit hyperactivity disorder (adhd) persists in adulthood in about two - third of patients, affecting 2.54.4% of adults. concomitant bd and adhd are frequent with 1021% of adults with bd having adhd and 520% of adults with adhd having bd. subjects with comorbid adhd and bd have poorer outcomes, and the management is further complicated by the risk of induction of (hypo) mania with the medications used for adhd treatment. here we report induction of hypomania by atomoxetine, a selective norepinephrine reuptake inhibitor when used for the treatment of comorbid adhd in a patient with bd.. a, a 22-year - old male, with diagnosis of bd (age of onset 20 years) and borderline personality disorder (bpd) was stable on sodium valproate 1000 mg / day and quetiapine 400 mg / day since a year. he was adherent with medications and had adequate serum valproate levels (95 g / ml). he then had an impulsive deliberate self - harm attempt following a stressor and was admitted for in - patient care. during the in - patient evaluation, he reported significant inattention since childhood (age of onset 67 years). on further exploration, he was found to have features suggestive of impulsivity and hyperactivity, causing significant impairment in his academic and interpersonal functioning. on structured evaluation, he fulfilled the dsm-5 criteria for adult adhd and scored 24 on the conners ' adult adhd rating scale. we educated the patient and his caregivers about adhd, and added tablet atomoxetine 18 mg / day to his treatment regimen and increased it to 25 mg / day after 5 days (0.4 mg / kg body weight). after the 2 day of receiving 25 mg / day of atomoxetine, he was noted to be more talkative than usual and irritable over minor issues. from the following day his need for sleep decreased and psychomotor activity increased. on rating with young 's mania rating scale, he scored 14. these symptoms were consistently noted for about 3 days while he was on atomoxetine 25 mg / day. after discussing with the patient and his family, atomoxetine was stopped and other medications were continued at the same dose. the patient also had family history of bd in a second - degree relative and history of manic switch with imipramine. the score on the naranjo adverse drug reaction probability was 8, suggesting that atomoxetine - induced hypomania was probable. there are no randomized controlled trials that have evaluated any treatment in adult patients with adhd - bd, and no definitive treatment recommendations are available. the consensus is to follow staged or hierarchical approach by treating bd first and then treat adhd. it is believed that mood stabilizers provide protection against the possible risk of (hypo) mania induction with the medications used for adhd. in the above scenario, our patient was euthymic and on adequate doses of two mood stabilizers when we commenced pharmacotherapy for adhd. several experts suggest avoiding these medications in bd patients, especially in those having risk factors such as family history of psychosis or bd. however, earlier reports in children and adolescents with adhd - bd suggest that the possibility of (hypo) manic switch is low when atomoxetine is used along with mood stabilizers or antipsychotics. in the patient described above, as the dose of atomoxetine was increased, we witnessed hypomanic symptoms. these hypomanic symptoms disappeared within few days of stopping atomoxetine. to our knowledge, this is the second report of atomoxetine - induced mania / hypomania in an adult patient. the first report was published in 1985, in a 46-year - old patient with major depressive disorder having family history of postpartum depression. an important point in this report is that the hypomania occurred while the patient was on treatment with two mood stabilizers at adequate doses. this is probably the first report of atomoxetine - induced hypomania in an adult patient stabilized on mood stabilizers. conversely, the literature on children and adolescents with adhd has several documented reports of atomoxetine - induced (hypo) mania. in an uncontrolled open trial, about one - third (33%) of adhd children receiving atomoxetine reported irritability, hypomania, mania, and aggression. in this study, the majority of the affected children experiencing above - mentioned adverse effects had family or history suggestive of mood disorder or symptoms. the salient features of published case reports on atomoxetine - induced (hypo) mania in children and adolescents are summarized in table 1. a family history of affective disorder was commonly seen in these case reports, similar to the findings from the trial described above. in addition, the findings from these case reports suggest atomoxetine - induced hypo (mania) to be a dose - dependent phenomenon with a higher probability at the dose range of least 0.81.7 mg / kg of body weight. the patient, in this case, developed hypomania at a dose of 0.4 mg / kg. however, it is to be noted that he was a known case of bd. the dose of atomoxetine required to induce hypo (mania) in adults with bd and the role of comorbid conditions such as bpd need further investigation. summary of published reports on atomoxetine induced (hypo) mania in children and adolescents with adhd atomoxetine carries the risk of induction of (hypo) mania even in stabilized patients with bd. further systematic research is required on the predictors and pathophysiology of atomoxetine - induced hypo (mania) in children and adults. | comorbidity of bipolar disorder (bd) with attention deficit hyperactivity disorder (adhd) is frequent. the management of comorbid adhd and bd is complicated by the risk of induction of (hypo) mania by the medications used for adhd treatment. earlier reports in children and adolescents with adhd - bd suggest that the possibility of (hypo) mania induction is low when atomoxetine is used along mood stabilizers or antipsychotics. here, we report induction of hypomania by atomoxetine when used for the treatment of comorbid adhd in a bd patient while on prophylactic treatment with mood stabilizers. this report indicates that atomoxetine carries the risk of induction of (hypo) mania even in stabilized bd patients. clinicians should closely monitor such patients for (hypo) mania symptoms. |
stress response mediator activating transcription factor 3 (atf3) engages in diverse oncogenic pathways including the androgen receptor signaling essential for prostatic proliferation. in line with frequent downregulation of atf3 expression in human prostate cancers, we have provided the first genetic evidence supporting the role of atf3 as a tumor suppressor in a subset of prostate cancers with pten dysfunction. |
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portal vein tumor thrombosis (pvtt) is one of the worst prognostic factors in patients with hepatocellular carcinoma (hcc) originating from liver cirrhosis. it can be observed in about 30% of hcc patients [1, 2 ], and the average survival of these patients after the diagnosis is very poor (2.7 - 4.0 months). death is often due either to a progressive increase in portal pressure, resulting in gastrointestinal bleeding, or to the decrease in portal vein flow leading to ascites, jaundice, encephalopathy and liver failure. until a few months ago, best supportive care was considered a reasonable choice for patients with such a critical prognosis, since transarterial chemoembolization entails a high risk of ischemic damage and chemotherapy was largely ineffective. some authors reported high survival rates in patients treated with a combination of transarterial chemoembolization and surgery with thrombectomy, but this procedure may be used only in highly selected patients. since sorafenib (sor) has been shown to improve survival in patients with advanced hcc, this drug has become the standard treatment even for patients whose disease was complicated by pvtt. so far, however, there is no clear evidence concerning the effect of sor in this subset of patients and their outcome. on the other hand, the well - known interference between tyrosine kinase inhibitors and the coagulation pathway calls for caution against their use in this setting. here, we describe a case of multifocal hcc with pvtt showing an unexpected favorable outcome after a short course of sunitinib (sun), a tyrosine kinase inhibitor which is currently under investigation for use in the treatment of intermediate - advanced hcc. a 72-year - old male patient affected by chronic hcv - related hepatitis underwent percutaneous alcohol injection for a nodule of the sixth segment of the liver. two years later, an abdominal ct scan demonstrated a massive thrombus involving the main portal vein and its branches, with infiltrative (not evaluable according to recist) neoplastic pattern of the sixth segment (fig. histologic and cytologic examination of a needle biopsy of the infiltrative lesion and a fine needle aspiration of the portal vein thrombus demonstrated a poorly differentiated trabecular hcc with tumor thrombosis. child - pugh score was a6 with meld score 10, and the tumor stage was bclc - c. serum albumin, ammonium, creatinine and coagulation parameters were normal ; blood chemistry documented only a slight increase in transaminases (alt 66 iu / l and ast 57 blood count showed mild anemia (hemoglobin 11.2 g / dl) and thrombocytopenia (84 10/mm). there was no ascites, and serum a - fetoprotein was 3 ng / ml. no distant metastases were detected on a whole - body ct scan and bone scintigraphy, and upper endoscopy showed no esophageal varices. f - fluorodeoxyglucose (fdg) pet - ct showed f - fdg uptake (fig. 2a) in the sixth segment of the right lobe (standard uptake value, suv, 4.6) and also at the liver hilum (suv 3.1.). 2005 - 005732 - 27) with sun (50 mg / die) on a standard 4-week - on/2-week - off regimen. after the first cycle, a 25% dose reduction was required because of grade iii thrombocytopenia. the other reported toxicities were grade ii fatigue, mild hypertension, grade i nausea and a cutaneous rash. the pet - ct scan performed after the first cycle showed metabolic stabilization of the disease according to eortc criteria, with a reduction in suv 1 year despite the interruption of sun at the beginning of the third cycle. this excellent outcome was accompanied by re - canalization of pvtt and liver function improvement. although the sensitivity of f - fdg - pet for hcc is about 50% and it is well known that f - fdg uptake is associated with tumor differentiation, f - fdg uptake is significantly higher in poorly differentiated hcc and may be a good predictor of outcome in these patients. a clinical response according to eortc criteria and recist is very rare in hcc. in the sharp study however, it must be considered that in our patient treatment was complicated by a potentially life - threatening side effect which required hospitalization in an intensive care unit. even if gastrointestinal bleeding is a very common adverse event in pvtt patients, we can not exclude a relationship between treatment and the complication due to the interaction between sun and the coagulation pathway. this case clearly stresses the narrow therapeutic window of multikinase inhibitors in hcc originating from liver cirrhosis. the same mechanism of action that interferes with tumor growth might affect the liver vasculature already impaired by cirrhosis. the brief exposure to sun needed in our patient to obtain both tumor response and gastrointestinal bleeding suggests further studies on the effectiveness of sun in this setting as well as on the duration of treatment with multikinase inhibitors in hcc. although sun treatment in advanced hcc has been evaluated only in a few phase - ii studies and resulted in poor outcome [12, 13 ], our case report may attract further interest in this drug, since the response rate with sor is very low despite its survival benefit ; moreover, no specific evaluation of patients with pvtt was made. targeting a different set of tyrosine kinase receptors, including vegfr1 and vegfr2, pdgfr and c - kit, sun, or other multikinase inhibitors, might exert a different effect both on cancer cells and the coagulation pathway compared to sor, which mainly interferes with the raf / mek / erk pathway. in conclusion, in this patient with advanced hcc, sun was highly effective not only in terms of metabolic and morphologic responses, but also of pvtt recanalization. as far as we know, this effect has never been reported using this tyrosine kinase inhibitor. | the prognosis of patients with advanced hepatocellular carcinoma (hcc) is very poor. the outcome of these patients is particularly bleak when the disease is complicated by portal vein tumor thrombosis (pvtt), since the increased portal pressure often causes serious gastrointestinal bleedings. before the introduction of sorafenib (sor), a tyrosine kinase inhibitor, no effective treatment was available for patients with advanced disease. sor is now considered the standard treatment even for patients with tumor thrombosis, although the well - known interference between tyrosine kinase inhibitors and the coagulation pathway calls for caution against their use in this setting. here, we report the case of a 74-year - old male patient with advanced hcc and pvtt treated with sunitinib (sun), another multikinase inhibitor. during the third cycle, our patient experienced a life - threatening hematemesis with hemorrhagic shock that required intensive care treatment and sun discontinuation. however, he completely recovered, and the pet / ct scan performed 1 year after the adverse effect demonstrated no evidence of the tumor together with portal vein recanalization. the short course of sun causing both tumor response and gastrointestinal bleeding warrants further studies on the effectiveness of sun in this setting as well as on the duration of treatment with multikinase inhibitors in patients with tumor thrombosis. |
the prevalence of osteoporotic fractures has increased with the recent acceleration of population ageing1). in particular, trochanteric fractures of the femur are osteoporotic fractures that mainly occur in the elderly with systemic problems, and are often treated surgically to prevent prolonged bed - ridden state caused by functional problems, such as hip joint pain, and the risk of complications2,3). although the surgeries used to treat trochanteric fractures of the femur, namely compression hip screw (chs) fixation, intramedullary nailing, and bipolar hemiarthroplasty (bha), result in successful treatment of stable fractures, they need to be carefully chosen for patients with unstable fractures due to the difficulties in reduction, maintenance, and early acquisition of stability4,5). compression hip screw fixation is widely used and has an advantage of facilitating bone reduction through compressive force induced by sliding of the compression screw6,7), but fixation failures have been reported such as perforation of the femoral head and excessive sliding of the screw8). meanwhile, bha is increasingly used because of its advantage of enabling early ambulation. however, this surgical method is associated with some problems, including difficulty in achieving early stability of the femoral stem due to posteromedial bone fragments causing instability, implant dissociation, infection, dislocation, subsidence of the femoral stem, and difficulty in surgical manipulation9,10,11). although several authors have reported the clinical results of chs fixation and bha in elderly individuals with unstable intertrochanteric fractures of the hip4,5,10,12,13,14), there has been no comparative studies of these surgeries in ao type a2 intertrochanteric fractures. therefore, we aimed to verify the hypothesis that there is no difference in clinical and radiological results between chs fixation and bha by retrospective analysis of outcomes of respective treatments in patients with ao type a2 intertrochanteric fractures. this study included 109 patients aged over 65 years, who received surgical management from march 2003 to december 2009 due to ao type a2 intertrochanteric fractures. we excluded 20 patients who were followed up for less than 1 year, and retrospectively examined a total of 89 cases (16 men and 73 women). the mean age was 77.7 years (range, 65 - 94 years), and the mean follow - up period was 5.9 years (range, 1 - 8.3 years). the mean body mass index (bmi) was 22.2 kg / m (range, 15.4 - 31.6 kg / m). bha was performed when severe osteoporosis or poor bone quality was anticipated on the basis of radiography in patients with substantial loss in trabecular bone mass in the proximal femur, or when reoperation was impossible due to the patient 's systemic state. in all other cases, the prosthetic components used were chs (synthes - stratec, oberdorf, switzerland) in all chs fixation patients, and cementless femoral stems in all bha patients : sl - plus (smith & nephew, memphis, tn, usa ; 41 cases), versys (zimmer, warsaw, in, usa ; 3 cases), and abg ii (stryker, newbury, uk ; 2 cases). in chs fixation patients, closed reduction under image intensifier was performed on the fracture table after anesthesia. when satisfactory anatomical reduction was achieved, a chs was fixed through a lateral approach. in case of anatomical reduction failure, fixation was performed using a chs and adjuvant devices such as a bone hook or a lowman clamp by requiring greater exposure of the fracture site. severely displaced and unstable lesser trochanter fragments were additionally fixed using cerclage wire. in bha, the fracture site was exposed using a posterolateral approach, and then femoral neck resection was performed directly under the femoral head. after the reduction of lesser trochanter fragments, fixation was attempted in the calcar femorale and posterior lesser trochanter by firmly tightening 2 or 3 metal cerclage wires. the femoral stem component was inserted into the femur after rasping the femoral canal, and then 1 or 2 metal cerclage wires were additionally used to fix greater trochanter fragments. for postoperative rehabilitation, quadriceps - strengthening exercises and passive exercises of the hip and knee joints were carried out. from the seventh postoperative day, patients were allowed partial weight - bearing using a walker. to evaluate the clinical outcome, we examined the merle d'aubign - postel score15) to assess pain and function, anesthesia time, operation time, the amount of postoperative transfusion, hospital stay, the starting time of partial weight - bearing, and systemic complications. anesthesia time (based on the anesthesia records) was defined as the period from admission into the operating room to completion of operation, and the operation time was defined as the period from the incision to completion. the transfusion volume was defined as the number of packed blood units used from incision to discharge from the hospital. radiological results were analyzed based on the pre-, post - operative and final follow - up radiographs (anterior - posterior and lateral) of the hip joint. to assess the failure rate, failures were defined as non - union, perforation, and sliding of > 15 mm in the chs group16), and dislocation, osteolysis around the femoral stem, and subsidence by > 5 mm in the bha group17). to identify systemic complications, we examined complications in the respiratory, circulatory, renal, urinary and gastrointestinal systems, and wound - related complications that occurred within 3 postoperative months. we used the wilcoxon rank - sum test for continuous variables and fisher 's exact test for categorical variables. there was no significant difference between the two groups in the demographic variables, which included the sex ratio, bmi, follow - up period, the number of associated diseases, and ao classification. the mean age was 75.56.4 years in the chs group and 79.76.5 years in the bha group. the average t - score was -2.61.0 in the chs group and -3.00.8 in the bha group. the score was more favorable in the chs group, but showed no statistically significant difference between the two groups (table 1). the amount of transfusion, general complications, and the merle d'aubigne and postel score were not significantly different between the two groups. according to the merle d'aubign - postel score, pain, function, ambulation ability and anesthesia time was 2.30.9 h in the chs group and 1.50.7 h in the bha group. although a significantly longer anesthesia time was found in the chs group (p 5 mm in one case (2.2%). park.24) found no difference in the bleeding volume and operation time between chs fixation and cemented bha. bha showed better clinical results and reduced the incidence of complications. in our study, there was no difference in transfusion volume between the two groups. however, the chs group showed longer anesthesia time, which was probably attributable to a longer time needed for preoperative bone reduction. in most cases, the time of weight - bearing is determined by the surgeon. however, despite the surgeon 's instructions, the actual starting time of weight - bearing seems to be different depending on the patient 's systemic state, compliance, and tendency. therefore, we allowed patients to freely perform postoperative weight - bearing and analyzed the actual starting time of weight - bearing as clinical results. early stability can be achieved by using a cementless implant, and early ambulation can be initiated by stable fixation of the lesser and greater trochanters. in a study by kayali.12), full weight - bearing was initiated by the fourth postoperative week (on average) in elderly patients who underwent arthroplasty using a cementless implant due to an unstable intertrochanteric fracture. this starting time was significantly earlier than that of patients with internal fixation. in the present study, patients were able to start partial weight - bearing using a walker at an average of 13.6 postoperative days. furthermore, ambulation was earlier in the bha group than in the chs group patients, who started weight - bearing at an average of 22.8 postoperative days. these findings imply that bha is more favorable for early ambulation and rehabilitation in patients with intertrochanteric fractures in comparison with chs fixation. first, the follow - up period was short, and we retrospectively investigated a relatively small number of patients. second, we recognize that a limitation of this study is the starting time of weightbearing, which may have been affected by surgeons ' instructions. since there may be individual differences depending on the patients ' muscular strength, the degree of postoperative recovery, and implant stability, we reviewed the actual starting time of weight - bearing to minimize the effect of surgeons ' intervention. third, this study was limited to patients classified as ao type a2 to minimize the difficulties in interpreting a large number of individual variables. moreover, surgery was performed by a single surgeon to prevent potential errors due to the differences in surgeons ' preference and experience. among elderly individuals with ao type a2 intertrochanteric fractures, patients treated with bha were able to perform earlier ambulation than patients treated with compression hip screw fixation. however, there was no significant difference in operation time, the amount of postoperative transfusion, clinical results, hospital stay and radiological failure rate between the bha and compression hip screw fixation groups. | purposethe purpose of this study was to compare the outcomes of osteosynthesis using compression hip screw fixation versus bipolar hemiarthroplasty in ao type a2 intertrochanteric fractures.materials and methodsfrom march 2003 to december 2009, 89 patients were included in this study. they were treated using compression hip screws (43 cases) or bipolar hemiarthroplasty (46 cases). the mean age of patients was 77.7 years (65 - 94 years) and the mean follow - up period was 5.9 years (1 - 8.3 years). for comparison of the outcomes in the two groups, statistical analyses were performed with parameters including anesthesia time, operation time, amount of transfusion, hospital stay, general complications, clinical outcome, time of partial weight - bearing using a walker, and radiological failure rate.resultsdifferences in the amount of transfusion, general complications, and clinical outcome (merle d'aubign and postel score) were not statistically significant between the two groups. the bipolar hemiarthroplasty group showed better results than the compression hip screw group for anesthesia time and the time of partial weight - bearing using a walker. radiological failures were observed in hips in one case (2.2%) of bipolar hemiarthroplasty, and in four cases (9.3%) of compression hip screw fixation.conclusionamong elderly individuals with ao type a2 intertrochanteric fractures, patients treated with bipolar hemiarthroplasty were able to perform early ambulation. however, no significant difference in operation time, amount of postoperative transfusion, clinical results, hospital stay, and radiological failure rate was observed between the bipolar hemiarthroplasty and compression hip screw fixation groups. |
repair of damaged tissues is a complex physiological process that results in the deposition of extracellular matrix (ecm) components by resident fibroblasts. although the deposition of ecm proteins is normally a transient event, repeated tissue injuries in chronic pathologies or dysregulation of this process can lead to fibrosis and, eventually, to organ dysfunction. fibrosis can affect almost all tissues and organs, including heart, liver, kidney, lungs, and skin, therefore representing a major health issue for which efficient therapies are needed. regardless of the specific fibrotic disease and organs affected, the mechanisms involved in the progression of this pathology are very similar. indeed, damaged tissue repair can be recapitulated in four overlapping phases, hemostasis, inflammation, proliferation, and remodeling in which several cell types, closely interconnected to each other, play an important role. during the phases of hemostasis and inflammation, platelets secrete cytokines, including platelet - derived growth factor (pdgf) and transforming growth factor- (tgf-) that, in turn, recruit macrophages, neutrophils, and natural - killer cells to the site of injury. these cells, besides removing dead cells, debris, and pathogens, release cytokines that trigger activation and proliferation of resident fibroblasts, thus affecting ecm production. for example, macrophages release tgf-1 that controls a wide spectrum of activities, such as promoting fibroblast differentiation into active myofibroblasts, inducing ecm protein expression [5, 6 ], and repressing the expression of matrix metalloproteinases (mmps), key proteins able to degrade several ecm components. in addition, macrophages release tumor necrosis factor- (tnf-) and interleukin-1 (il-1) that promote fibroblast activation and fibrotic tissue deposition. tissue damage and inflammation increase reactive oxygen species (ros) production, which, in turn, contributes to fibrosis, enhancing the secretion of fibrogenic factors. acylated and unacylated ghrelin are circulating peptide hormones encoded by the ghrelin gene which are mainly released from the stomach during fasting. the 117-amino acid preproghrelin undergoes proteolytic cleavages leading to the mature ghrelin peptides and to another biological active peptide named obestatin. the acylated form, through high affinity binding to the growth hormone secretagogue receptor type 1a (ghsr-1a), induces gh release and promotes food intake, adiposity, and positive energy balance [1113 ]. alongside its role in feeding and energy homeostasis, ghrelin exerts also many other biological activities, including cardioprotection and enhancement of cardiac function, a strong anti - inflammatory activity, antioxidant activity on several cell types and tissues such as liver, heart, and lung [1619 ], and neuroprotective activities. the acylated ghrelin anti - inflammatory function mainly depends on its direct effect on t lymphocytes and monocytes, in which it inhibits the expression of proinflammatory cytokines such as il-1, il-6, and tnf-. acylation of ghrelin is essential for its binding to ghsr-1a, since the unacylated form does not activate this receptor, unless administered at very high concentrations, in which case it acts as a functional agonist [2225 ]. however, both acylated and unacylated ghrelin share high affinity binding sites in a number of cell lines and tissues, where they mediate several activities, such as protection from apoptosis and oxidative injury [2632 ], stimulation of cell differentiation [3336 ], induction of proliferation [30, 3739 ], and protection of skeletal muscles from wasting [4042 ]. these effects suggest the presence of a not yet identified common receptor of both acylated and unacylated ghrelin. in addition, some biological activities are elicited only by the unacylated but not the acylated form of ghrelin, suggesting the existence of a specific receptor for unacylated ghrelin [39, 4345 ]. circulating levels of acylated and unacylated ghrelin are often altered in pathological states associated with fibrosis and this suggests a role for these hormones in tissue homeostasis and/or in etiology of these conditions ([4652 ], table 1). the massive deposition of collagen in the heart that occurs upon several stimuli, such as cardiomyocyte death, inflammation, hypertension - induced enhanced workload, hypertrophy, or chemotherapy with doxorubicin, plays a crucial role in cardiac remodeling after heart injury and may contribute to ventricular arrhythmias, left ventricular dysfunction, heart failure, and sudden cardiac death. together with inflammation, cardiac fibroblasts, the most abundant cells in the heart, are the main players in cardiac remodeling : upon injury they undergo proliferation and synthesize collagen to replace the necrotic or apoptotic cardiomyocytes. due to the antiapoptotic and anti - inflammatory activity of ghrelin, several researchers investigated the antifibrotic effect of acylated and unacylated ghrelin in different models of cardiac injury. doxorubicin, an antibiotic used in chemotherapy, alters cardiomyocytes energy metabolism and induces their apoptosis, thus determining myocardial fibrosis, which eventually results in cardiomyopathy and congestive heart failure. accordingly with the in vitro data on the cardioprotective effect of acylated and unacylated ghrelin against doxorubicin - induced apoptosis of cardiomyocytes, it has been recently demonstrated that both peptides are effective in inhibiting the cardiotoxicity of this drug also in vivo [55, 56 ]. unacylated ghrelin displays antiapoptotic effects on cardiomyocytes through the activation of the prosurvival erk1/2 and pi3k / akt signaling pathways ([26, 55 ], figure 1). acylated ghrelin seems to play an important role in the regulation of autophagy, a cellular pathway involved in protein and organelle degradation. although this cellular pathway is normally a protective mechanism, excessive autophagy can destroy essential cellular components and eventually induce apoptosis. doxorubicin treatment induces oxidative stress, autophagy, apoptosis, and, finally, cardiac dysfunction and collagen deposition in the heart [56, 57 ]. in this experimental model of cardiac injury, acylated ghrelin inhibits ros - induced autophagy and cardiomyocyte death through the inhibition of ampk and activation of p38-mapk pathway (, figure 1), thus leading to a decrease of doxorubicin - induced fibrosis and cardiac dysfunction. the antifibrotic effects of acylated and unacylated ghrelin have been demonstrated in other experimental models of cardiac injury, such as isoproterenol administration, myocardial infarction (mi), and spontaneous or diabetes - associated hypertension [5862 ]. the subacute injection in rats of the -adrenergic agonist isoproterenol induces myocardial injury and fibrosis and increases myocardial ghrelin expression and plasmatic acylated ghrelin levels [58, 59 ]. in this model, acylated ghrelin treatment ameliorates myocardial function and reduces fibrosis, although the mechanisms of such a protection have not been elucidated. the unacylated form of the peptide displays similar effects, suggesting that the antifibrotic activity of ghrelin is mediated by both ghsr - dependent and ghsr - independent pathways. ghrelin has a positive effect on cardiac remodeling and cardiac function also in rats undergoing mi by coronary artery ligation. mi induces a strong increase in tissutal il-1 and tnf- that is inhibited by the chronic administration of ghrelin. ghrelin also blunts the induction of mmp-2 and mmp-9 that could be viewed as an inhibition of overall fibroblasts activity. however, in spontaneously hypertensive rats, the synthetic gh - secretagogue hexarelin prevents cardiac fibrosis by inducing, rather than by inhibiting, mmp-2 and mmp-9 activity. notably, unacylated ghrelin, despite reducing cardiac fibrosis in diabetic mice, has no effect on other mmps involved in cardiac fibrosis development such as mmp-8 and mmp-13. the effect of unacylated ghrelin treatment was in fact investigated also in db / db diabetic mice compared to nondiabetic mice, since cardiac fibrosis is also observed in diabetic patients without hypertension. in this model of diabetic mice, unacylated ghrelin impairs collagen accumulation by upregulating adiponectin cardiac expression, which is known to prevent myocardial hypertrophy and fibrosis [64, 65 ]. in liver, hepatitis c or b viral infections, autoimmune diseases, alcohol abuse, and nonalcoholic fatty liver disease (nafld) can progress to a severe fibrotic disease in which parenchymal tissue is replaced by nonfunctional fibrotic tissue, a condition defined as cirrhosis. removal of the causative agent, such as viral infections, could revert liver fibrosis, but in the case of autoimmune diseases and nafld the causative agent is not clearly defined and the identification of new agents that could modulate this process is of pivotal importance. in patients with alcoholic hepatitis and chronic hepatitis c, plasmatic ghrelin levels are lower than in healthy subjects and negatively correlate with the severity of fibrosis (, table 1). circulating ghrelin levels also correlate with other hepatic fibrotic diseases ; however, in the case of patients with nafld, a worsening of the fibrotic stage is associated with high plasmatic concentration of both acylated and unacylated ghrelin (, table 1). interestingly, a screening of mirnas expression in visceral adipose tissue of nafld patients revealed that mir-132, of which the ghrelin gene is a predicted target, is downregulated in nonalcoholic steatohepatitis (nash) compared to non - nash patients, although a biological validation of this relationship still needs to be performed. although the causative relationship between ghrelin circulating levels and nafld is not defined, ghrelin might have a therapeutic potential in this and other hepatic pathologies, as demonstrated in several experimental models. the most used models to induce hepatic fibrosis include ccl4 or thioacetamide (taa) administration to rodents, which lead to oxidative stress - mediated liver cirrhosis. another model to induce liver fibrosis consists in bile duct ligation (bdl), which causes accumulation of hydrophobic bile acids in the liver, leading to ros formation, oxidative damage, inflammatory cell accumulation, and the increase of serum proinflammatory cytokines. in addition, nafld may be reproduced in rats by feeding animals with a high - fat diet, thus inducing liver fat accumulation, inflammation, and cellular necrosis. in this model, ghrelin treatment blunts the induction of tnf- and il-6 expression, counteracts hepatic oxidative stress, and inhibits hepatic cell apoptosis. the beneficial effects of ghrelin on liver injury and fibrosis have been pointed out by other studies as well. indeed, in rats with chronic hepatic fibrosis caused by bdl, ghrelin administration prevents hepatic damage by blunting the bdl - induced increase of tnf-, il-1, and il-6 plasma levels. moreover, ghrelin treatment impairs neutrophil infiltration and diminishes the amount of myofibroblast accumulation in the injured liver [48, 73 ]. accordingly, ghrelin downregulates the expression of collagen-1 and tgf-1 in primary hepatic stellate cells (hsc), the main hepatic fibrogenic cells, resulting in a diminished collagen deposition [48, 73 ]. ghrelin features anti - inflammatory and antifibrotic effects also in taa - induced hepatic injury in rats where it attenuates liver injury and collagen deposition through inhibition of hepatic cell apoptosis and antioxidative activity, in a way partially mediated by the induction of nitric oxide (no). finally, the physiological role of the ghrelin gene in the establishment of liver fibrosis was investigated exploiting ghrelin knock - out mice that display much more severe ccl4-induced liver injury and fibrosis compared to wild type animals, suggesting that endogenous ghrelin is required for a proper response to liver damage. ghrelin is expressed in kidneys and its expression is altered in pathological conditions such as glomerulopathies, in particular in the proliferative form, in which the immunoexpression of ghrelin is abated. moreover, the expression of ghrelin negatively correlates with the profibrotic protein endothelin-1 and interstitial inflammatory cell infiltration, suggesting that the loss of ghrelin could contribute to the development of renal interstitial fibrosis, which is the common feature of different end - stage renal diseases. the renin - angiotensin system (ras) is a well - known regulator of blood pressure and contributes to the development of target organ damage due to hypertension. angiotensin - ii (angii) is the main mediator of ras - induced chronic kidney damage through multiple mechanisms, including promotion of inflammation, fibrosis, oxidative stress, and senescence. indeed, in the experimental model of chronic kidney disease induced by angii infusion, the kidneys display increased ros and an accelerated tissue senescence [76, 77 ]. in addition, treated mice express higher levels of tgf- and plasminogen activator inhibitor-1 (pai-1) than saline - infused animals. in this model, ghrelin impairs renal tubular damage, fibrosis development, and senescence by both reducing the oxidative stress and maintaining the redox state. this is mediated by the induction of ucp2 and pgc1 that affect ros production and mitochondriogenesis, respectively (, figure 1). the antifibrogenic activity of ghrelin was demonstrated also in a rat model of renal damage obtained by unilateral ureteral obstruction (uuo), which results in tubular injury and cell death, with interstitial macrophage infiltration. in this model, ghrelin protects renal tubular cells from apoptosis, impairs macrophage infiltration, and reduces the induction of the proinflammatory cytokines il-1, tnf-, and monocyte chemoattractant protein-1 (mcp-1). moreover, this work demonstrates that ghrelin attenuates renal fibrosis by inhibiting fibroblast differentiation and by blocking epithelial mesenchymal transition (emt), thus stabilizing the epithelial phenotype. the mechanisms through which ghrelin elicits its antifibrotic activity involve the reduction of collagen i / iii, fibronectin, and -sma expression via inhibition of the tgf-1/smad3 signaling pathway. lung fibrosis occurs as a consequence of acute lung injury leading to persistent respiratory failure. lung fibrosis is usually differentiated into distinct types, including diffuse fibrosing alveolitis, diffuse interstitial fibrosis, and idiopathic pulmonary fibrosis, which is considered the most common and severe form of pulmonary fibrosis. currently, there are no therapies to counteract acute lung injury progression and lung transplantation remains the only possible intervention in end - stage disease. acute lung injury is characterized by the damage of the alveolar capillary barrier, neutrophil accumulation, and the induction of proinflammatory cytokines, followed by devastating lung fibrosis [82, 83 ]. in particular, the exfoliation of alveolar epithelial cells from alveolar septa leads to the activation of fibroblasts and the subsequent massive ecm deposition. cecal ligation and puncture (clp), the most used technique to induce peritonitis and sepsis, also induces lung injury and fibrosis as direct consequence of hypoxemia, neutrophilic inflammation, and alveolar edema. in clp - treated rats, ghrelin attenuates acute lung injury and mortality through inhibition of nuclear factor- (nf-) b activity (, figure 1). nf-b is a transcription factor that regulates gene expression of several cytokines, including tnf-, il-6, il-1, and il-8. accordingly, treatment with ghrelin reduces pulmonary levels of tnf- and il-6 in clp - treated rats. another experimental model used to induce acute lung injury in rodents is the intratracheal injection of bleomycin that promotes massive cell death, neutrophil and lymphocyte infiltration, cytokine production, and fibrosis [83, 86 ]. in bleomycin - treated mice, ghrelin administration improves animal survival in a dose - dependent manner and maintains lung architecture by reducing fibrosis. this antifibrotic activity is due to the impairment of neutrophil infiltration and accumulation in bronchoalveolar lavage fluid and through the inhibition of proinflammatory cytokines and of igf-1 release, which promotes collagen production by fibroblasts. in addition, the inhibition of alveolar epithelial cell death, observed in ghrelin - treated mice, represents another mechanism that contributes to ghrelin antifibrotic effects, since the prevention of the denudation of alveolar membranes impairs the subsequent fibrosis establishment. in the same model of lung fibrosis, the traditional japanese herbal medicine rikkunshito, known to stimulate a strong secretion of ghrelin, reduces lung fibrosis and ameliorates the systemic cachectic condition. however, rikkunshito effects are only partially due to the associated ghrelin increase, since it maintains its protective effects also in mice devoid of the ghrelin gene. systemic sclerosis, or scleroderma, is an autoimmune chronic connective tissue disease characterized by extensive fibrosis of the skin and internal organs, including lungs, gastrointestinal tract, kidneys, and heart. plasmatic levels of acylated and unacylated ghrelin are lower in systemic sclerosis patients than in healthy controls and even lower in patients with interstitial lung disease, suggesting that acylated ghrelin levels inversely correlate with tissue fibrosis (, table 1). consistently, acylated ghrelin treatment of fibroblasts isolated from systemic sclerosis patients reduces tgf-1 expression and collagen production. skin scleroderma might be experimentally induced in mice by subcutaneous injections of bleomycin that result in increased dermal thickness, a higher number of -sma - positive myofibroblasts, and greater infiltration of inflammatory cells. taken together, these data suggest that restoring normal circulating acylated and unacylated levels might efficiently contrast the fibrosis induced by systemic sclerosis. fibrosis is an intrinsic response to chronic injury, maintaining organ integrity when extensive necrosis or apoptosis occurs. with protracted damage to date, no satisfactory treatments are available. anti - inflammation strategies are one of the possible therapeutic approaches to fibrosis. acylated ghrelin has a potent anti - inflammatory activity and its ability to inhibit proinflammatory cytokines expression and release has been demonstrated by a large number of studies, both in vitro and in vivo. most of the studies on the antifibrotic effects of acylated and unacylated ghrelin agree that the mechanism of action includes the reduction of inflammation. however, also their effect on oxidative stress reduction plays a crucial role in repressing the formation of fibrosis, and their broad antiapoptotic activity surely contributes in maintaining organ structure and function. this has, however, raised a doubt that if they inhibit apoptosis also in myofibroblasts, this could help, instead of hinder, fibrosis. circulating levels of ghrelin are often altered in pathologies characterized by the presence of fibrosis ; however, it is difficult to discern a causative effect between ghrelin levels and fibrosis, as it is plausible that alterations in ghrelin levels reflect body mass and/or body energy metabolism. this is particularly possible in pathologies co - occurring with cachexia, such as heart and renal failure, in which the increase of ghrelin may represent a compensatory mechanism of the organism in the attempt at re - establishing optimal energetic balance or the establishment of ghrelin resistance. however, in pathologies such as scleroderma, in which fibrosis affects the gastrointestinal tract, it can not be excluded that the altered levels of ghrelin are a direct consequence of the altered gut condition. based on the studies reviewed herein, ghrelin, both in its acylated and unacylated forms, acts at least at two different levels. on one side, ghrelin peptides reduce the infiltration of inflammatory cells in the injured tissue and the subsequent release of cytokines responsible for fibroblast activation. on the other side, they directly affect fibroblast activity by reducing collagen production through the inhibition of tgf- signaling pathway. in conclusion, ghrelin peptides and their analogues appear to be promising in the treatment of fibrosis, although their safety and efficacy in long - term use still need to be elucidated. | fibrosis can affect almost all tissues and organs, it often represents the terminal stage of chronic diseases, and it is regarded as a major health issue for which efficient therapies are needed. tissue injury, by inducing necrosis / apoptosis, triggers inflammatory response that, in turn, promotes fibroblast activation and pathological deposition of extracellular matrix. acylated and unacylated ghrelin are the main products of the ghrelin gene. the acylated form, through its receptor ghsr-1a, stimulates appetite and growth hormone (gh) release. although unacylated ghrelin does not bind or activate ghsr-1a, it shares with the acylated form several biological activities. ghrelin peptides exhibit anti - inflammatory, antioxidative, and antiapoptotic activities, suggesting that they might represent an efficient approach to prevent or reduce fibrosis. the aim of this review is to summarize the available evidence regarding the effects of acylated and unacylated ghrelin on different pathologies and experimental models in which fibrosis is a predominant characteristic. |
the prevalence of obesity in japanese children and adolescents has increased from 5% to more than 10% over the last two decades. this has become a serious public health concern, as childhood obesity, which is the leading cause of hyperinsulinemia and insulin resistance, is associated with adult diseases such as hypertension (ht), dyslipidemia, type 2 diabetes mellitus (t2 dm) and metabolic syndrome (ms) in adulthood. the latter factors, the so - called lifestyle factors, are more closely related to the development of adult diseases than genetic factors. this has been demonstrated by the recent rapid increase in fat consumption in japan. leading an adequately healthy lifestyle from childhood will reduce the likelihood of developing obesity and prevent certain adult diseases. therefore, pediatricians should be concerned with the health education of children and adolescents in order to prevent the possibility of obesity in later life. in 2004, (dohad), which considers that intrauterine growth restriction (iugr) is associated with an increased risk of developing ms and its related diseases in later life (1). the supply of nutrients to the fetus is the major determinant of fetal growth and is dependent on the mother s size, body composition, nutrient store, diet during pregnancy, and the transport of nutrients to the placenta and their subsequent transfer across the placental membrane. dohad suggests that health education for children and adolescents is important to prevent them from bearing offspring who will be at risk of developing ms and its related diseases because maintaining a healthy lifestyle is necessary for mothers to prevent iugr. in this paper, we briefly review several epidemiological studies of japanese children, adolescents and adults and discuss the applicability of dohad in present day japan (table 1table 1 epidemiological studies of the relation of birth weight and risks factors to cardiovascular disease in japan). as dohad was shown to be applicable in japan, the need for appropriate health education for both children and adolescents is emphasized. previously, we reported the relationship of birth weight and current body weight with blood pressure (bp) in 195 healthy 3-yr - old japanese children from niigata, japan (2). systolic blood pressure (sbp) was inversely correlated with weight at birth and positively correlated to weight at 3 yr of age. children at 3 yr of age whose birth weights were more than 3,520 g had a mean sbp of 3.0 mmhg below that of those whose birth weight was 2,999 g or less. mean sbp at 3 yr of age for children whose weight at 3 yr exceeded 16.8 kg was 9.4 mmhg higher than that of age - matched children whose weight was 14.2 kg or less. we also found an increase of 0.12 mmhg in the sbp of children for each increment of 1 mmhg in the sbp of their mothers. the sbp at 3 yr of age in children of mothers who had experienced pretibial edema during pregnancy (101.0 8.8 mmhg) was significantly higher than that in children whose mothers did not have edema (96.6 9.6 mmhg). reported the relationship of birth weight or weight velocity (weight gain / year) with risk factors of atherosclerosis in 330 japanese children (187 boys, 143 girls) between 7 and 12 yr of age who underwent screening for lifestyle - related diseases in okinawa, japan (3). birth weight was significantly correlated with serum levels of adiponectin (r=0.239, p=0.000), uric acid (r=0.247, p=0.000) and high - density lipoprotein cholesterol (hdl - c ; r=0.117, p=0.034) after adjusting for age, sex and body mass index (bmi) percentile. however, weight velocity was a stronger predictive variable than birth weight for both adiponectin and uric acid. miura. reported the relationship of birth weight, current weight, rate of height increase from 3 yr to 20 yr of age and bp with serum total cholesterol (tc) level in 4,626 japanese (2,198 men, 2,428 women) at 20 yr of age and born between 1965 and 1974 in ishikawa, japan (4). using multiple linear regression analysis, the authors estimated that a higher birth weight (+ 1 standard deviation) was significantly associated with sbp (lower by 1.6 mmhg in men and by 1.0 mmhg in women) and serum tc (lower by 0.07 mmol / l in women) after adjusting for current weight and rate of height increase. reported the relationship of birth weight and bp with serum lipid levels in 299 (207 men, 92 women) medical students of dokkyo university school of medicine who had a mean age of 23 yr (5). in this case, male birth weight was inversely correlated with serum tc and triglyceride (tg) levels in young adulthood and was independent of current bmi. these findings suggest the relation of birth weight with bp, tc, hdl - c, uric acid and adiponectin. however, a study reported that birth weight was not associated with these risk factors of cardiovascular disease (5). to reveal the relationship of fetal and infant growth with these risk factors, tamakoshi. reported the relationship of birth weight with adult ht in a japanese workplace population (2,303 males, 804 females) aged 35 to 66 yr (6). multiple logistic regression analysis adjusted for age, sex, bmi, parental history, and lifestyle revealed that the adjusted odds ratios for ht were 1.26 (95% confidence interval (ci) : 0.881.80), 1.00 (reference), 0.89 (95%ci : 0.731.08) and 0.70 (95%ci : 0.491.00) for subjects who were in the birth weight categories of 3,500 g, respectively (p - value=0.009). reported the relationship of birth weight with serum adiponectin levels in 2,277 subjects (1,661 men, 616 women) aged 35 to 66 yr (7). after adjusting for potential confounders, including age, sex, current bmi, smoking status, alcohol consumption and exercise activity level, the geometric mean adiponectin levels were 6.63, 6.45, 6.86, 7.05, 6.75 and 7.22 mg / ml for subjects with birth weights that were 3,500 g, respectively. consequently, a positive association was found between birth weight and adiponectin concentration (p<0.002). these findings suggest that the relations of weight and height at birth and postnatal weight increases with the risk factors of cardiovascular disease in middle - aged japanese adults. we investigated the association of birth weight with metabolic derangements in obese children in niigata, japan (8,9,10,11). we reported the relationships of birth weight and current visceral fat accumulation with hyperinsulinemia and insulin resistance in 967 obese japanese children and adolescents (650 boys, 317 girls) aged 6 to 15 yr (9). we divided the subjects into 4 groups on the basis of their birth weight or standard deviation (sd) score and compared the following anthropometric measurements : maximum preperitoneal fat thickness of the abdominal wall (pmax), bp, serum insulin levels, homeostasis model assessment - insulin resistance (homa - r) and quantitative insulin sensitivity check index (quicki) among the quartiles. the fasting serum insulin levels and homa - r were highest in the quartile with the lowest birth weight or sd score. birth weight and sd score were inversely related to the serum insulin levels and homa - r but positively related to quicki after adjusting for pmax. these findings suggest that both the intrauterine environment and current visceral fat accumulation are related to hyperinsulinemia, insulin resistance and the subsequent development of ms in obese japanese children. we also reported the relationship of birth weight with serum insulin, adiponectin, glutamic pyruvic transaminase (gpt), tg, hdl - c and low density lipoprotein cholesterol (ldl - c) levels (10). multiple linear regression analyses adjusted for sex, age, and current waist circumference revealed a correlation of birth weight with serum adiponectin, insulin and gpt levels (table 2). on the other hand, the serum tg, hdl - c, and ldl - c levels showed no relation to birth weight.table 2 multiple regression analyses of adiponectin, insulin and gpt with birth weight and waist circumference adjusted for sex and age among 600 obese boys and 283 obese girls from japanindependent variablesdependent variablesadiponectininsulingptpppmodel 1waist circumference0.235<0.00010.591<0.00010.453<0.0001birth weight0.09<0.010.139<0.00010.131<0.0001model 2waist circumference0.236<0.00010.595<0.00010.457<0.0001birth weight sd score0.087<0.010.150 < 0.00010.14<0.0001gpt, glutamic pyruvic transaminase. moreover, we have reported on the relationships of current weight - to - birth weight ratio (wbwr) with bp, elevated gpt and hyperinsulinemia in 126 obese japanese children and adolescents (97 boys, 29 girls ; age range 912 yr) (11). the subjects were divided into an ms group and a non - ms group on the basis of criteria proposed for diagnosing japanese children with ms. although there were no significant differences in age or anthropometric measurements between the two groups, the birth weight of the ms group was lower than that of the non - ms group, whereas the wbwr of the ms group was higher than that of the non - ms group. systolic and diastolic blood pressure, serum insulin and gpt levels correlated positively with birth weight after adjusting for sex, current height and weight. we then investigated the association of birth weight and current waist circumference with the prevalence of ms and serum insulin levels in obese children and adolescents. we divided 416 obese children (261 boys, 125 girls) into 9 groups on the basis of low, medium or heavy birth weight, or small, medium or large waist circumference. the group with the low birth weight and large waist circumference had the highest prevalence of ms and mean serum insulin level among the 9 groups (tables 3 and 4).table 3 prevalence of metabolic syndrome and the mean levels of fasting serum insulin in 261 japanese obese boys according to birth weight and waist circumference after being divided into 3 groupswaist circumference (cm)birth weight (g)1,7403,1203,1303,4253,4304,875total64.082.04 (10.8%)0 (0.0%)1 (3.8%)5 (5.6%) 12.7 11.4 11.4 11.9 3727269082.588.57 (29.2%)3 (10.3%)6 (18.2%)16 (18.6%) 17.4 13.4 12.8 14.3 2429338689.0116.09 (33.3%)8 (26.7%)5 (17.9%)22 (25.9%) 30.8 22.5 20.2 24.4 27302885total20 (22.7%)11 (12.8%)12 (13.8%)43 (16.5%) 19.5 16 14.7 16.8 888687261upper stand : number with metabolic syndrome (%). middle stand : the mean level of fasting serum insulin (u / ml). table 4 prevalence of metabolic syndrome and the mean levels of fasting serum insulin in 125 japanese obese girls according to birth weight and waist circumference after being divided into 3 groupswaist circumference (cm)birth weight (g)1,8453,0403,0443,3753,3904,000total69.080.50 (0.0%)0 (0.0%)0 (0.0%)0 (0.0%) 15 16.4 17.8 16.4 1219114286.085.53 (20.0%)1 (11.1%)0 (0.0%)4 (10.0%) 20.5 20.3 19.6 20.1 159164086.0114.511 (73.3%)1 (7.7%)2 (13.3%)14 (32.6%) 32.8 23.7 21.5 26.1 15131543total14 (33.3%)2 (4.9%)2 (4.8%)18 (14.4%) 23.3 19.6 19.8 20.9 424142125upper stand : number with metabolic syndrome (%). middle stand : the mean level of fasting serum insulin (u / ml). middle stand : the mean level of fasting serum insulin (u / ml). middle stand : the mean level of fasting serum insulin (u / ml). these findings suggest that birth weight is related to metabolic derangements in obese japanese children and adolescents. ideally, epidemiological studies should be performed in other regions of japan, because the presented studies have only performed in some regions. according to epidemiological studies, interactions between birth weight and early postnatal growth have been associated with the development of diabetes in later life (12). recent studies of pima indians and schoolchildren in taiwan revealed a u - shaped relationship between birth weight and the risk of developing t2 dm (13, 14). reported the associations of birth weight and clinical characteristics with childhood - onset t2 dm (15, 16). the frequencies of low and high birth weights were higher among patients with t2 dm than among those in a control group, producing a u - shaped distribution (p<0.05). low - birth - weight patients had a low prevalence of a family history of diabetes. in contrast, high - birth - weight patients had a higher prevalence of diabetic mothers who used medication that included insulin therapy (p<0.05). these findings supported the notion that fetal growth is related to the development of t2 dm in japanese children and adolescents. a poor intrauterine environment, such as that of a mother with malnutrition, a smoking habit or an inappropriate diet, will result in fetal growth retardation. when the maternal - placental nutrient supply fails to match the fetal nutrient demand, the fetus adapts to the poor nutrition by changing its metabolism, altering its secretion of hormones and the sensitivity of tissues to these hormones, redistributing its blood flow to protect key organs, especially the brain, and slowing its growth rate. although this adaptation called programming is beneficial for fetal survival, it permanently alters the structure and function of both the tissues and the body of the newborn. the downstream effects of poor fetal nutrition include insulin resistance in muscle, liver and adipose tissue, poor development of pancreatic -cell mass and function, decreased replication of kidney cells and reduced activity of placental 11-hydroxysteroid dehydrogenase (11-hsd) type 2 (17). this programming is considered to be the individual s adaptation to the mismatch of nutrition during growth and development in the fetal stage. moreover, the second mismatch of the prenatal versus the postnatal environment will contribute to an increase in risk factors for developing adult disease. the underlying mechanisms of this innate programming involve epigenetic modifications to the non - imprinted genes and are induced by aspects of the developmental environment, which modify gene expression without altering the dna sequence (18). in japan, as of 2005, the average birth weight has decreased to < 3,000 g, while the incidence of low - birth - weight infants has increased to 9.5%. the maternal diet has become insufficient, as the average daily energy intake is around 1,7001,880 kcal and is not increased during pregnancy. such a poor nutritional condition, which is often associated with a western lifestyle, is expected to result in a trend of increased risk for present day japanese children developing adult diseases in later life. pediatricians and schoolteachers should therefore understand the concept and importance of dohad and educate both children and their families regarding appropriate diet to reduce the likelihood of developing adult diseases in later life. | the origins of adult disease are considered to relate to fetal undernutrition, and this concept is termed developmental origins of adult health and disease (dohad). here, we describe several epidemiological studies performed in japan and discuss whether dohad is applicable to children in present day japan. in a study of healthy children and young adults, it was found that systolic blood pressure, total cholesterol and adiponectin were associated with birth weight. hyperinsulinemia, high blood pressure, elevated transaminase levels and prevalence of metabolic syndrome in obese children were inversely correlated with birth weight and positively correlated with current weight and waist circumference. birth weight was related to the development of type 2 diabetes in children. dohad is therefore considered to be applicable in japan. the key considerations of dohad are the following two mismatches. the first mismatch pertains to growth and development in response to environmental influences, especially those of nutrition. the second mismatch pertains to the prenatal versus postnatal environment. we consider that the chance of children in present day japan developing adult diseases is determined by the above mismatches. pediatricians and schoolteachers should therefore understand the concept of dohad, so that they can educate both children and their families regarding an appropriate diet to reduce the likelihood of developing adult diseases in later life. |
she had no surgical history and presented with paroxysmal periumbilical abdominal pain, accompanied by nausea and vomiting. biological examinations showed leucocytes at 17 040 and c - reactive protein at 9.2. urine cytology examinations and the vaginal swab were sterile. the patient was treated with tocolysis with parenteral nicardipine, corticotherapy with intramuscular betamethasone and anti - biotherapy with amoxicillin and gentamicine. the digestive symptoms worsened over the following 48 hours with the absence of stools or gas emissions and refractory faecal vomiting. the abdominal - pelvic ct - scan showed a pre - splenic left position of the caecum and right colon, with no part of the colon on the right. the radiologist concluded that the patient was suffering from a complication of intestinal malrotation (i m) (figs 1, 2, 3). figure 1:abdominal - pelvic ct - scan, non - contrast enhanced, in the sagittal plan, tenth dorsal vertebra level. figure 2:abdominal - pelvic ct - scan, non - contrast enhanced, in the sagittal plan, eleventh dorsal vertebra level. figure 3:abdominal - pelvic ct - scan, non - contrast enhanced, in the sagittal plane. abdominal - pelvic ct - scan, non - contrast enhanced, in the sagittal plan, tenth dorsal vertebra level. abdominal - pelvic ct - scan, non - contrast enhanced, in the sagittal plan, eleventh dorsal vertebra level. abdominal - pelvic ct - scan, non - contrast enhanced, in the sagittal plane. exploration revealed incomplete i m and ladd 's bands with internal hernia of the whole small intestine. treatment of the hernia consisted in extracting the small intestine from the hernial sac, and then resection of the hernial sac by electrocoagulation followed by repositioning of the intestines. cases of obstruction syndromes during pregnancy are rare and a major difficulty in such cases is diagnosis. the risks of obstruction increase from 16 to 24 wa, with passage of the uterus to the abdominal - pelvic position at 36 wa, the descent of the foetus into the pelvis and in the post - partum period during uterine involution. the more advanced into the pregnancy, the greater the risk of obstruction : 6% of obstructions occur during the first trimester, 27% during the second, 44% during the third and 21% post - partum. other symptoms like the absence of stools and gas emissions, the intensity of the abdominal pain and refractory vomiting should alert the practitioner. two principal etiologies of obstruction stand out in our case and in cases in the literature : obstruction of the small intestine due to adhesions, and volvulus of the sigmoid (tables 1 and 2). among obstruction syndromes, the prognosis in volvulus of the sigmoid is more severe, with numerous foetal deaths, necrosis of the sigmoid, often two - step surgery (colostomy) and hospitalization in intensive care for severe sepsis [2, 46 ] (table 2). table 1cases of small intestine occlusion in the literaturearticlenumber of casestermtype occlusiontreatmentmaternal complicationsoutcomes foetusmeyerson. nine casessecond trimester = 4, 3 trimester = 4, post - partum = 1adhesionadhesiolysisone case of intestinal necrosisthree foetal deathsnajih. three cases17 waadhesionadhesiolysis_vaginal delivery at term35 waadhesionadhesiolysis_caesarean 35 wa35 waadhesionadhesiolysis_caesarean 35 wa table 2cases of colon occlusion in the literaturearticlenumber of casestermtype of occlusiontreatmentmaternal complicationsoutcomes foetusnarjis one case24 wavolvulus of the sigmoidresection of the sigmoid and colostomynecrosis of the sigmoidin utero foetal death at 24 wakolusari. four cases7 wa, 31 wa, 32 wa, post - partumvolvulus of the sigmoidresection of the sigmoid with colostomy, and anastomosis in the case at 7 waone death, four cases of necrosis of the sigmoidin utero foetal death at 31 waribeiro nascimento. one case33 wavolvulus of the sigmoidleft colectomy and colostomyperitonitis with septic shock following necrosis of the sigmoid + haemorrhage of the after birth with hysterectomy for haemostasisin utero foetal death, caesarean 33 wakhan and rehman one case30 wavolvulus of the sigmoidcolectomy and ileostomymaternal death due to necrosis of the sigmoid and post - operative septic shockin utero foetal death at 30 wanajih. two cases16 watumour of the coloncolostomy_caesarean at 40 wa37 wavolvulus of the sigmoidresection of the sigmoid and double ostomynecrosis of the sigmoidin utero foetal death cases of small intestine occlusion in the literature cases of colon occlusion in the literature in our patient, it was an exceptional case of internal hernia in a context of i m. i m is an embryonic malformation due to the interruption of normal rotation of the primitive gut tube at 90 in the 4th week. the small intestine is therefore on the right and the colon in its entirety is on the left. it is extremely rare to discover i m in an adult : 0.20.5% of cases, in which the condition is asymptomatic and can be discovered on a volvulus of the small intestine. abdominal - pelvic ultrasonography is the first - line diagnostic examination as it is harmless, it provides useful information (dilation or signs of intestinal ischaemia, intraperitoneal effusion) and it rules out differential diagnoses (appendicitis, ovarian torsion). it can also rule out intestinal obstruction with sensitivity of 89% and specificity of 100%. the use of a plain abdominal x - ray could have been discussed to search for hydroaeric levels. however, given the advanced term and the normal findings of the first abdominal us - scan, a ct - scan seemed to be the most useful examination. the abdominal - pelvic ct - scan is the gold - standard in the exploration of obstruction syndromes and can be used if the expected benefits outweigh the risks of foetal irradiation [3, 8 ]. mri is an interesting examination in the diagnosis of obstruction syndromes in pregnant woman, because no ionizing radiation is used. mri, however, is still difficult to obtain in an emergency. in our case, the aetiology of i m could have been suspected given the absence of a surgical history. the intestine was not ischaemic and the clinical context allowed foetal pulmonary maturation at 32 wa and maintenance of normal haemodynamics in the mother [2, 5 ] (table 1). in cases of sub - obstruction, medical treatment via a nasogastric tube and the perfusion of analgesics may make it possible to avoid surgery and reduce the risk of foetal prematurity. laparotomy is often unavoidable to save the mother, whatever the term, because the maternal - foetal prognosis depends on the speed of the surgery. before 28 wa, between 28 and 32 wa, the balance between the benefits and risk must be considered ; the mother 's clinical condition and the risks associated with induction of a highly premature delivery must be taken into account. from 32 wa onwards, extraction of the foetus before the digestive surgery must be considered whenever possible, after pulmonary maturation, depending on the clinical picture of the mother and foetus. obstruction syndromes in pregnant women are difficult to diagnose, and the difficulty increases with the term. plain abdominal x - ray and us - scans must be used in cases of doubt. the surgery, performed by a double team, must be done as soon as possible after the diagnosis to improve maternal - foetal survival and to diminish complications. | we present the case of a 33-year - old female who presented intestinal obstruction at the 7th month of pregnancy. she was complaining of abdominal pain with paroxysms, nausea and vomiting. when the symptoms worsened, we performed a ct - scan, which revealed suspected intestinal obstruction in a context of intestinal malrotation (i m). conservative therapy failed and she required emergency laparotomy beginning with a caesarean section. the surgery revealed a peritoneal defect due to the i m through which the jejunum and ileum were incarcerated and strangulated. the incarcerated bowel was reduced and intestinal colour quickly returned to normal. primary closure of the peritoneal defect was thus performed and the post - operative course was uneventful for the mother and the baby, who was admitted to the neonatal intensive care unit. |
parkinsonism is a common symptom, but early diagnosis of parkinson s disease (pd) and dementia with lewy bodies (dlb) is very difficult even for neurologists because clinical history or physical examination is nonspecific. metaiodobenzylguanidine (mibg) is a physiological analog of noradrenaline (norepinephrine) and has been used to evaluate postganglionic cardiac sympathetic innervation. many studies have reported that cardiac mibg uptake is reduced in the case of lewy body disease (lbd). the studies have proven that the heart to mediastinum (h / m) ratio of i - mibg cardiac scintigraphy is a useful diagnostic tool for indicating the significant reduction in myocardial mibg uptake in lbd as compared with a control group. mibg scintigraphy is performed twice in the early phase (from 10 to 30 min after injection of the radioisotope) and in the delayed phase (from 3 to 4 h after injection of the radioisotope). all previous studies on lbd examined the patients twice, and the h / m average count ratio was obtained for both early and delayed images mainly using planar imaging. however, only the delayed h / m ratio was used to evaluate the diagnostic performance in most of the previous studies 2. to our knowledge, there are no reports comparing the diagnostic performance of h / m ratio statistically between early and delayed phases 3,4. therefore, the potential role of early h / m ratio has not been established. the aim of our study was to compare the diagnostic performance of the h / m ratio in early - phase i - mibg scintigraphy with that in delayed - phase imaging. if the diagnostic performance of the h / m ratio in the early phase is found comparable to that of the delayed phase, the total time taken by the examination to evaluate lbds would be much shorter. our institutional review board approved the study and the need for informed consent was waived. between april 2012 and december 2013, 217 consecutive patients with suspected degenerative parkinsonism underwent i - mibg cardiac scintigraphy. the exclusion criteria for this study were a history of tricyclic antidepressive medication or heart disease. the latter included coronary heart disease, infarction, and heart failure. the final diagnosis of lbd that is, pd or dlb was made by neurologists who took into account the patient s history, the clinical presentation of key features, and a diagnostic brain mri to exclude symptomatic parkinsonism such as multiple arteriosclerotic changes or hydrocephalus. the uk pd society brain bank criteria 5 were used for the clinical diagnosis of pd, and the third report of the dlb consortium 3 was used to make the clinical diagnosis of dlb. patient characteristics all patients underwent i - mibg myocardial scintigraphy after an intravenous injection of 111 mbq of i - mibg (fujifilm ri pharma co. ltd, tokyo, japan). anterior images of the chest were obtained after 15 min and again after 4 h using a dual - headed gamma camera (toshiba e.cam duet ; toshiba medical systems, kawasaki, japan) equipped with low - to medium - energy general - purpose collimators. the cardiac uptake of i - mibg was determined by manually drawing a region of interest (roi) that encircled the left ventricle. the mediastinal uptake was determined by setting a rectangular roi drawn by a skilled medical radiation technologist in the upper mediastinum. the h / m ratio was calculated as the ratio of the average counts per pixel in the heart roi divided by that in the mediastinum roi. we used software for transport and analysis of myocardial planar images (stamp ver1.3 ; fujifilm ri pharma co. ltd) to calculate the h / m ratio. normality of the h / m ratios of the early and delayed images was examined by means of the shapiro the difference in h / m ratios between the early and delayed images was examined for statistical significance using the mann we conducted conventional receiver - operating characteristic (roc) analysis for assessing the diagnostic performance of the h / m ratio in the early and delayed phases. the area under the curve (auc) was calculated for each phase and the difference in auc was analyzed. the optimal cutoff value of the h / m ratio for both early and delayed phases was chosen to maximize the youden index (sensitivity+specificity1). our institutional review board approved the study and the need for informed consent was waived. between april 2012 and december 2013, 217 consecutive patients with suspected degenerative parkinsonism underwent i - mibg cardiac scintigraphy. the exclusion criteria for this study were a history of tricyclic antidepressive medication or heart disease. the latter included coronary heart disease, infarction, and heart failure. the final diagnosis of lbd that is, pd or dlb was made by neurologists who took into account the patient s history, the clinical presentation of key features, and a diagnostic brain mri to exclude symptomatic parkinsonism such as multiple arteriosclerotic changes or hydrocephalus. the uk pd society brain bank criteria 5 were used for the clinical diagnosis of pd, and the third report of the dlb consortium 3 was used to make the clinical diagnosis of dlb. all patients underwent i - mibg myocardial scintigraphy after an intravenous injection of 111 mbq of i - mibg (fujifilm ri pharma co. ltd, tokyo, japan). anterior images of the chest were obtained after 15 min and again after 4 h using a dual - headed gamma camera (toshiba e.cam duet ; toshiba medical systems, kawasaki, japan) equipped with low - to medium - energy general - purpose collimators. the cardiac uptake of i - mibg was determined by manually drawing a region of interest (roi) that encircled the left ventricle. the mediastinal uptake was determined by setting a rectangular roi drawn by a skilled medical radiation technologist in the upper mediastinum. the h / m ratio was calculated as the ratio of the average counts per pixel in the heart roi divided by that in the mediastinum roi. we used software for transport and analysis of myocardial planar images (stamp ver1.3 ; fujifilm ri pharma co. ltd) to calculate the h / m ratio. normality of the h / m ratios of the early and delayed images was examined by means of the shapiro the difference in h / m ratios between the early and delayed images was examined for statistical significance using the mann we conducted conventional receiver - operating characteristic (roc) analysis for assessing the diagnostic performance of the h / m ratio in the early and delayed phases. the area under the curve (auc) was calculated for each phase and the difference in auc was analyzed. the optimal cutoff value of the h / m ratio for both early and delayed phases was chosen to maximize the youden index (sensitivity+specificity1). the h / m ratios of the early and delayed images did not show a normal distribution. the h / m ratio of the lbd group ranged from 1.1 to 3.88 (median 1.8) in the early phase and from 0.97 to 4.7 (median 1.45) in the delayed phase. the h / m ratio of the nondiseased group ranged from 1.17 to 4.18 (median 2.93) in the early phase and from 0.85 to 4.77 (median 3.18) in the delayed phase. both early and delayed h / m ratios of the lbd group there was no normality in the h / m ratios in the early and delayed images. (a) the comparison of early h / m ratio for lbd and nondiseased patients. early h / m ratios of lbd were significantly lower than those of the nondiseased group (p<0.0001). (b) the comparison of delayed h / m ratio for lbd and nondiseased patients. delayed h / m ratios of lbd were significantly lower than those of the nondiseased group (p<0.0001). the boxes are bound on the top by the third quartile, and on the bottom by the first quartile. the upper whisker extends from the third quartile to the third quartile+1.5iqr (interquartile range), and the other extends downward from the first quartile to the first quartile1.5iqr. h / m, heart to mediastinum ratio ; iqr, interquartile range ; lbd, lewy body disease. the roc curves for both phases are shown in fig. the auc for the early phase was 0.871 and that for the delayed phase was 0.893. the optimal cutoff value of the h / m ratio for the early and delayed phases was 2.28 and 1.91, respectively. these are higher because our collimator and the way of drawing rois were different from the past report. the sensitivity and specificity of the early phase were 80.2% (65/81) and 91% (101/111), respectively. the sensitivity and specificity for the delayed phase were 81.4% (66/81) and 96.7% (106/111), respectively. in contrast, five nondiseased participants were classified correctly as nondiseased only with the early phase (their h / m ratio was greater than the cutoff value). receiver operating characteristic curves of the h / m ratio in the early and delayed phases. the auc for the early phase was 0.871 and that for the delayed phase was 0.893. auc, area under the curve ; h / m, heart to mediastinum ratio. to our knowledge, this is the first research to statistically compare the diagnostic performance of early and delayed phases. although previous studies 810 have reported the sensitivity and specificity of early and delayed phases, they did not compare the difference statistically. our results reveal that the diagnostic performance of i - mibg scintigraphy was not significantly different between early and delayed phases. the results for i - mibg cardiac scintigraphy in the evaluation of lbd have been obtained both in the early (1530 min) phase and in the delayed (35 h) phase. however, the delayed phase was weighted heavily in the meta - analysis part of a previous study 2. early myocardial uptake of mibg reflects the integrity and distribution of the presynaptic sympathetic system 11. however, the neuronal accumulation of mibg uptake reaches its peak 34 h after injection and the delayed uptake may reflect the functional status such as the relative level of neuronal uptake or the degree of washout of norepinephrine from sympathetic nerve terminals 8. 12 reported that the h / m ratio in the lbd group was significantly lower in the delayed phase than in the early one, and the h / m ratio in the control group was higher in the delayed phase than in the early one. this means that the difference in h / m ratio between the lbd and control groups was larger for the delayed phase than for the early one. regardless of these explanations, the previous studies actually revealed that the h / m ratio in the early phase was also significantly lower in the lbd group than in the control group 1,9,11. 8 conducted an roc analysis for each of the two phases and showed similar aucs (0.86 for the early phase and 0.85 for the delayed phase), although they were not statistically compared. therefore, once the optimal cutoff value of the h / m ratio for the early phase is determined, early - phase imaging can provide comparable diagnostic performance to delayed - phase imaging. the comparable diagnostic performance of early - phase imaging could change the protocol for patients suspected of having lbd. at present, it takes about 4 h to complete the procedure because patients have to wait to undergo delayed - phase imaging. early - phase imaging would shorten the time from injection to completion of imaging to less than 1 h and would make testing available to patients who can not tolerate long hours because of their parkinsonism or dementia. nowadays, i - ioflupan dopamine transporter visualization using single photon emission computed tomography (spect) is being increasingly used to assist in the evaluation of parkinsonism. low dopamine transporter uptake in the basal ganglia is one of the suggestive features of parkinsonism according to the third report of the dlb consortium, whereas abnormally low uptake in mibg scintigraphy is only a supportive feature 3. this means that i - ioflupan spect is a more specific examination for lbd compared with i - mibg cardiac scintigraphy. the sensitivity and specificity of i - ioflupan spect for pd were 86.5 and 93.6%, respectively, and that for dlb were 98 and 67% 13,14. therefore, the diagnostic performance of i - ioflupan spect for lbd relative to that of i - mibg cardiac scintigraphy may be somewhat superior but not overwhelmingly so. second, setting rois on i - ioflupan spect images has not been standardized and the optimal cutoff value for diagnosis has not been established. third, it is reported that mibg can differentiate lbd from multiple systemic atrophy or progressive supranuclear palsy, but i - ioflupan can not 13,14. in addition, i - ioflupan spect takes longer than early - phase i - mibg scintigraphy ; that is, the data of i - ioflupan are usually collected from 3 to 6 h after injection of the radioisotope. therefore, we believe that cardiac i - mibg scintigraphy will not be completely replaced by i - ioflupan spect. it is retrospective in nature and the results of i - mibg scintigraphy could have affected the clinician s diagnosis. currently, an roi is placed automatically over the heart by the new software smart mibg (fujifilm ri pharma co. ltd). although an experienced technician placed the roi in this study, third, the patients were referred to the university hospital, and hence pretest probability may be high. early h / m ratio has almost the same diagnostic performance as delayed h / m ratio. early - phase i - mibg scintigraphy may be optional for patients who can not tolerate a long interval from injection to completion of imaging. | objectivesthe aim of the study was to compare the diagnostic performance of early - phase 123i - metaiodobenzylguanidine (mibg) scintigraphy with that of delayed - phase imaging in lewy body disease (lbd).methodsa retrospective cohort study of 123i - mibg scintigraphy was carried out in 192 patients who were suspected of having lbd. clinical diagnosis was obtained using the uk parkinson s disease brain bank criteria in some cases or the third report of the dementia with lewy bodies consortium in others. the participants consisted of 81 patients with lbd and 111 nondiseased patients. an injection of 111 mbq of 123i - mibg was used. planar images were obtained in an early phase and again in a delayed phase and the heart to mediastinum count ratio was calculated for both phases. diagnostic performance was compared using a receiver - operator characteristic analysis. the cutoff value was chosen to maximize the youden index. the sensitivity and specificity of each phase were calculated from the optimal cutoff value.resultsthe heart to mediastinum ratio of the lbd group (median 1.8 and 1.45 for early and delayed phases, respectively) was significantly lower than that of the nondiseased group (median 2.93 and 3.18 for early and delayed phases, respectively). the area under the receiver - operating characteristic curve was not significantly different between the early and delayed phases (0.871 vs. 0.893 ; p=0.0914). sensitivity and specificity were 80.2 and 91% for early - phase imaging (cutoff value at 2.28) and 81.5 and 95.5% (cutoff value at 1.91) for delayed - phase imaging, respectively.conclusionthe diagnostic performance of 123i - mibg scintigraphy was not significantly different between early - phase and delayed - phase imaging. |
hepatosplenic t - cell lymphoma (hstcl) is a rare peripheral t - cell lymphoma with predominant occurrence in young male adults and characterized by extranodal infiltration of mature malignant post - thymic t - lymphocytes into sinusoids of the liver and spleen without lymphadenopathy and significant cytopenias. increased numbers of normal t - cells can be found in patients with chronic antigen stimulation, autoimmune disease, post solid organ transplantation (kidney, liver) and splenectomy. the hstcl represents < 5% of peripheral t/ nk - lymphomas, and is less common than peripheral t cell lymphoma. only 150 cases have been described in literature since the initial description by farcet and gaulard in 1990. a 20-year old patient was admitted to the department of haematology for clarification of haematomata in both hands, hepatosplenomegaly and pancytopenia. she has never taken any contraceptives or other medication and previously she was healthy. during pregnancy her blood work was normal. two months before admittance to our department she first noticed haematomata in extremities, and more recently she had become tired, dizzy and pale. clinical examination revealed an enlarged liver and spleen (15 cm) besides paleness and haematomata in her extremities. blood test revealed pancytopenia (red blood cells (rbc) 3,510/ l, haemoglobin (hb) 91 g / l, reticulocytes 8510/l, white blood cells (wbc) 3,410/l with 7% of atypical lymphocytes, platelets 2810/l, elevated total bilirubin (28 mol / l) and c reactive protein (22 mg / l), low cholinesterase (che) 92 kat / l and prothrombin time 0,55 e, other blood tests (aspartate transaminase (ast), alanine transaminase (alt), urea, creatinine, lactate dehydrogenase (ldh), vitamin b12, folates, electrolytes, tumour markers and immunological markers) were within normal limits. viral and bacterial serology was performed (toxoplasmosis, cytomegalovirus, tularaemia, hiv, wassermann test, yersinia, human herpesvirus, epstein - barr virus (ebv), hepatitis) and showed the presence of antibodies to ebv (table 1). ultrasound and abdominal computed tomography (ct) showed a slightly enlarged liver with a homogeneous pattern and markedly enlarged spleen reaching into the pelvis, bulging the anterior abdominal wall, pressing onto stomach and left kidney and pushing the left liver lobe away. because of the suspicion of a blood disease, we performed a bone marrow phenotyping, which showed a population of cd3 lymphocytes expressing cd56 +, cd16 partly, cd38, cd8 low, cd2, cd7 but not cd5 or tdt. of the additionally tested antigens, cd45ro and cd161 were positive, however the intracellular granzyme b was negative (figure 2). this population with aberrant expression of antigen cd5 and low expression of antigen cd8 (in figure 3 appearing red) presented 16% of leukocytes, 70% of all lymphocytes and 77% of t - lymphocytes in the bone marrow sample. the immunophenotyping results caused suspicion for the involvement of the extremely rare hepatosplenic lymphoma that was later confirmed by liver puncture. the liver puncture revealed that the liver was diffusely infiltrated with lymphoma cells which were typically distributed in the sinusoids instead of in the portal tracts as seen in usual lymphomas. the nuclei of the lymphoid cells were round, ovoid, occasionally irregular with quite dense chromatin, and a small amount of pale to clear cytoplasm (figure 3). the patient was treated with high - dose chemotherapy (chop) and autologous bone - marrow transplant. signs of disease progression (pancytopenia, spleen - growth reaching into pelvis) appeared after one year with b - signs. table 1patient 's laboratory data.parameter, unitspatientreference valueswbc, e10/l3,44,010,0blood smear, % segmented neutrophils454060banded neutrophils303lymphocytes362040monocytes928rbc, e10/l3,54,26,3hb, g / l91120180mcv, fl798194reticulocytes, e10/l85,12194platelets, e10/l28140340biochemistryast, kat / l0,1500,58alt, kat / l0,2300,74che, kat / l118117317total bilirubin, mol / l300 17urea, mmol / l5,32,87,5creatinine, mmol / l714497folic acid, mol / l12,96,132,6b12, pmol / l114132857crp, mg / l40 5ldh, kat / l1,0504,13prothrombin time, e0,550,71,2ana, anca, aca, amanegativenegativeca 19 - 9, ca 15 - 3, cea, fpnegativenegativeserological teststoxoplasmosis, cmv, yersinia, human herpesvirus, ebvebv (igg 1 : 160, igm 1:10,ebna 1 : 10), other negativehepatitis a, b, c, hivnegativenegativewasserman testnegativenegativemcv, mean corpuscular volume ; ana, antinuclear antibody ; anca, anti- neutrophil cytoplasmatic antibody ; aca, anti - centromere antibody ; ama, anti - mitochondrial antibody ; cmv, cytomegalovirus ; ebv, epstein - barr virus. mcv, mean corpuscular volume ; ana, antinuclear antibody ; anca, anti- neutrophil cytoplasmatic antibody ; aca, anti - centromere antibody ; ama, anti - mitochondrial antibody ; cmv, cytomegalovirus ; ebv, epstein - barr virus. figure 1computer tomography of the patient 's abdomen, transverse scan. l, left ; r, right ; s, very enlarged spleen ; liv, enlarged liver computer tomography of the patient 's abdomen, transverse scan. l, left ; r, right ; s, very enlarged spleen ; liv, enlarged liver figure 2flow cytometry in our patient : population of 77 % cd3 t - lymphocytes are marked red on the cd 45 - ssc diagram (phenotype cd56 +, cd16 partially, cd38, cd8 low, cd2, cd7, cd5, tdt, cd45ro, cd161, intracellular granzyme b negative). flow cytometry in our patient : population of 77 % cd3 t - lymphocytes are marked red on the cd 45 - ssc diagram (phenotype cd56 +, cd16 partially, cd38, cd8 low, cd2, cd7, cd5, tdt, cd45ro, cd161, intracellular granzyme b negative). figure 3liver histology of our patient shows lymphoma cells (arrow) between hepatocytes (star) (haemotoxylin - eosin stain, 400). liver histology of our patient shows lymphoma cells (arrow) between hepatocytes (star) (haemotoxylin - eosin stain, 400). although the aetiology of the hstcl is still unknown, approximately 10% to 20% of affected patients have a previous history of immunocompromise, including solid organ transplant (renal, liver, heart), inflammatory bowel disease (mostly crohn 's disease), hepatitis b infection, or the patients have been treated with immunosuppressive therapy (e.g. due to rheumatoid arthritis, systemic lupus erythematosus). it has been established that during pregnancy the placenta contains more nk cells, t cells and t - cells, which express more cytotoxic molecules than t - cell intracellular antigen 1, granzyme b and especially perforin. it is assumed that during pregnancy the high progesterone concentration might affect the perforin expression and that maternal immunity and hormonal changes during pregnancy and presumably delivery might eventually provide a chance for decidual lymphocytes to transform and develop hstcl. the leading sign of hstcl is hepatosplenomegaly and cytopenia, while lymph node enlargement excludes the disease. however, other clinical features like fatigue, coombs negative haemolytic anaemia, jaundice due to hepatic involvement and purpura due to thrombocytopenia may occur. the main sign of the disease is blood cell reduction, ranging from isolated reduction of one lineage to pancytopenia as a consequence of hypersplenism and/or suppression of bone marrow precursor cells by cytokines released by neoplastic cells. the blood smear is usually normal, a leukemic picture or lymphocytosis can yet be found, or, as in our case, a minor population of atypical lymphocytes. all the above mentioned clinical and laboratory tests are non - specific and if not recognized they can lead to misdiagnosis of virus infection (mostly ebv), immune thrombocytopenia or acute lymphoblastic leukaemia. to diagnose hstcl, flow cytometric immunophenotyping is extremely helpful in diagnosing, however both, the diagnostic and the examiner, should be experienced enough to recognize clonal changes of t - lymphocytes. unlike b - lymphocytes, t - lymphocytes do not have an efficient indicator of clonality on the membrane, so they can be recognized by flow cytometry based only on aberrant expression of usually present antigens of the t - cell and nk - cell subsets. malignant - changed t - lymphocytes, including the rare - ones, can often be recognized, since the antigen of the t - cell subset can be completely absent or its intensity of expression has changed when compared to other normal - polyclonal t lymphocytes. there are also exceptions to the common phenotype, since expression of cd5, cd7, cd8, cd16 and cd56 is variable. antigens characteristic for b - lymphocytes (cd19, cd20, cd21, and cd22), immunoglobulins, tcr - chain, tdt, cd10, cd15, cd25, cd33, cd34, cd41, and cd68 are not expressed. a special subcategory shows tcr expression as well as clinical and pathologic features that resemble those of hstcl. based on the flow cytometry of our patient 's bone marrow aspirate we were able to establish the phenotype of cells - suspicious for hstcl in a few hours, which was later additionally confirmed by the bone and liver biopsy. since the bone marrow examination with regular staining does not show the cells typical for this disease (however the flow cytometry does reveal the phenotype suspicious for hstcl), it is recommended to additionally perform immunohistochemical staining tests for t - lymphocytes, which reveal a hypercellular bone marrow with a sinusoidal infiltration of atypical, medium sized lymphoid cells with abundant light and basophilic cytoplasm and multiple granulations. both, liver and spleen puncture, reveal sinusal infiltration with atypical lymphocytes. the splenic white pulp is reduced or completely lost. since the normal t - lymphocytes have special homing receptors for splenic sinuses it is most likely that the hstcl originates in the splenic red pulp. mostly in male patients cytogenetic changes can occur (isochromosome 7q, trisomy 8 or absent y - chromosome), but this is not sufficient to define the disease. differential diagnosis should exclude other rare t - cell lymphomas / leukaemia (t - lymphoblastic lymphoma expressing tcr, peripheral cytotoxic t - cell lymphomas and unusual forms of t chronic lymphocytic / promyelocytic leukaemia with cd8 phenotype) ; however all these entities usually express tcr chains. hstcl is an extremely aggressive disease with median survival rate of 8 months to a maximum of 2 years, with possible remission due to treatment. at the present time an effective treatment of hstcl is lacking, as treatment modalities for other lymphoma appear to be ineffective in most patients. a wide array of therapies have been used, including steroid or alkylating treatment, purine analogs, splenectomy and high dose chemotherapy with or without allogeneic stem cell transplantation. prolonged disease free survival has been reported in patients who underwent allogeneic stem cell transplantation, receiving combined treatment with cytostatics and alemtuzumab, as pralatrexate and romidepsin for progressive or relapsed hstcl. hstcl is a rare entity of t - cell lymphoma, uncommon in female patients and not reported after delivery so far. flow cytometry is essential in diagnosing hstcl rapidly, and should additionally be confirmed by bone marrow examination and liver and/or spleen puncture. since it is a rare disease the examiner should have enough experience to recognize clonal changes of t - lymphocytes. the patient 's fate, however, lies in good cooperation between departments, diagnostic laboratory for flow cytometry and eventually the pathologist. cooperation is the only way to diagnose this rare condition soon enough to help the patient. | hepatosplenic t - cell lymphoma (hstcl) is a very rare peripheral t - cell lymphoma characterized by extranodal infiltration of mature malignant post - thymic t - lymphocytes into sinusoids of the liver and spleen without lymphadenopathy and significant cytopenias. the aetiology of the disease is unknown. we describe the case of a female patient in whom hstcl developed after delivery and who was previously without disease. flow cytometry and liver puncture are essential for diagnosing hstcl, especially in patients with unexplained pancytopenia and hepatosplenomegaly. since phenotypic results can easily be misinterpreted as non - malignant, the examiner should have enough experience to recognize clonal changes of t - lymphocytes. namely, in contrast to b - lymphocytes, t - lymphocytes do not have an efficient indicator of clonality and are recognized by flow cytometry based only on aberrant expression of commonly present antigens of t - cell and nk - cell subsets. at present, there is no known cure for hstcl with a maximum survival up to 2 years. |
epithelioid hemangioendothelioma (ehe) is an uncommon malignant vascular tumor and characterized by an often unpredictable clinical course. there is no definitive association of this disease with any causative factor except for oral contraceptives in hepatic ehe. the incidence of this tumor is less than 3% of all tumors in the small intestine. due to its variable clinical course, it has been difficult to identify and diagnose this disease in the early phase. computed tomography (ct) scan has shown some promising investigation in identifying this disease, as described in our case. however, final diagnosis can be made only by the aid of histopathological and immunohistochemical studies. due to inability to diagnose this disease in the early stage, it can lead to high mortality and the 5-year survival rate for those patients suffering from ehe is 33.5%. here, we report such a patient in the early stage when searching for the etiology of gastrointestinal tract bleeding by multiphase ct examination. to our knowledge, no similar case has been described in the literature yet. a 47-year - old man was admitted to our hospital with intermittent black stool over the past 2 weeks. the patient denied taking any non - steroidal anti - inflammatory drugs. except for the positive fecal occult blood test and microcytic, hypochromic anemia with decrease hemoglobin values down to 5.5 g / l, all the other laboratory values including tumor markers were in the normal range. an upper gastrointestinal endoscopy and colonic endoscopy did not reveal any cause or site of gastrointestinal tract bleeding. abdominal contrast - enhanced ct scan showed a hypervascular lesion with persistent enhancement and contrast extravasations in the ileum (figs. regional bowel wall thickening associated with a small area of ulceration was found in the ileum about 150 cm proximal to the ileocecal valve, and a short segmental resection was done with a functional end - to - end anastomosis. on histopathological examination, resected specimen was confirmed to be malignant hemangioendothelioma with regional lymphnode metastasis (figs. 4 and 5). he refused for chemotherapy or antiangiogenic agents, and died on the post - operative follow - up of 18 months. ehe is an uncommon endothelial tumor that most frequently arises in soft tissue, liver, lung and skeleton. malignant ehe can be seen in most age groups and has its peak frequency in the 2nd and 3rd decades. the tumor affects males and females equally and develops in whites disproportionately more often than members of other races. although ehe has an equal sex distribution, involvement of the lung and liver is more frequently observed in women. there are no good modalities for diagnosing this disease prior to surgery due to its uncertain presentations. but radiological diagnosis can sometimes be helpful, especially contrast - enhanced ct scan on the basis of its specific constituent components, even if there is no obvious mass formation. ehe is frequently angiocentric and is associated with a medium sized vessel, especially a vein. no definite etiological association has been ascribed to this tumor till now, except an association with oral contraceptives in ehe of liver. injury and radiation therapy has also shown virtual association in the development of this tumor according to few literatures. ehe is pale tan in color, and lacks the red, hemorrhagic appearance of conventional hemangiomas. microscopically, it is poorly defined as an infiltrative tumor, characterized by nests and cords of spindle to epithelioid cells embedded in a hyaline, myxoid, chondroid or collagenous stroma. red blood cells may be present within some of these vacuoles (reminiscent of primitive vascular channels). the tumor cells express the full spectrum of immunohistochemical endothelial markers including factor viii, cd34 and cd31 and, like epithelioid endothelial cells in general, may also exhibit intense and extensive positive staining for keratin and epithelial membrane antigen. the tumor cells usually do not stain with antibodies to s-100 protein, and desmin. differential diagnoses should be kept in mind during establishing the diagnosis which includes other epithelioid vascular neoplasms, particularly epithelioid hemangioma and angiosarcoma. although a standard systemic treatment for malignant ehe has not been fully established, complete surgical excision is strongly recommended if feasible. multifocal lesions may be difficult to excise and may require radiation, chemotherapy or thermal ablation. the results of a small number of single case report of ehe patients treated with cytotoxic chemotherapy, such as doxorubicin or liposomal formulation, have been far from ideal. as new anticancer agents, antiangiogenic agents and an inhibitor of the tyrosine - kinase, such as bevacizumab, lenalidomide, thalidomide and sorafenib, provide encouraging evidence of the potential benefit in the treatment of those patients with ehe. ehe has an overall mortality rate of about 13%, but this can be as high as 65% for the patients the 5-year survival rate for the patient with ehe is only 33.5%, mainly because the condition is often diagnosed late. if our patient would have received chemotherapy or antiangiogenic agents like sorafenib, the patient could have probably lived more than 18 months. it is convincing that ehe carries a grave prognosis due to lack of diagnosis in the early stage however contrast - enhanced ct is helpful in identifying ileal ehe timely in the early stage, even when there is no obvious mass formation. surgical excision in combination with antiangiogenic agents is probably a promising therapy in the future. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. | introductionmalignant epithelioid hemangioendothelioma (ehe) is an uncommon and grave vascular tumor. ehe is frequently angiocentric and is associated with a medium sized vessel, especially a vein. no definite etiological associations have been ascribed to this tumor so far, except an association with oral contraceptives in ehe of liver.presentation of casea 47 year old man presented with the complaint of intermittent black stool over the past two weeks. occasionally, he experienced pain in left lower abdomen. on computed tomography (ct), it showed hypervascular lesion in the ileum with persistent enhancement. an exploratory laparotomy was performed with short segmental resection and functional end - to - end anastomosis. it was diagnosed finally with the histopathological and immunohistochemical analysis as a malignant ehe.discussionehe is an uncommon endothelial tumor that most frequently arises in soft tissue, liver, lung and skeleton. it behaves biologically in between benign epithelioid hemangioma and the more aggressive epithelioid angiosarcoma. although a standard systemic treatment for malignant ehe has not been fully established, complete surgical excision is strongly recommended if feasible.conclusionehe has a variable presentation and ct is helpful in identifying ileal ehe timely in the early stage, even when there is no obvious mass formation, however the diagnosis can be confirmed only after histopathological and immunohistochemical studies. |
strains and plasmids the e. coli strains and plasmids used are described in supplemental table 1, and the primers used for construction of plasmid vectors are described in supplemental table 2. protein overexpression and purification all the proteins used in this study were purified as fusion proteins with a six - histidine tag from e. coli overexpressing strains according to the protocols given in the supplemental material. pulldown assays for pulldown assays, proteins were overexpressed with a c - terminal s - tag for use with the cognate protein that was his - tagged. membrane pellets, dissolved in detergent, or cell supernatant containing the s - tagged prey protein were mixed with 2 mg of purified his - tagged bait - protein. the mixture was loaded onto a ni - charged hitrap chelating column (ge healthcare), so that the bait - protein could be immobilized on the column along with the prey - protein if they interact. the column was then washed with 15 - 20 column volumes of tris buffer containing 75 - 100 mm imidazole and triton x-100 (0.2% (v / v)) to eliminate any false - positive results because of nonspecific interactions. the bait - prey protein complex was eluted with 500 mm imidazole tris buffer, containing triton x-100 (0.2% (v / v)), and sds - page was used to visualize the bait- and prey - proteins. a western blot was performed with anti - s - tag antibodies to confirm the presence of the s - tagged prey - protein. coli cells, of strain kam3(de3), harboring plasmids were grown at 37 c, with shaking at 200 rpm, until the cell density gave an a600 of 0.5, when 0.05 - 0.1 mm isopropyl 1-thio--d - galactopyranoside was added to the cells. the cells were grown for a further 3 h, when the cells were diluted with 2 yt media containing 100 g / ml erythromycin, and the growth curve was recorded over the next 12 h. for experiments to measure the loss in growth because of erythromycin, cells were grown for 3 h in the absence and presence of 50 g / ml erythromycin, and the growth loss is the ratio of the a600 values. analytical ultracentrifugation sedimentation equilibrium measurements were performed using a beckman optima xl - a analytical ultracentrifuge equipped with both absorbance and interference optics. 100 l of macb in buffer 1 (20 mm tris, ph 8.0, 150 mm nacl, 1% w / v glycerol, and 0.006% (w / v) ddm) supplemented with 10, 25, or 50% d2o was placed in the sample compartment of a epon double - sector centerpiece, and 110 l of buffer 1 was placed in the reference compartment. the final protein concentrations used in the runs were between 0.5 and 1.0 mg / ml. the d2o was used to match the density of the solvent to the density of the detergent as described previously (25) the samples were centrifuged at 283 k (10 c) and 10,000, 15,000, and 25,000 rpm using an an60-ti rotor. scans were acquired using the absorbance optical system 15 h after the start of the experiment and in 1-h intervals until equilibrium was attained. sedimentation velocity measurements were performed using the same hardware at 55,000 rpm at 10 c in buffer 1. figure 1.maca interacts with both macb and tolc. a, overexpression and purification of maca, macb, and tolc. an sds - polyacrylamide gel of purified macb (lane 1), maca (lane 2), and tolc (lane 3) is shown. the purified his - tagged macb and tolc proteins were used as bait, immobilized on a ni - agarose column, over which a slurry of either detergent - solubilized membranes (e.g. from strains overexpressing s - tagged maca or tolc) or soluble proteins (e.g. from strains overexpressing s - tagged 20maca) was passed to test whether the cognate proteins from the tripartite pump could be pulled out of this complex mixture of proteins. 1st and 2nd lanes, sds - polyacrylamide gel of immobilized his - tagged macb (1st lane) and the detergent - solubilized membranes from cells overexpressing the s - tagged maca (2nd lane). 3rd to 11th lanes, western blot using antibodies to the s - tag (1:5000 dilution) on maca. the pulldown assay was performed with his - tagged macb immobilized on a ni - agarose column, over which a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed (7th to 9th lanes). the flow - through (9th lane), 100 mm imidazole wash (8th lane), and the 500 mm imidazole elution (7th lane) were tested for the presence of maca, which was now also detected in the elution fraction, indicating that it was bound to macb. a negative control experiment was performed in the absence of immobilized macb in which maca was passed through a ni - agarose column (3rd to 5th lanes), and the flow - through (5th lane), 100 mm imidazole wash (4th lane), and the 500 mm imidazole elution (3rd lane) were tested for the presence of maca, which was only found in the flow - through (5th lane), establishing that s - tagged maca does not bind to the column. these results indicate that macb can pull maca from a complex mixture of membrane proteins. purified his - tagged macb did not cross - react with the antibodies to the s - tag (10th lane). c, pulldown of maca by tolc. an sds - polyacrylamide gel (lanes 1 - 8) for the pulldown of s - tagged maca by his - tagged tolc and the corresponding western blot (lanes 1-8) probed with antibodies (1:5000 dilution) to the s - tag on maca. purified his - tagged tolc was immobilized on a ni - agarose column (lanes 1 and 1) ; a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed through the column and the proteins in the flow - through (lane 2 and 2), released by washing the column with 100 mm imidazole (lanes 3 and 3) and eluted with 500 mm imidazole (lanes 4 and 4), were detected. as a negative control, s - tagged maca was passed through the column (lanes 6 and 6), in the absence of immobilized tolc, and the column was washed with 100 mm (lanes 7 and 7) and 500 mm (lanes 8 and 8). comparing lane 4 and 8 demonstrates that maca is only bound to the column in the presence of tolc, indicative of its interaction with tolc. an sds - polyacrylamide gel (lanes 1 - 4) for the pulldown of s - tagged tolc by his - tagged macb and the corresponding western blot (lanes 1-4) probed with antibodies (1:5000 dilution) to the s - tag on tolc. purified his - tagged macb was immobilized on a ni - agarose column (lanes 1 and 1), and a slurry of detergent - solubilized membranes from cells overexpressing s - tagged tolc was passed through the column (lane 3 and 3), which was then washed with 75 mm imidazole (lanes 4 and 4), and bound proteins were eluted with 500 mm imidazole (lanes 5 and 5). a weak band, which was not present in the absence of immobilized macb, was apparent in lane 5, indicative of a weak interaction between macb and tolc. a control experiment was performed in the absence of immobilized macb in which tolc was passed through a ni - agarose column and the flow - through (lane 6), the 100 mm imidazole wash (lane 7), and the 500 mm imidazole elution (lane 8) were tested for the presence of tolc, which was only found in the flow - through (lane 6), establishing that s - tagged tolc does not bind to the column. an sds - polyacrylamide gel shows the his - tagged macb (lane 1) that was immobilized on a ni - agarose column (lane 1), a slurry of cytoplasmic proteins released by disruption of cells overexpressing s - tagged 20-maca (lane 2), which was passed through the column, over immobilized macb, and the proteins in the flow - through detected (lane 3) ; the proteins were released by washing the column with 100 mm imidazole (lane 4) ; and the proteins were eluted with 500 mm imidazole (lane 5). a western blot was performed on each of the corresponding protein fractions (indicated with 1-5) using antibodies to the s - tag (1:5000 dilution) to detect s - tagged maca. the elution of macb yields an extra, low mr, band on the sds - polyacrylamide gel that corresponds to that expected for maca (lane 5) and was identified as such by western blotting (lane 5). a control experiment was performed in the absence of immobilized macb in which 20maca was passed through a ni - agarose column and the flow - through (lane 7), the first and second washes with 100 mm imidazole (lanes 8 and 9, respectively), and the 500 mm imidazole elution (lane 10) were tested for the presence of maca, which was only found in the flow - through and first wash (lane 7 and 8, respectively), establishing that s - tagged maca does not bind to the column. these results indicate that maca does not require the n - terminal -helix, which anchors it to the inner membrane, to interact with macb. an sds - polyacrylamide gel of purified macb (lane 1), maca (lane 2), and tolc (lane 3) is shown. the purified his - tagged macb and tolc proteins were used as bait, immobilized on a ni - agarose column, over which a slurry of either detergent - solubilized membranes (e.g. from strains overexpressing s - tagged maca or tolc) or soluble proteins (e.g. from strains overexpressing s - tagged 20maca) was passed to test whether the cognate proteins from the tripartite pump could be pulled out of this complex mixture of proteins. b, pulldown of maca by macb. 1st and 2nd lanes, sds - polyacrylamide gel of immobilized his - tagged macb (1st lane) and the detergent - solubilized membranes from cells overexpressing the s - tagged maca (2nd lane). 3rd to 11th lanes, western blot using antibodies to the s - tag (1:5000 dilution) on maca. the pulldown assay was performed with his - tagged macb immobilized on a ni - agarose column, over which a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed (7th to 9th lanes). the flow - through (9th lane), 100 mm imidazole wash (8th lane), and the 500 mm imidazole elution (7th lane) were tested for the presence of maca, which was now also detected in the elution fraction, indicating that it was bound to macb. a negative control experiment was performed in the absence of immobilized macb in which maca was passed through a ni - agarose column (3rd to 5th lanes), and the flow - through (5th lane), 100 mm imidazole wash (4th lane), and the 500 mm imidazole elution (3rd lane) were tested for the presence of maca, which was only found in the flow - through (5th lane), establishing that s - tagged maca does not bind to the column. these results indicate that macb can pull maca from a complex mixture of membrane proteins. purified his - tagged macb did not cross - react with the antibodies to the s - tag (10th lane). c, pulldown of maca by tolc. an sds - polyacrylamide gel (lanes 1 - 8) for the pulldown of s - tagged maca by his - tagged tolc and the corresponding western blot (lanes 1-8) probed with antibodies (1:5000 dilution) to the s - tag on maca. purified his - tagged tolc was immobilized on a ni - agarose column (lanes 1 and 1) ; a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed through the column and the proteins in the flow - through (lane 2 and 2), released by washing the column with 100 mm imidazole (lanes 3 and 3) and eluted with 500 mm imidazole (lanes 4 and 4), were detected. as a negative control, s - tagged maca was passed through the column (lanes 6 and 6), in the absence of immobilized tolc, and the column was washed with 100 mm (lanes 7 and 7) and 500 mm (lanes 8 and 8). comparing lane 4 and 8 demonstrates that maca is only bound to the column in the presence of tolc, indicative of its interaction with tolc. an sds - polyacrylamide gel (lanes 1 - 4) for the pulldown of s - tagged tolc by his - tagged macb and the corresponding western blot (lanes 1-4) probed with antibodies (1:5000 dilution) to the s - tag on tolc. purified his - tagged macb was immobilized on a ni - agarose column (lanes 1 and 1), and a slurry of detergent - solubilized membranes from cells overexpressing s - tagged tolc was passed through the column (lane 3 and 3), which was then washed with 75 mm imidazole (lanes 4 and 4), and bound proteins were eluted with 500 mm imidazole (lanes 5 and 5). a weak band, which was not present in the absence of immobilized macb, was apparent in lane 5, indicative of a weak interaction between macb and tolc. a control experiment was performed in the absence of immobilized macb in which tolc was passed through a ni - agarose column and the flow - through (lane 6), the 100 mm imidazole wash (lane 7), and the 500 mm imidazole elution (lane 8) were tested for the presence of tolc, which was only found in the flow - through (lane 6), establishing that s - tagged tolc does not bind to the column. an sds - polyacrylamide gel shows the his - tagged macb (lane 1) that was immobilized on a ni - agarose column (lane 1), a slurry of cytoplasmic proteins released by disruption of cells overexpressing s - tagged 20-maca (lane 2), which was passed through the column, over immobilized macb, and the proteins in the flow - through detected (lane 3) ; the proteins were released by washing the column with 100 mm imidazole (lane 4) ; and the proteins were eluted with 500 mm imidazole (lane 5). a western blot was performed on each of the corresponding protein fractions (indicated with 1-5) using antibodies to the s - tag (1:5000 dilution) to detect s - tagged maca. the elution of macb yields an extra, low mr, band on the sds - polyacrylamide gel that corresponds to that expected for maca (lane 5) and was identified as such by western blotting (lane 5). a control experiment was performed in the absence of immobilized macb in which 20maca was passed through a ni - agarose column and the flow - through (lane 7), the first and second washes with 100 mm imidazole (lanes 8 and 9, respectively), and the 500 mm imidazole elution (lane 10) were tested for the presence of maca, which was only found in the flow - through and first wash (lane 7 and 8, respectively), establishing that s - tagged maca does not bind to the column. these results indicate that maca does not require the n - terminal -helix, which anchors it to the inner membrane, to interact with macb. mass spectrometry analyses were performed in a nanoflow es mass spectrometer q - tof2 (micromass). the following experimental parameters were used to record mass spectra of 2 mg / ml macb in the q - tof2 instrument : needle voltage of 1.5 kv and mcp 2350 v. atomic force microscopy macb was diluted to a final concentration of 1 g / ml, and 45 l of the sample was allowed to adsorb to freshly cleaved mica. imaging in air was performed with a multimode atomic force microscope (digital instruments, santa barbara, ca) in tapping mode. the silicon cantilevers containing a diamond - like extratip had a drive frequency of 300 khz and a specified spring constant of 40 newtons / m (mikromasch, portland, or), and the applied imaging force was kept as low as possible (target amplitude 1.6 - 1.8 v and amplitude set - point 1.3 - 1.5 v). the molecular volumes of the protein particles were determined from particle dimensions based on afm images (see supplemental material). atpase assays an enzchek phosphate assay kit (invitrogen) was used to determine the atpase activity of macb hydrolyzing mgatp to release phosphate, when the reactants were mixed in a stopped - flow device (see supplemental material). generally, 2.3 m protein was mixed with varying concentrations of atp, up to 4 mm, in the presence of 6 mm mgcl2, to ensure that all the atp was complexed with mg. in control experiments, generally, for macb alone, the hydrolysis of atp was characterized by a pi burst, which was of near equivalence to the macb concentration, consistent with the atpase activity being attributable to macb, rather than any contaminant proteins. quantification of erythromycin binding to affinity - purified mac proteins the equilibrium binding of [n - methyl - c]erythromycin to purified mac proteins was determined by rapid filtration and quantification of the radioactivity remaining on 0.2-m filters as outlined in the supplemental material. maca interacts with both macb and tolc via its periplasmic domain interactions between e. coli maca, macb, and tolc were tested using detergent - solubilized proteins for pulldown assays (fig. 1b), which we confirmed by cross - linking the proteins (supplemental fig. n - terminal truncated maca (20-maca) interacted with macb (fig. 1e), indicating that it is the periplasmic domains of these proteins that interact. the fact that in each case the cognate pump protein could be pulled out of a complex mixture of detergent - solubilized proteins from membranes or cells indicated that the interactions are specific. figure 2.macab-tolc form a tripartite complex that confers resistance to erythromycin. a, growth curves for e. coli cells, of strain kam3 (de3), harboring the plasmids petduet (), petduet - macb (), petduet - macb / maca (), petduet - macb / tolc (), petduet - macb / maca / tolc () and petduet - macb / giii - ss-20maca / tolc () grown in the presence of 100 g / ml erythromycin. b, bar chart showing the extent of inhibition of the growth of e. coli cells in response to 50 g / ml erythromycin, of strain kam3(de3), harboring the plasmids petduet (blank), petduet - macb (macb), petduet - macb / maca (macab) or no plasmid (wild). for each strain the a600 was determined after growth for 3 h in the absence and presence of erythromycin, and the growth inhibition was determined as the ratio of these measurements. cells expressing both maca and macb suffered less from erythromycin growth inhibition than those expressing only macb, suggesting that maca confers elevated resistance to erythromycin on the macb strain. macab - tolc form a tripartite complex that confers resistance to erythromycin. a, growth curves for e. coli cells, of strain kam3 (de3), harboring the plasmids petduet (), petduet - macb (), petduet - macb / maca (), petduet - macb / tolc (), petduet - macb / maca / tolc () and petduet - macb / giii - ss-20maca / tolc () grown in the presence of 100 g / ml erythromycin. b, bar chart showing the extent of inhibition of the growth of e. coli cells in response to 50 g / ml erythromycin, of strain kam3(de3), harboring the plasmids petduet (blank), petduet - macb (macb), petduet - macb / maca (macab) or no plasmid (wild). for each strain the a600 was determined after growth for 3 h in the absence and presence of erythromycin, and the growth inhibition was determined as the ratio of these measurements. cells expressing both maca and macb suffered less from erythromycin growth inhibition than those expressing only macb, suggesting that maca confers elevated resistance to erythromycin on the macb strain. e. coli maca and macb form a functional complex with tolc the simultaneous expression of maca, macb, and tolc in the e. coli acrab strain kam3 (26) conferred resistance to erythromycin, indicative of the formation of a functional complex (fig. cells expressing macb with tolc conferred modest resistance to erythromycin in comparison with cells expressing macb, tolc, and maca, indicating that maca is required to couple macb to tolc (fig. we sought to test whether the n terminus of maca, which incorporates an -helix that could interact with macb, is required for the functional assembly of the complex. a construct in which the giii - signal sequence was fused to truncated maca, targeting it to the periplasm, was capable of conferring resistance to erythromycin (fig. 2a), indicating that the n - terminal domain is not essential for the assembly of the functional complex. this is consistent with a report that a truncated lipid - deficient acra derivative was functional as judged by resistance of the cells to erythromycin (27). macb alone conferred elevated resistance to erythromycin, probably because of its ability to pump the antibiotic into the periplasm, but we consistently found that expressing macb with either tolc or maca conferred greater resistance. consequently, we sought to test if maca could enhance this ability. to overcome the difficulty in comparing the growth of cells overexpressing multiple proteins that tend to grow at different rates, we monitored the growth of cells in the presence and absence of erythromycin and determined the growth loss (for cells growing in the presence of erythromycin in comparison with cells growing in the absence of erythromycin) (fig. this revealed a significant loss in growth of the cells expressing macb compared with those expressing macab, indicating that the simultaneous expression of maca and macb increases the resistance of the cells to erythromycin (fig. 2b), suggesting that maca enhances the ability of macb to confer antibiotic resistance. similarly, a previous study reported that macab, but not macb alone, conferred resistance to macrolides (3). macb forms dimers macb has an atypical structure for an abc transporter as it is predicted to have an n - terminal cytoplasmic nbd, which is connected to a four - helix transmembrane domain, with a large periplasmic domain formed by the loops connecting helices 1 and 2 (3, 23). if macb resembles other abc transporters that use a pair of nbds to bind atp, then it should function as a dimer. however, many transporters, including abc transporters, have 12 membrane - spanning helices ; macb could adopt a similar topology by forming trimers. furthermore, acrb (4, 5) and tolc (9), which assemble into a tripartite complex with acra, clearly form trimers. if the trimeric arrangement of the periplasmic domains in acrb forms a necessary scaffold for binding of acra, so that it can effectively interact with tolc, then by analogy the periplasmic domain of macb might also be forced into forming trimers when interacting with maca and tolc. size - exclusion chromatography indicated that it forms higher order oligomers consistent with a dimer (data not shown), but such measurements are not only dependent upon the molecular weight but also the shape of the protein. furthermore, there is a need to determine the number of detergent molecules complexed by the protein. consequently, to determine whether the detergent - solubilized macb was dimeric, we added a cross - linker to trap the oligomers ; when we ran the cross - linked protein on an sds - polyacrylamide gel, the most predominant band ran between the 120- and 160-kda markers, indicative of a dimer, which has a calculated molecular mass of 145.8 kda (supplemental fig. a representative sedimentation equilibrium profile from one of the runs (two different velocities of the same sample) is shown. experimental data (dots) and fitted model for a 162.6-kda particle (solid line) is shown for each. b, auc sedimentation velocity profiles of macb are consistent with the formation of a stable dimer. the upper panel shows sedimentation profile curves at different time points, and the lower panel presents a c(s) size distribution analysis with solutions of the lamm equation. the sedimentation coefficient is 6.8 s corresponding to an apparent molecular mass of 160 kda, consistent with a dimer with about 16 detergent molecules bound. the mass spectrum indicated a molecular mass for macb of 145.96 kda, which is consistent with a dimer. a representative sedimentation equilibrium profile from one of the runs (two different velocities of the same sample) is shown. experimental data (dots) and fitted model for a 162.6-kda particle (solid line) is shown for each. b, auc sedimentation velocity profiles of macb are consistent with the formation of a stable dimer. the upper panel shows sedimentation profile curves at different time points, and the lower panel presents a c(s) size distribution analysis with solutions of the lamm equation. the sedimentation coefficient is 6.8 s corresponding to an apparent molecular mass of 160 kda, consistent with a dimer with about 16 detergent molecules bound. the mass spectrum indicated a molecular mass for macb of 145.96 kda, which is consistent with a dimer. to further confirm the basic oligomeric unit as a dimer, we used two other techniques, analytical ultracentrifugation (auc) and electrospray mass spectrometry (es - ms). for the auc experiments, we reduced the ddm concentration to just below the critical micelle concentration to avoid the formation of micelles. to determine the detergent contribution in the buoyant mass of the protein - detergent complex, we used a series of different density buffers prepared using a range of d2o concentrations. the apparent molecular mass for macb was determined from sedimentation equilibrium measurements to be 162.6 kda (fig. 3a) and from sedimentation velocity measurements to have a sedimentation coefficient of 6.8 s, corresponding to a molecular mass of 160.0 kda (fig. 3b). this molecular mass is greater than expected for monomeric and less than expected for trimeric macb, complexed with bound detergent, but it is highly consistent with a macb dimer to which about 16 ddm molecules are bound. although the amount of detergent bound to the macb dimer appears to be lower than reported for rnd (28) and mf (29) transporters, this reflects the fact that in our experimental set - up the detergent contribution was actively suppressed using a solvent density matching technique (25). our independent measurement of bound detergent using a calorimetric assay (30) indicated that, when the detergent concentration was close to the critical micelle concentration, the amount of bound detergent was similar to that of other membrane proteins (e.g. macb solubilized in 0.05% w / v ddm bound 1.2 g of ddm / g of macb, which is equivalent to a ddm : macb molar ratio of 164:1). electrospray - mass spectrometry (es - ms) was used in nontandem configuration to determine accurately the molecular mass of the protein, under conditions that would dissociate the ddm, revealing a peak with a molecular mass of 145,961.25 20.57 da that is consistent with a macb dimer (fig. two populations were revealed with average molecular volumes of 118 and 238 nm (fig. these would accommodate proteins of about 60 - 70 and 130 - 140 kda, respectively. furthermore, the larger particles could clearly be seen at higher resolution to consist of two protein domains that are highly suggestive of a dimer. we found that in the presence of amp - pnp, a nonhydrolysable analogue of atp, the ratio of dimers to monomers on the afm grids increased (fig. 4c), which would be consistent with the nucleotide stabilizing the interaction between monomers. our findings are novel because detergent molecules tend to impair the resolution of afm studies of detergent - solubilized membrane proteins ; this implies that for macb, we may be able to get information on the topology and stoichiometry of its assemblies with maca and tolc. indeed, under coincubation of maca and macb, we could clearly distinguish a significant distribution of particles with molecular volumes larger than those corresponding to macb dimers, which is consistent with multiprotein complexes formed between both proteins (data not shown). such promising data paves the way toward further characterization of membrane multiprotein complexes and could prove a powerful instrument for determining the stoichiometry of the tripartite assembly. maca regulates the atpase activity of macb macb retained atpase activity when detergent - solubilized, but this was detergent - dependent, being active in triton x-100 but not in ddm (data not shown). the time course for hydrolysis of mgatp by macb was determined in a stopped - flow spectrophotometer, using the dye 2-amino-6-mercapto-7-methylpurine riboside to monitor the production of inorganic phosphate (pi). the time course was characterized by a burst in pi production, during the first 20 s, followed by a slower steady - state rate (fig. this kinetic behavior is consistent with the atp being rapidly hydrolyzed, to produce pi and adp, but further turnovers are rate - limited either by a subsequent conformational change or the slow release of products. because previous studies have established that the atpase activity of macb is inhibited by vanadate (24), which stabilizes bound adp, this indicates that pi is released before adp, suggesting that adp release is rate - limiting. the rate constant for the hydrolysis step was determined by fitting the burst phase to an exponential function, yielding a kcat value of 0.24 s, whereas the amplitude of the burst phase was 2.2 m for 1 mm atp (fig. when the maca and macb concentrations were increased to 3.5 m, the amplitude of the burst phase increased to 3.3 m (data not shown), indicating that the burst is approximately equivalent to the macb concentration and that both nbds within the macb dimer are functional. we did not notice any deviation from a single exponential that would indicate that these nbds turn over atp differentially. the steady - state phase was characterized by a rate of pi production that increased in a hyperbolic manner with the atp concentration (fig. 5b) ; fitting the steady - state rate data to a hyperbolic function yielded a maximal specific activity of 8.9 nmol of atp / min / mg macb and a km of 374 m. a progressive reduction in the amplitude of the burst phase for atp concentrations below the steady - state km value hindered analyses because the pre - steady - state phase tended to merge with the steady - state phase ; consequently, we only used the steady - state rates determined at atp concentrations of 0.1 mm and above. for comparison, the lipid a transporter msba, an half - abc transporter, was characterized by a vmax of 37 nmol of atp / min / mg and a km of 878 m (31). figure 4.afm analyses, afm imaging of macb. a, three - dimensional picture of a low magnification image of macb acquired in air in tapping mode with a diamond - like extra tip of resonant frequency 300 khz and spring constant of 40 newtons / m. m and d show particles that belong to the first and second peak in b, respectively. b, frequency distribution of molecular volumes of macb. the curve indicates a fitted gaussian function. the m and d peaks correspond to volumes of 118 1 nm (n = 1642) and 238 5 nm (n = 665), consistent with the monomer and dimer, respectively. c, frequency distribution of molecular volumes of macb that had been incubated with the nonhydrolysable atp analogue amp - pnp. the peaks correspond to volumes of 112 3 nm (n = 94) and 218 14 nm (n = 116). these data indicate an increase in the dimer : monomer ratio in the presence of amp - pnp. d, high resolution images of structures where two small particles (m+m) are attached to one another, clearly indicative of dimer formation. afm analyses, afm imaging of macb. a, three - dimensional picture of a low magnification image of macb acquired in air in tapping mode with a diamond - like extra tip of resonant frequency 300 khz and spring constant of 40 newtons / m. m and d show particles that belong to the first and second peak in b, respectively. b, frequency distribution of molecular volumes of macb. the curve indicates a fitted gaussian function. the m and d peaks correspond to volumes of 118 1 nm (n = 1642) and 238 5 nm (n = 665), consistent with the monomer and dimer, respectively. c, frequency distribution of molecular volumes of macb that had been incubated with the nonhydrolysable atp analogue amp - pnp. the peaks correspond to volumes of 112 3 nm (n = 94) and 218 14 nm (n = 116). these data indicate an increase in the dimer : monomer ratio in the presence of amp - pnp. d, high resolution images of structures where two small particles (m+m) are attached to one another, clearly indicative of dimer formation. when maca was added to macb, at an equivalent or higher concentration, with both proteins in detergent, 5a). considering that the detergent was present for both the atpase assays with macb and macab suggests that the burst phase is mechanistically important and can not be attributed to the detergent modifying the behavior of macb. interestingly, although there was no pi burst by macb in the presence of maca, there was a lag in pi production ; this could signify that a conformational change that precedes the hydrolysis step becomes rate - limiting for the first turnover. the steady - state rate of atp hydrolysis by macb, in the presence of maca, was enhanced (fig., this behavior might appear consistent with maca increasing the rate of adp dissociation from macb, so that this step was no longer rate - limiting. however, although maca increased the specific activity of macb from 8.9 to 12.3 nmol of atp / mg macb / min, which would correspond to an increase in kcat from 0.011 to 0.015 s, the steady - state rate did not exceed the rate for hydrolysis in the absence of maca (e.g. 0.24 s) (fig., the increase in the steady - state rate, which we attribute to product release, or a conformational change preceding this, would not be rapid enough to prevent the phosphate burst. it seems more likely that maca also retards the rate of atp hydrolysis, so that this process becomes slower than the rate of product release. indeed, we found that the affinity of macb for atp was increased by more than 5-fold, from 374 to 72 m, in the presence of maca (fig. if the effect of maca was simply to enhance product release to a rate faster than that for nucleotide hydrolysis, then we would have expected a decrease in affinity for atp. on the other hand, if maca simply retarded nucleotide hydrolysis to a rate slower than product release, then the maximal steady - state rate would have decreased. our data are consistent with maca increasing the rate of product release, while decreasing the rate of hydrolysis to a similar rate to product release. in contrast to a previous study, which indicated that the atpase activity of reconstituted macb was not activated by n - terminal truncated maca (24), we found that when macb was mixed with 20-maca no pi burst was apparent (data not shown). in the same study, and consistent with our findings, n - terminal truncated maca was shown to interact with macb ; however, in contrast to our findings, when macb was expressed with n - terminal truncated maca in erythromycin - susceptible cells, there was no increase in erythromycin resistance (24). the reason for the difference with our own findings is unclear ; however, in the previous study the signal sequence of ompa was used to target maca, truncated at position 32, to the periplasm (24), raising the possibility that the shorter maca sequence and/or the ompa signal sequence interfered with the ability of maca to affect the atpase activity of macb. figure 5.maca regulates the atpase activity of macb. a, time course for the change in pi concentration, corresponding to the absorbance change of the 2-amino-6-mercapto-7-methylpurine riboside in a, where 2.3 m macb was mixed with 1 mm atp in the absence (upper trace) and presence (lower trace) of an equivalent concentration of maca. in the absence of maca, macb produced a phosphate (pi) burst, with a rate and amplitude of 0.235 (0.001) s and 2.20 (0.01) m, respectively. b, steady - state rate of pi production by macb as a function of the atp concentration in the absence (lower curve) and presence (upper curve) of an equivalent concentration of maca. the data are characterized by vmax and km values of 8.9 (0.7) nmol of atp / mg macb / min and 374 (126) m, respectively, for macb alone ; and of 12.3 (0.5) nmol of atp / mg macb / min and 72 (22) m, respectively, for macb in the presence of an equivalent concentration of maca. maca regulates the atpase activity of macb. a, time course for the change in pi concentration, corresponding to the absorbance change of the 2-amino-6-mercapto-7-methylpurine riboside in a, where 2.3 m macb was mixed with 1 mm atp in the absence (upper trace) and presence (lower trace) of an equivalent concentration of maca. in the absence of maca, macb produced a phosphate (pi) burst, with a rate and amplitude of 0.235 (0.001) s and 2.20 (0.01) m, respectively. b, steady - state rate of pi production by macb as a function of the atp concentration in the absence (lower curve) and presence (upper curve) of an equivalent concentration of maca. the data are characterized by vmax and km values of 8.9 (0.7) nmol of atp / mg macb / min and 374 (126) m, respectively, for macb alone ; and of 12.3 (0.5) nmol of atp / mg macb / min and 72 (22) m, respectively, for macb in the presence of an equivalent concentration of maca. purified proteins (50 g of maca or macb, or 25 g of maca plus 25 g of macb) were incubated in the presence of [n - methyl - c]erythromycin at concentrations as indicated (1, 5, or 10 m), after which drug binding was measured by rapid filtration. the bars represent the erythromycin bound by maca (left, black), macb (middle, light gray), and macab (right, dark gray). purified proteins (50 g of maca or macb, or 25 g of maca plus 25 g of macb) were incubated in the presence of [n - methyl - c]erythromycin at concentrations as indicated (1, 5, or 10 m), after which drug binding was measured by rapid filtration. the bars represent the erythromycin bound by maca (left, black), macb (middle, light gray), and macab (right, dark gray). the data indicate that maca enhances the binding of erythromycin to macb. in accord with previous studies (24), we could not detect an effect of erythromycin on the kinetics of macb atpase either in the absence or presence of maca (data not shown). recent studies have established that the atpase activity of pdr5 is uncoupled from substrate binding ; this basal atpase activity might be required to constantly cycle the transporter between conformations, so as to maintain the accessibility of the cytosolic substrate - binding site (32). in the case of macb, its atpase activity might instead be stimulated by tolc, to reset its conformation following drug transfer to tolc. maca increases the capacity of macb to bind erythromycin although we could not detect any effect of erythromycin on the atpase activity of macb, we could detect the binding of [c]erythromycin to detergent - solubilized macb (fig. importantly, we found that the macab complex bound more erythromycin than macb alone (which binds considerably more than maca alone) and that the amount bound increased in a concentration - dependent manner, as would be expected if maca increased the affinity of macb for erythromycin. however, because of the insolubility of the antibiotic in aqueous solutions, it was not possible to test a full range of erythromycin concentrations that might saturate macab. consequently, we can not exclude the possibility of the formation of additional sites within the macab complex that are not apparent in either macb or maca alone. our data indicate that maca not only modulates the atpase activity of macb but also enhances its capacity to bind erythromycin (be this due to an increase in affinity of macb for drugs or the formation of additional drug - binding sites within the macab complex). gram - negative bacteria possess tripartite pumps that facilitate the extrusion of protein toxins and cytotoxic compounds, such as antibiotics, from the cell. in these tripartite assemblies the imp is coupled to an omp by a periplasmic mfp, forming a pump that can translocate molecules across both the inner and outer membranes. intriguingly, gram - positive bacteria that lack an outer membrane also possess mfps, suggesting that they play a role in addition to stabilizing the interaction of the imp with the omp. indeed, we have noted in some of these mfps a large deletion corresponding to the coiled - coil hairpin that would interact with the omp (supplemental fig., we sought to determine the role of maca, the mfp that couples the imp macb, an atp - driven transporter, with the omp tolc in e. coli to extrude macrolide antibiotics (3). we established that maca interacts with both macb and tolc (fig. 1) to form a functional tripartite complex (fig. however, we also found that maca enhanced the resistance to erythromycin conferred by macb alone (fig. 2b), suggesting that it modulated the transport activity of macb, which is consistent with our data demonstrating that maca regulates the drug binding and atpase activity of macb (figs. 5 and 6). the role of the mfp besa in the activity of the besabc pump in borrelia burgdorferi, the causative agent of lyme disease, has recently been highlighted (33). it is quite remarkable that in this system besa also lacks the -helical hairpin. as such this periplasmic protein is unable to unlock the periplasmic entry site of the omp, a function that is attributed to the hairpin (11). to compensate, the omp has evolved to be constitutively leaky. the reason for the retention of besa in the borrelia system is most likely associated with its function in the activation of the inner membrane component, in that case an rnd transporter. it is conceivable then, that a similar role is also present in the abc transporters and their associated mfps, and it could explain the preservation of the hairpin - lacking mfps in gram - positive bacteria, such as s. aureus (supplemental fig. although e. coli macb has an nbd, which incorporates walker a and b motifs and an abc signature sequence that are characteristic features of members of the abc superfamily, it is not a classic abc transporter. it has a large periplasmic domain, reminiscent of that found in rnd transporters, which form trimers in which these domains form substantive sites of contact between the protomers. consequently, we sought to determine the oligomeric state of macb and in doing so have developed a novel es - ms approach to unambiguously establish that macb forms dimers (fig. es - ms is now widely accepted as a powerful method to determine accurately the stoichiometry of intact protein complexes (34). however membrane proteins, solubilized by detergent or adsorbed in micelles, have remained difficult to analyze under similar ms conditions. the development of strategies to tackle this field is challenging, primarily because the large quantities of detergent suppress the protein signal, whereas the poor solubility of membrane proteins in aqueous buffers often causes the electrospray needle to block. to date only a few ms studies have reported the observation of membrane proteins or their complexes by ms (35 - 39). here we report on the use of a miniaturized form of es with reduced flow rates (nano - es), and a high collision energy that facilitates the desolvation process and induces dissociation of detergent - proteins clusters, to determine the oligomeric state of an integral homomeric membrane protein. in our protocol we used lower quantities of ddm, than have been reported previously, without apparent detrimental effects on the stability of the protein. under our experimental conditions, we have successfully maintained the noncovalent subunit interactions, such that the oligomeric state of the protein complex could be determined without ambiguity. as would be expected for an abc transporter in which the nbds interact, our biophysical studies of macb indicate that it forms dimers (figs. 3 and 4 and supplemental fig.. how does dimeric macb interact with trimeric tolc and the number of maca molecules needed to stabilize the tripartite complex ? extension of our afm studies will be vital for determining the stoichiometry of the interactions to provide an understanding of the assembly of the tripartite complex, which may be difficult to address by other methods, such as crystallization of the complex. our findings and previous studies, which have shown that disruption of the walker a and b motifs not only inhibits the atpase activity but also blocks the capacity of macb to confer macrolide resistance (24), suggest that macb operates by a similar mechanism to typical abc transporters. our understanding of how abc transporters couple atp hydrolysis to transport is still rudimentary, but the determination of the crystal structures of several atpase subunits and complete abc transporters has suggested conservation of key steps in the molecular mechanism. the binding of atp to both soluble atpase subunits (40), solubilized nbds from abc transporters (41, 42), and to the nbds within a complete abc transporter (43, 44) has been shown to promote dimerization as the atp is bound at the interface of these nucleotide - binding sites, sandwiched between the walker a motif of one nbd and the abc signature motif of the other nbd. our afm studies indicated that there is a higher proportion of macb dimers in the presence of the nonhydrolysable nucleotide amp - pnp (fig., the binding of atp causes the transporter to adopt a conformation in which the substrate - binding site is outward - facing, because atp bridges the two nbds, closing off the inward - facing substrate - binding site (45). conversely, the release of the hydrolysis products adp and phosphate is thought to promote an inward - facing conformation, as the structural constraint imposed by binding of the nucleotide is released. consistent with this proposal, several studies indicate that nbd dimerization can not be induced by adp (46), because interactions between the -phosphate of atp and the signature sequence catalyze these events (47). 5), and so our kinetic data have clear implications in terms of such a mechanism. most significantly, maca increases the apparent affinity of macb for atp, while decreasing the rate of atp hydrolysis, so as to promote and stabilize the atp binding conformation, which we presume to be the conformation in which the antibiotic - binding site is outward - facing. in this manner, maca would play a direct role in driving antibiotic translocation between macb and tolc. during the course of our studies another investigation reported on the effect of maca on the steady - state atpase activity of macb (24). in this study, macb, solubilized in triton x-100, appeared to have a specific activity that was an order of magnitude higher than we had found ; however, a discontinuous assay was used to determine the atpase activity, and this could well have been influenced by pooling the time points from both the burst and steady - state phases. indeed, kcat was marginally slower than the hydrolysis rate determined from the pi burst phase in our experiments (e.g. 0.17 versus 0.24 s). possibly for similar reasons, this assay did not detect the effect of maca on macb solubilized in triton x-100. however, they did find that macb reconstituted into liposomes was characterized by a reduced kcat (e.g. kcat = 0.10 s) and decreased affinity of macb for atp (e.g. km = 2.30 mm). co - reconstitution of macb with maca had the effect of increasing both kcat (e.g. kcat = 0.78 s) and the affinity of macb for atp (e.g. km = 0.38 mm). this behavior is similar to the effect of maca on the steady - state kinetics of the atpase activity of solubilized macb in our study. to know if the reconstituted protein behaves in an identical manner to the solubilized protein would require the reconstitution of sufficient amounts of macb to define any pi burst, which is technically demanding. consequently, although these earlier studies support our conclusion that maca affects the atpase activity of macb, they do not give the detailed insight into the atpase mechanism provided by our pre - steady - state analyses. our studies established that conformational changes can be propagated from the periplasmic domain of maca to the cytoplasmic nbd of macb (figs. 1 and 2), and consequently, it is plausible that tolc, by interacting with maca, can detect the nucleotide state of macb. the binding of atp to macb would stimulate maca to interact with tolc, inducing the latter to adopt the open state. because atp and maca stabilize in the outward - facing conformation of macb, drug transfer from macb to tolc would be facilitated. tolc would then communicate with the nbd of macb (again, such communication is probably conveyed via maca) to stimulate atp hydrolysis, which would be required to reset macb in the inward - facing conformation. if atp hydrolysis is controlled by tolc, so that the macb conformation is only reset after the interaction with tolc and productive transfer of drugs, this would provide an explanation of the apparent insensitivity of the atpase activity of macb to drugs. indeed, without such a feedback control mechanism of drug export, it would appear that drug stimulation of the atpase activity of macb would be counter - productive because the macb conformation could be reset before drug transfer to tolc. there is an analogy in the mechanism of operation of abc transporters that work in conjunction with a periplasmic binding protein (47). in the e. coli maltose transporter, the binding of atp to the atpase subunit malk induces conformational changes, detected by tryptic digestion, in the periplasmic loops of the membrane subunits malf and malg (48), whilst epr studies revealed that atp binding, but not adp binding, caused an increase in affinity between the transporter and the maltose - binding protein (mbp), and forcing open the bound mbp to release its substrate (49). the transporter is reset in its original inward - facing conformation by atp hydrolysis, which is stimulated by the mbp (50). in our model tolc replaces the mbp and, because the open state requires disruption of the second selectivity filter and subsequent twisting of the helices of the tolc channel to keep it fully open, there is a need for the maca hairpins to stabilize this conformation, which could not be achieved by interaction with macb alone. although we have interpreted our findings in terms of an alternating conformation model for macb, in which the drug - binding sites are inwardly and outwardly exposed, such a model could easily be refined to account for drug binding to a fixed periplasmic site, in analogy to acrb, with atp binding and hydrolysis coupled to conformational changes that induce tolc association and dissociation. clearly the work presented here provides an important framework for further studies that will enable the elucidation of the mechanism underlying the dynamics of assembly of the macabtolc tripartite pump in the future. | gram - negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (imp), a periplasmic membrane fusion protein, and an outer membrane channel. we have investigated the assembly and function of the macab / tolc system that confers resistance to macrolides in escherichia coli. the membrane fusion protein maca not only stabilizes the tripartite assembly by interacting with both the inner membrane protein macb and the outer membrane protein tolc, but also has a role in regulating the function of macb, apparently increasing its affinity for both erythromycin and atp. analysis of the kinetic behavior of atp hydrolysis indicated that maca promotes and stabilizes the atp - binding form of the macb transporter. for the first time, we have established unambiguously the dimeric nature of a noncanonic abc transporter, macb that has an n - terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. structural studies of abc transporters indicate that atp is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate - binding site is outward - facing. consequently, our data suggest that in the presence of atp the same conformation of macb is promoted and stabilized by maca. thus, maca would facilitate the delivery of drugs by macb to tolc by enhancing the binding of drugs to it and inducing a conformation of macb that is primed and competent for binding tolc. our structural studies are an important first step in understanding how the tripartite complex is assembled. |
self - renewal activity and the ability to differentiate into various functional cells are two major characteristics of stem cells. stem cells have thus been considered among the therapeutic biomaterials of use in regenerative medicine, which can restore the function of damaged cells, tissues, or organs. before clinical application, however, a number of difficulties in manipulating stem cells should be overcome, and a variety of alternative technologies have been suggested to control stem cell fate and function. recent studies have demonstrated that conventional stem cell technologies do not meet the minimal requirements to precisely regulate stem cell function. to overcome this limitation, several scientists have taken an interest in regulating stem cells under a specific microenvironment, which uses artificial extracellular matrices (ecms) to control the stem cell self - renewal and differentiation activity. the use of artificial ecms for creating three - dimensional (3d) microenvironments would yield several benefits, mimicking the in vivo microenvironment of the undifferentiated or differentiated period at both the cell and tissue levels. biophysical microenvironments, including the stiffness of the substrate, nanotopography of the adhesion surface, microgeometric forces, and extracellular forces, must be constructed prior to the use of this 3d system, and the influence of stem cell fate in vitro through this artificial microenvironment can efficiently prevent the direct genetic manipulation of stem cells, which would otherwise limit the feasibility of clinical application [5 - 8 ]. having access to artificial ecms thus accelerates niche - related studies on manipulating stem cell self - renewal and differentiation ; the data obtained from 3d cultures can be comparable to those obtained from conventional two - dimensional (2d) cultures. as an initial step, most studies have employed embryonic stem cells (escs) in both experimental animals and humans. however, data from the culture of stem cells under conventional 2d systems provide a wealth of information that should assist in the development of innovative 3d culture systems. therefore, careful consideration of stem cell culture in 2d systems is a prerequisite for developing 3d culture systems. escs have generally been co - cultured with feeder cells such as xenogenic embryonic fibroblasts to maintain self - renewal activity. the feeder cells used for stem cell culture are able to supply growth factors, cytokines, and other extracellular matrix components such as leukemia inhibitory factor (lif), activin, wnt, bone morphogenetic proteins (bmps), insulin - like growth factor (igf), laminin, and vitronectin for maintaining the undifferentiated state of escs, which can create a suitable 2d environment. when applying these 2d systems to human escs, animal - derived feeder cells can cause unexpected disadvantages such as uncertain data outcomes and xenotransmission of unknown pathogens. to avoid these handicaps, studies on the development of feeder - free cultures and defined culture systems have been strongly encouraged. since the initial studies of richards., human feeder cells such as neonatal foreskin fibroblasts, fetal muscle, fetal skin, adult fallopian tube epithelial cells, adult muscle, adult skin, marrow - derived stromal cells, amniotic fluid fibroblasts, placenta - derived fibroblasts, and human esc (hesc)-derived fibroblasts have been employed to provide a suitable cellular niche for hescs without the use of xenogenic cells. a positive outcome of the replacement was reported, although batch differences in feeder cell - based culture methods is considered another uncertainty that impedes the establishment of stable culture conditions. to further develop defined stem cell culture systems, the use of non - cellular niches can subsequently be considered. an initial attempt has been made to employ non - cellular niches for the development of defined esc culture systems, and artificial ecms were thus employed. instead of feeder cells, xu. escs were successfully cultured with the use of fibroblast - conditioned medium, to which co - culture technology was applied for the culture of other stem cell lines. each medium conditioned with different cells had a different capacity to maintain esc self - renewal. however, some showed negative results in attempts to support the long - term culture of escs, which demonstrates their unsuitability as a standardized culture regime. such a limitation directly encourages the development of a defined culture medium and the refinement of escs for suitable esc culture methods. first reported the successful use of knockout serum for esc culture as a replacement for bovine serum, although this semi - defined serum replacement contains xenogenic or undefined substances such as bovine serum albumin. subsequently, a culture medium containing human - originated recombinant proteins was developed (x - vivo 10 medium). as suitable supplements for hesc culture, basic fibroblast growth factor (b - fgf), stem cell factor (scf), recombinant human fms - like tyrosine tesr1 using recombinant proteins and purified material from humans was also suggested as a culture supplement for hesc culture. discovery of the alternative material matrigel was developed and the single use of laminin, one of the matrigel components, was successful for the culture of undifferentiated hescs. it has also been reported that laminin isoforms 111, 332, and 511, vitronectin, and e - cadherin support the adhesion and proliferation of hescs [27 - 29 ]. escs have generally been co - cultured with feeder cells such as xenogenic embryonic fibroblasts to maintain self - renewal activity. the feeder cells used for stem cell culture are able to supply growth factors, cytokines, and other extracellular matrix components such as leukemia inhibitory factor (lif), activin, wnt, bone morphogenetic proteins (bmps), insulin - like growth factor (igf), laminin, and vitronectin for maintaining the undifferentiated state of escs, which can create a suitable 2d environment. when applying these 2d systems to human escs, animal - derived feeder cells can cause unexpected disadvantages such as uncertain data outcomes and xenotransmission of unknown pathogens. to avoid these handicaps, studies on the development of feeder - free cultures and defined culture systems have been strongly encouraged. since the initial studies of richards., human feeder cells such as neonatal foreskin fibroblasts, fetal muscle, fetal skin, adult fallopian tube epithelial cells, adult muscle, adult skin, marrow - derived stromal cells, amniotic fluid fibroblasts, placenta - derived fibroblasts, and human esc (hesc)-derived fibroblasts have been employed to provide a suitable cellular niche for hescs without the use of xenogenic cells. a positive outcome of the replacement was reported, although batch differences in feeder cell - based culture methods is considered another uncertainty that impedes the establishment of stable culture conditions. to further develop defined stem cell culture systems, the use of non - cellular niches can subsequently be considered. an initial attempt has been made to employ non - cellular niches for the development of defined esc culture systems, and artificial ecms were thus employed. instead of feeder cells, xu. escs were successfully cultured with the use of fibroblast - conditioned medium, to which co - culture technology was applied for the culture of other stem cell lines. each medium conditioned with different cells had a different capacity to maintain esc self - renewal. however, some showed negative results in attempts to support the long - term culture of escs, which demonstrates their unsuitability as a standardized culture regime. such a limitation directly encourages the development of a defined culture medium and the refinement of escs for suitable esc culture methods. first reported the successful use of knockout serum for esc culture as a replacement for bovine serum, although this semi - defined serum replacement contains xenogenic or undefined substances such as bovine serum albumin. subsequently, a culture medium containing human - originated recombinant proteins was developed (x - vivo 10 medium). as suitable supplements for hesc culture, basic fibroblast growth factor (b - fgf), stem cell factor (scf), recombinant human fms - like tyrosine kinase 3 ligand (rhflt3l), and lif were recommended. tesr1 using recombinant proteins and purified material from humans was also suggested as a culture supplement for hesc culture. discovery of the alternative material matrigel was developed and the single use of laminin, one of the matrigel components, was successful for the culture of undifferentiated hescs. it has also been reported that laminin isoforms 111, 332, and 511, vitronectin, and e - cadherin support the adhesion and proliferation of hescs [27 - 29 ]. among 3d scaffolds being constructed with various biocompatible biomaterials, hydrogel - based ecms have been employed for the culture of stem cells. the hydrogel is a cell - friendly, 3d macromolecule platform formed by the crosslinking of hydrophilic polymers, which can absorb h2o molecules to over five hundred times its own weight. in response to the salt concentration, ph, temperature, and crosslinking method, the chemical structure of the hydrogel can change in unlimited ways, so many biomedical and pharmaceutical applications may be possible, such as drug delivery, artificial tissue, membrane fabrication, and matrix construction. to date, hydrogel - based, protein - like materials such as matrigel, collagen, fibrin, and silk, polysaccharide - like materials such as hyaluronic acid, alginate, dextran, chitosan, and agarose, and dna have been used. in most of these cases, however, difficulties with manipulating their mechanical properties, the degradation rate, and the reproducibility occurred due to their undefined protein composition, which limited the modification of binding domains or sites in cellular activity and immune responses. to overcome this limitation, a hydrogel - based, synthetic (chemically defined) material has subsequently been suggested, which has a number of advantages including easy manipulation of mechanical properties, degradation, shape, and reproducibility. many biological processes can be regulated by the use of synthetic hydrogel components, which further makes it possible to develop transplantable biomaterials. hydrogels polymerized with peptide or poly (lactic - co - glycolic acid) (plga) have subsequently been developed, and recently, the use of poly (ethylene glycol) (peg)-based hydrogel systems has drawn attention. this newly developed hydrogel complex yields many benefits because of its conjugating activity, and it is difficult to induce bio - degradation into the monomers by natural metabolites, or cosmetic and medical substances. to create a cell - friendly microenvironment, a hydrogel polymerized by polyethylene glycol (peg) has been suggested. in nature, the polymerization of diverse functional groups and functionalized peg was conducted under cell - unfriendly conditions, which resulted in cellular toxicity. to avoid this insufficiency, a remodeled 3d scaffold system has been suggested (figure 1). in this system, vinyl sulfone (vs)-functionalized peg, which contains dicystein - containing peptide having matrix metalloproteinase (mmp)-specific cleavage sites (hereafter referred to as a combining region of the matrices with crosslinker), is employed. therefore, conjugation of the sulfhydryl (sh) group of cystein with the vs group of peg is conducted spontaneously through a michael - type addition reaction in a cell - friendly environment. based on these previous achievements, employing vs - functionalized hydrogel is now being employed in the design of artificial niches for regulating stem - cell function and activity. using peg - vs and a crosslinker, an ecm analog of an acellular niche has been developed and the microenvironmental influence of this 3d niche on self - renewal was evaluated in mouse escs (mescs). the initial study evaluated the transcriptional, translational, and functional activity of integrin subunits and subsequently identified the expression of integrin 51, v5, 61, and 91 in the membranes of undifferentiated mescs. these integrin heterodimers play important roles in esc self - renewal and select rgdsp, ttswsq, and aeidgiel peptide motifs to co - activate integrin 51, v5, 61, and 91 (figure 2). in next study, the effectiveness of peg - based hydrogels of different mechanical properties, which were modulated by the ratio of (vs) and (sh) concentration (hereafter referred to as the stoichiometric ratio), was evaluated. the mechanical properties of the hydrogel with a 0.7 stoichiometric ratio significantly stimulated mesc self - renewal (figure 3). combinatorial conjugation of the optimized ecm analogs with selected motifs showed that four integrin heterodimers promoted self - renewal of mescs. subsequently, strong expression of stemness - related genes, which was similar to the expression under a conventional 2d cellular niche, was detected. self - renewal of mescs under the designed 3d acellular biomimetic niche was successfully maintained without stimulation of lif signaling, and stat3 activation by exogenous lif is no longer the rate - limiting factor of stem cell self - renewal under a designated niche. instead, akt1/smad signaling was significantly activated in the mescs maintained under the 3d microenvironment (figure 4). these data clearly indicate that a different pathway of esc self - renewal is activated to adapt to a different microenvironment. although significant changes in morphological properties were detected, this signal shift under a different niche does not cause any initial change in esc self - renewal activity. in the case of hescs, a 3d acellular niche showed the potential to support hesc self - renewal without ecm analogs. the optimal properties of 3d peg - based hydrogel for the h9 hesc line was examined and the mechanical properties of the hydrogel were modified by the vs - functionalized peg multiarm number and peg concentration. it was found that hesc self - renewal and stemness were the highest after encapsulation in 8-multiarm peg hydrogel consisting of 10% (wt / v) peg (figure 5). this optimized peg - hydrogel was successfully applied to the 3d culture of h1 and novo hesc lines. the 3d scaffold reduced the difference in the proliferative activity among three hesc lines evaluated. however, it does not seem that this optimized condition is sufficient for long - term maintenance of hescs. among 3d scaffolds being constructed with various biocompatible biomaterials, hydrogel - based ecms have been employed for the culture of stem cells. the hydrogel is a cell - friendly, 3d macromolecule platform formed by the crosslinking of hydrophilic polymers, which can absorb h2o molecules to over five hundred times its own weight. in response to the salt concentration, ph, temperature, and crosslinking method, the chemical structure of the hydrogel can change in unlimited ways, so many biomedical and pharmaceutical applications may be possible, such as drug delivery, artificial tissue, membrane fabrication, and matrix construction. to date, hydrogel - based, protein - like materials such as matrigel, collagen, fibrin, and silk, polysaccharide - like materials such as hyaluronic acid, alginate, dextran, chitosan, and agarose, and dna have been used. in most of these cases, however, difficulties with manipulating their mechanical properties, the degradation rate, and the reproducibility occurred due to their undefined protein composition, which limited the modification of binding domains or sites in cellular activity and immune responses. to overcome this limitation, a hydrogel - based, synthetic (chemically defined) material has subsequently been suggested, which has a number of advantages including easy manipulation of mechanical properties, degradation, shape, and reproducibility. many biological processes can be regulated by the use of synthetic hydrogel components, which further makes it possible to develop transplantable biomaterials. hydrogels polymerized with peptide or poly (lactic - co - glycolic acid) (plga) have subsequently been developed, and recently, the use of poly (ethylene glycol) (peg)-based hydrogel systems has drawn attention. this newly developed hydrogel complex yields many benefits because of its conjugating activity, and it is difficult to induce bio - degradation into the monomers by natural metabolites, or cosmetic and medical substances. to create a cell - friendly microenvironment, a hydrogel polymerized by polyethylene glycol (peg) has been suggested. in nature, the polymerization of diverse functional groups and functionalized peg was conducted under cell - unfriendly conditions, which resulted in cellular toxicity. to avoid this insufficiency, a remodeled 3d scaffold system has been suggested (figure 1). in this system, vinyl sulfone (vs)-functionalized peg, which contains dicystein - containing peptide having matrix metalloproteinase (mmp)-specific cleavage sites (hereafter referred to as a combining region of the matrices with crosslinker), is employed. therefore, conjugation of the sulfhydryl (sh) group of cystein with the vs group of peg is conducted spontaneously through a michael - type addition reaction in a cell - friendly environment. based on these previous achievements, employing vs - functionalized hydrogel is now being employed in the design of artificial niches for regulating stem - cell function and activity. using peg - vs and a crosslinker, an ecm analog of an acellular niche has been developed and the microenvironmental influence of this 3d niche on self - renewal was evaluated in mouse escs (mescs). the initial study evaluated the transcriptional, translational, and functional activity of integrin subunits and subsequently identified the expression of integrin 51, v5, 61, and 91 in the membranes of undifferentiated mescs. these integrin heterodimers play important roles in esc self - renewal and select rgdsp, ttswsq, and aeidgiel peptide motifs to co - activate integrin 51, v5, 61, and 91 (figure 2). in next study, the effectiveness of peg - based hydrogels of different mechanical properties, which were modulated by the ratio of (vs) and (sh) concentration (hereafter referred to as the stoichiometric ratio), was evaluated. the mechanical properties of the hydrogel with a 0.7 stoichiometric ratio significantly stimulated mesc self - renewal (figure 3). combinatorial conjugation of the optimized ecm analogs with selected motifs showed that four integrin heterodimers promoted self - renewal of mescs. subsequently, strong expression of stemness - related genes, which was similar to the expression under a conventional 2d cellular niche, was detected. self - renewal of mescs under the designed 3d acellular biomimetic niche was successfully maintained without stimulation of lif signaling, and stat3 activation by exogenous lif is no longer the rate - limiting factor of stem cell self - renewal under a designated niche. instead, akt1/smad signaling was significantly activated in the mescs maintained under the 3d microenvironment (figure 4). these data clearly indicate that a different pathway of esc self - renewal is activated to adapt to a different microenvironment. although significant changes in morphological properties were detected, this signal shift under a different niche does not cause any initial change in esc self - renewal activity. in the case of hescs, a 3d acellular niche showed the potential to support hesc self - renewal without ecm analogs. the optimal properties of 3d peg - based hydrogel for the h9 hesc line was examined and the mechanical properties of the hydrogel were modified by the vs - functionalized peg multiarm number and peg concentration. it was found that hesc self - renewal and stemness were the highest after encapsulation in 8-multiarm peg hydrogel consisting of 10% (wt / v) peg (figure 5). this optimized peg - hydrogel was successfully applied to the 3d culture of h1 and novo hesc lines. the 3d scaffold reduced the difference in the proliferative activity among three hesc lines evaluated. however, it does not seem that this optimized condition is sufficient for long - term maintenance of hescs. the possibility of maintaining stem cell self - renewal was found under a defined 3d niche, which is different from a conventional 2d niche. the peg - based hydrogel using peg - vs and crosslinkers containing both sh group and mmp - specific cleavage sites greatly contributes to establishing a cell - friendly 3d non - cellular niche system, which is capable of constructing diverse niches by mimicking different biomechanics and patterning after diverse ecm analogs. probably, the suggested, hydrogel - nased system can be useful for creating cell - friendly environment in vitro. however, further trials to improve this 3d non - cellular niche for esc self - renewal should be conducted for field and clinical application. there have been only a few applications using any 3d culture system, but more extensive studies about the characterization of stem cell niches in vivo. attempts to synthesize ecm analogs or recombinant proteins, or to discover conjugating peptides will suggest a variety of alternative technologies for stem cell manipulation. | to overcome the difficulty of controlling stem cell fate and function in applications to regenerative medicine, a number of alternative approaches have been made. recent reports demonstrate that a non - cellular niche modulating the biophysical microenvironment with chemical factors can support stem cell self - renewal. in our previous studies, early establishment was executed to optimize biophysical factors and it was subsequently found that the microgeometry of the extracellular matrix made huge differences in stem cell behavior and phenotype. we review here a three - dimensional, non - cellular niche designed to support stem cell self - renewal. the characteristics of stem cells under the designed system are further discussed. |
the deep neck flexor is made up of the longus colli, longus capitis, and rectus capitis anterior, and the superficial neck flexor is made up of the sternocleidomastoid (scm) and scalene muscle1. unlike the superficial neck flexor muscle, the deep neck flexors, which are attached to the cervical spine, support the cervical lordosis directly and give stability2. previous studies reported that 70% of patients with neck pain and headache showed a decrease in strength and endurance of the scm and scalenus1. after applying craniocervical flexion exercise to patients with neck pain using a pressure biofeedback unit (pbu), there has been a significant increase in the strength of the deep neck flexor muscle in the forward head posture3. another study claimed that when the thorax is not fixed during craniocervical flexion exercise, contraction of the scm and anterior scalenus increased, among other superficial neck flexors4. on the other hand, the same study did not report conclusive results on whether manipulation of the thorax during craniocervical flexion exercise affects muscle activation. this study examined the effects of manipulation of the thorax and the intensity of pbu on the superficial cervical flexors muscle during craniocervical flexion exercise. thirty - three subjects participated in the experiment. this study also selected ten healthy people without any orthopedic history. information on the study was provided to all subjects prior to their participation and written informed consent according to the ethical standards of the declaration of helsinki, to participate in the project was obtained. the average ages, heights, and weights were 29.91 4.4 years old, 171.55 7.78 cm, and 62.82 8.57 kg in the experimental group, respectively. hixson, usa), which was adopted in a previous study, was used to apply an increasing pressure of 20, 30, and 40 mmhg2. the subjects could monitor the pressure applied to cervical vertebra 3 of the craniocervical junction by markings on pbu. craniocervical flexion exercise was performed for 20 seconds per pressure, and two minutes of rest was allowed after exercise to reduce muscle fatigue. the subjects were instructed to flex both of their hip joints and knee joints to limit the contraction of rectus abdominis in the supine position. the subjects pulled their chins downward during the exercise to contract the lungus colli and longus capitis. care was taken to minimize neck bending by contraction of the superficial muscles, such as the scm and ant. the thorax of the subjects was fixed using a manual belt. to reduce the discomfort by pressure caused by the manual belt, a towel was placed between the belt and thorax to ensure that the subjects experienced no discomfort prior to the experiment. in this study, electromyography (qemg-4 system, lxm 3204 laxtha, korea) was used to measure the change in the values of muscle activation in the superficial muscles. electromyogram electrodes were attached to the following areas5 : (1) scm, between mastoid process and sternal notch ; and (2) ant. scalene, behind the scm, above the clavicle, and triangle shape formed by the upper trapezius. the electrodes were attached to the superficial muscles and the subjects bent their cervical vertebra for 20 seconds while their muscle activation value was recorded. the distance between the electrodes was 2 cm, and the sampling rate and band pass filter were set to 1,024 hz and 20450 hz, respectively. in addition, the signals collected from each muscle were normalized to the maximal voluntary isometric contraction (% mvic). spss win version 12.0 was used for statistical analysis. to compare the change in superficial muscle activation by the pbu intensity of the chest fixed group and non - fixed group, 3 2 two - way repeated anova was used, and a paired t - test was conducted to compare the difference in muscle activation between the two groups. significant differences in the post - training gains in the scm and scalene were observed between the thorax fixation group and thorax non - fixation group (p a, bnon - fixation6.83 3.059.32 9.78 11.80 10.93c > ascalenusfixation6.37 1.837.46 2.5410.06 4.47c > a, bnon - fixation8.21 3.599.27 4.3612.33 patients with neck pain exhibit reduced cervical region control ability due to a weakening of the deep neck flexor muscles6. the scm and anterior scalene, superficial muscles, are activated excessively in the early stages, limiting the bending of the cervical region. such abnormal movement extends the chin forward, shortening the cervical muscle that is between the occipital region and cervical vertebra 1, and weakening the lungus colli and longus capitis7. applied craniocervical flexion exercise to the normal cervical spine and witnessed an increase in muscle activation of the lungus colli and longus capitis8. as a result, during craniocervical flexion exercise using pbu, the cervical region superficial muscle activation was lower when the thorax was fixed than when it was not. a previous study reported that lower muscle activation of the superficial neck flexor during craniocervical flexion9. this is because when thorax is fixed, activation of the scm and scalenus decreases, which was also observed in the present study9. therefore, craniocervical flexion exercise performed by neck pain patients with their thorax fixed is a clinically effective method that reduces the superficial neck flexor muscle activation10. | [purpose ] this study examined the effects of manipulation of the thorax and the intensity of the pressure biofeedback unit on the superficial cervical flexors muscle during craniocervical flexion exercise. [subjects and methods ] thirty three subjects participated in the experiment. thirty three healthy people without any orthopedic history were also selected. the subjects could monitor the pressure applied to cervical vertebra 3 of the craniocervical junction by markings on the pressure biofeedback unit. craniocervical flexion exercise was performed for 20 seconds per pressure, and two minutes of rest was allowed after exercise to reduce muscle fatigue. [results ] significant differences in the post - training gains in the sternocleidomastoid and scalene were observed between the thorax fixation group and thorax non - fixation group. the thorax fixation group showed that muscle activation of the sternocleidomastoid and scalene was increased when the pressure biofeedback unit intensity was 40 mmhg than when pressure biofeedback unit intensity was 20 mmhg and 30 mmhg in the post - hoc result. the thorax non - fixation group showed that muscle activation of the sternocleidomastoid and scalene was higher when the pressure biofeedback unit intensity was 40mmhg compared to that when the pressure biofeedback unit intensity was 20mmhg in the post - hoc result. [conclusion ] craniocervical flexion exercise is a clinically effective method that reduces the superficial neck flexor muscle activation. |
the level of turnout at elections is often seen as an indicator of the health of a democracy (fieldhouse., 2007), yet there is a general trend towards declining rates of electoral participation in western europe (e.g. aarts and wessels, 2005 ; blais and rubenson, 2007 ; franklin., 2004). this has led to fears that democratic legitimacy may decline as elections increasingly fail to act as the institutional connection (topf, 1995a) between citizens and the state. in light of these developments, it has been suggested that the minimum voting age should be lowered to 16 (e.g. power commission, 2006 ; votes at 16, 2008 ; hart and artkins, 2011). supporters of such a reform argue that lowering the voting age would have a positive impact on electoral participation. this is because young people under 18 are likely to still be in school and live with their families, two factors that have been shown to encourage turnout through a variety of socialisation mechanisms (franklin, 2004 ; highton and wolfinger, 2001 ; bhatti and hansen, 2010). in the long term, this higher level of participation at a young age may then facilitate the early development of a habit of voting (e.g. plutzer, 2002 ; franklin, 2004). of course, lowering the voting age is not only justified as a way to stop the decline in turnout. for example, it is also seen as a way to ensure that the interests of young citizens are represented in the political system (votes at 16, 2008).4 however, the proposed reform is not without its critics. the main argument made against lowering the voting age is that young people under 18 lack the ability and motivation to participate effectively in the electoral process (chan and clayton, 2006). it is suggested that this will lead to low turnout rates, comparable to if not even lower than those observed among citizens aged 1825 (electoral commission, 2004). a further consequence would be that citizens under 18 might not make use of their vote as effectively as older voters. while they might vote for the sake of voting thus, their vote choice would be driven more strongly by expressive instead of instrumental considerations (tka, 2009), and their policy views would not be well - represented by political actors. in this paper are young people under 18 less able and motivated to participate effectively in politics ? and do these factors influence whether and how they use their right to vote ? if the answer to these questions is yes, then lowering the voting age could indeed have negative consequences for the health of democracy. if the answer is no, then critics are arguably left with fewer arguments why we should oppose lowering the voting age. instead, we might consider potential positive consequences of the reform, such as tying young people to the democratic process, encouraging the development of a habit of voting and ensuring the representation of their interests. we examine the choices made by young people under 18 using data from austria, where in 2007 the voting age at national elections was lowered to 16. specifically, we use a survey carried out in the run - up to the european parliament (ep) elections 2009 which over - sampled young people under 26. austria 's reform allows us to examine for the first time whether the critics of lowering the minimum voting age are right. before, the only possible empirical strategies were either to extrapolate about the behaviour of citizens under 18 from that of voters just over 18 or to study the potential electoral behaviour of young people under 18 in a context where they did not have the vote. our survey indicates that the intention to turn out was indeed relatively low among citizens under 18 in the 2009 ep election. using the self - assessed likelihood of voting on a scale of 010, under-18s have a low average intention of turning out, with a mean score of 5.91. this is lower than among respondents aged between 18 and 21 (6.24) and between those aged between 22 and 25 (6.98), while respondents over 30 have a mean score of 7.38. is this pattern due to the fact that austrians under 18 are particularly unable or unwilling to participate in politics ? first, measures of political interest, knowledge and non - electoral participation indicate that young people under 18 are not particularly unable or unwilling to participate in political life. second, these factors do not help to explain their lower turnout rates, so we can not say that young citizens fail to vote for reasons that are particularly troubling for democratic legitimacy. finally, there is no evidence that the quality of vote choices among citizens under 18 is any worse than that of older voters. we begin this paper by discussing in greater depth existing arguments regarding the political behaviour of citizens under 18 and the potential effects of lowering the voting age in terms of democratic legitimacy, focussing on turnout and the quality of vote choice. after describing the survey, we provide a brief descriptive account of young people 's motivation and ability to engage in politics. we then turn to a multivariate analysis that explores the reasons behind turnout decisions of citizens under 18. in the scholarly debate democratic legitimacy includes two dimensions : input and output legitimacy (scharpf, 1999). [p]olitical choices are legitimate if they reflect the will of the people that is, if they can be derived from the authentic preferences of the members of a community (scharpf, 1999 : 6). input legitimacy requires citizens who are motivated and competent and who engage in reasoned arguments in collective decision - making processes. as a result, input legitimacy may be negatively affected by lowering the voting age if this only serves to extend suffrage to citizens who are not motivated or able to participate in decision - making in this way. simply put, the central question is whether citizens under 18 have the ability and motivation to participate effectively in elections. chan and clayton (2006) argue that young people under 18 are simply not politically they measure the political maturity of young people under 18 using political interest, party identification, political knowledge and attitudinal consistency. according to chan and clayton (2006), those under 18 fail to score high enough on any of these indicators. they suggest that these differences can not be explained by the fact that in the uk those under 18 do not yet have the vote and therefore have no incentive to become involved in politics. instead, citing dawkins and cornwell (2003), they argue that the teenage brain may simply not be ready to vote at 16. however, hart and artkins (2011) point out that so far no neurological evidence has been put forward to prove this point, while steinberg. (2009) show that teenage citizens possess the same cognitive sophistication as young adults. it is perhaps more likely that these age differences may exist due to a universal life - cycle effect, with younger voters simply not yet having developed the political interest, knowledge and sense of duty that comes with age (aarts and wessels, 2005). thus, from this critical perspective young citizens under 18 lack the ability and motivation to engage effectively in politics. since our aim is to test the arguments made by critics of lowering the voting age, our hypotheses are as follows : h1a : young citizens under 18 are less able to participate in politics effectively than older voters.h1b : young citizens under 18 are less motivated to participate in politics effectively than older voters. young citizens under 18 are less able to participate in politics effectively than older voters. young citizens under 18 are less motivated to participate in politics effectively than older voters. enlarging suffrage to include young people under 18 may have consequences for the level of turnout. on the one hand, some scholars argue that turnout numbers may improve, especially in the longer term, as young people under 18 are more easily and more lastingly mobilised to vote due to socialisation effects (e.g. franklin, 2004). on the other hand, critics put forward the argument that it could also be that young people under 18 simply mirror the low levels of turnout found among those aged between 18 and 21 (e.g. electoral commission, 2004). however in this paper, we are not concerned with the levels of turnout themselves. for one, to examine the development of a habit of voting requires a longer - term perspective than can not be achieved just two years after the voting age was lowered. moreover, looking exclusively at the level of turnout should not be the only way to address whether declining electoral participation is worrying. as pointed out, it is particularly concerning when decisions not to vote are a reflection of disenchantment, indifference or a lack of capabilities (chan and clayton, 2006).5 lower levels of turnout among citizens under 18 do not automatically indicate that this pattern is due to a lower ability and motivation to participate. other reasons may underlie this decision. first, young voters may privilege new modes of political participation over traditional forms of electoral participation (topf, 1995b), bypassing the electoral routes (franklin, 2002 : 165). electoral participation is not the only way that a democratic bond between citizens and the political system can be created (e.g. topf, 1995b ; franklin, 2002 ; fuchs and klingemann, 1995 ; dalton, 2009). young voters may be particularly likely to choose other forms of participation due to longer schooling years, exposure to other forms of informal civic education, higher information levels, new information channels and a decrease in party affiliation (e.g. thomassen, 2005). second, young voters may simply see voting itself as less of a civic duty (e.g. blais, 2000 ; dalton, 2009 ; wattenberg, 2008). they may have a more individual calculus of the utility of voting and rely more heavily on the assessment of the importance of election outcomes (thomassen, 2005).6 thus, analysing only turnout rates per se is not enough to provide a good picture of the status of input legitimacy as we also need to take the underlying motives into account. in other words, we need to know whether citizens under 18 fail to vote because of a lower ability and motivation to participate effectively. if this is the case, then this undermines input legitimacy ; if not, then lower turnout is perhaps less worrying.7 in sum, we argue that the quality of the electoral participation of citizens under 18 is particularly unsatisfactory if low turnout can be explained by a low willingness and motivation to engage in politics. we will therefore test the following two hypotheses : h2a : the lower turnout of young people under 18 can be explained by their lower ability to participate in politics.h2b : the lower turnout of young people under 18 can be explained by their lower motivation to participate in politics. the lower turnout of young people under 18 can be explained by their lower ability to participate in politics. the lower turnout of young people under 18 can be explained by their lower motivation to participate in politics. just because citizens go to the polls does not mean that they will be well - represented by those they elect. argue : [v]otes freely given are meaningless unless they accurately reflect a citizen 's true preferences citizens should be able to select accurately between political actors and make a choice that is consistent with their own views, attitudes and preferences (e.g. lau and redlawsk, 1997). if voters under 18 take choices that do not reflect their interests and attitudes, then this will limit their substantial representation (pitkin, 1967). the arguments presented earlier that citizens under 18 may lack the requisite ability and motivation to participate (chan and clayton, 2006) would also lead them to be less inclined to think carefully about their decision and therefore choose parties that do not reflect their preferences. thus, there would also be negative consequences for democracy if the choices made by voters under 18 are less well - linked to their actual preferences than those of older voters. on the other hand, if the decisions of voters under 18 reflect their preferences as well as they do in older age groups, then the critics ' arguments have no empirical basis. we would have no reason to believe that the interests and preferences of voters under 18 would be less well - represented. our final hypothesis therefore tests this last argument by critics of lowering the voting age and reads as follows : h3:the quality of vote choice among voters under 18 is lower than among older voters. the quality of vote choice among voters under 18 is lower than among older voters. until now, empirical research on the effects of lowering the voting age has had to take one of two unsatisfactory approaches. the first method has been to assume that under-18s are little different from those just over 18, justifying the use of evidence from the voting behaviour of young citizens aged 18 and older (e.g. electoral commission, 2004).8 the second approach uses data on citizens under 18 before they have the right to vote (e.g. chan and clayton, 2006). studying electoral participation for those who do not have the right to vote has a considerable flaw : without the right to cast a ballot, there is no rational incentive for citizens to increase their interest and knowledge in politics. simply having voting rights may encourage people to gather information and become politically active in other ways (rubenson. correctly whether the electoral participation of under-18s matches the quality of that of their older peers, we therefore need a case where such young citizens have the right to vote. austria is the only country in europe that has a voting age of 16 for national elections.9 the reform was passed by the austrian parliament in 2007, and since then, young people under 18 have cast ballots at a series of elections, including for the national parliament in 2008, the european parliament in 2009 and the presidential elections in 2010. austria thus provides the first opportunity to examine the political participation of under-18s in a nation - wide election, at least in a stable advanced industrial democracy. the specific data used in this paper are from a pre - election survey (n = 805) conducted at the end of may and the beginning of june 2009, so in the weeks directly before the european parliament election (kritzinger and heinrich, 2009).10 voters between 16 and 25 were over - sampled for this survey (n = 263), making this dataset particularly suitable to our research questions. we take advantage of the over - sampled segment of austrian voters to compare 16- and 17-year olds to voters between 18 and 21, 22 and 25, 26 and 30 and to voters over 31.11 we assess the ability and motivation to participate effectively in politics using three measures.12 the ability to engage in politics is evaluated using political knowledge, which we measure by assessing whether respondents correctly place the social democrats (sp) to the left of the two far - right parties (fp and bz) and the people 's party (vp). we measure the motivation to participate effectively in politics using political interest and the willingness to consider various forms of non - electoral participation. the respondents ' interest in politics is measured as the average of answers to eight questions tapping attention to politics in general and the ep campaign in particular. the variable was rescaled to range from 0 to 1, and the alpha reliability coefficient of this scale is 0.81. we measured non - electoral political participation by asking respondents to rate on a four - point scale their hypothetical willingness to engage in a series of political activities : contacting a politician, collecting signatures, working for a non - governmental organization, taking part in a legal demonstration and working on a campaign. we also create an overall index for non - electoral political participation using the average answer to the five questions. it is always difficult to measure turnout using survey questions due to the problems of over - reporting, sample selectivity, social desirability bias and the stimulus effects of pre - elections interviews (e.g. aarts and wessels, 2005 ; bernstein., 2001 ; karp and brockington, 2005).13 there is evidence that the pre - election turnout intention questions are the best available predictor of whether a person is likely to vote (bolstein, 1991). respondents might be more honest regarding their actual intention to turn out when presented with a scale in which people can indicate uncertainty and reluctance without declaring directly that they might abstain. therefore, we use turnout intention as our dependent variable. we measure propensity to turn out with a question asking respondents to state their certainty of voting in the upcoming ep election on a scale of 010. in our sample, 54.1% of respondents gave a vote intention score of 8 or higher and 41% a score of 9 or higher.14 this compares favourably to the 46% who actually voted on 7 june 2009.15 examining the intention to turn out in an ep election gives us also the advantage of studying an election with lower overall turnout ; this could reduce the social desirability bias as people might be less reluctant to declare that they will not vote when abstention is a more common phenomenon. we take into account the specific eu nature of the election by including eu - specific versions of core variables in our regression models and by including a control variable concerning views on european integration. first, we present descriptive findings on the ability and motivation to participate in politics among young people under 18. next, we examine the causes underlying turnout decisions before finally examining the quality of vote choice. critics of lowering the voting age argue that citizens under 18 have a lower motivation and ability to engage in politics than older citizens. we test this by considering three measures widely used in the literature to capture these constructs (e.g. fieldhouse., 2007) : we see, first, that interest in politics is by no means particularly low among under-18s ; indeed, it is the second - highest average of the four age groups under 30. however, in spite of their apparent interest in politics, political knowledge is somewhat lower among under-18s compared to the other three groups of young voters. however, it is worth noting that this difference is significant in a two - tailed t - test only for the comparison with 22- to 25-year - olds. moreover, a cautious interpretation of these results is required since we only have one knowledge question. this may be due to the fact that young citizens do not yet have the experience necessary to place parties correctly on a left there is thus some support for h1a, i.e. that citizens under 18 are less able to participate in politics. citizens can also engage in politics outside of elections. in fig. 2, we present the average scores for each age group across all five activities we asked about, with the top - left panel presenting the results for the overall index. it is clear that the youngest citizens ' willingness to participate in non - electoral politics is relatively high and no different from the overall mean. young citizens are particularly likely to say that they would take part in a demonstration. overall, citizens under 18 are just as motivated to take part in political life as older age groups. finally, we further illustrate the attitudes of young people under 18 using measures of democratic disaffection and alienation, specifically the three indicators institutional trust, satisfaction with democracy and the perceived impact of politics on one 's life (fig. surprisingly, our data show that trust in institutions among citizens under 18 is significantly higher than the overall mean among all citizens, so there is no indication at all of disaffection using this classical measure. in addition, satisfaction with national and european democracy among citizens under 18 is actually higher (and often significantly so) than among older citizens. turning to the bottom - right panel, younger citizens do not differ from older citizens in the proportion who say that either the national parliament or the ep has a strong impact on them personally. given this evidence and their satisfactory level of political interest, there is strong descriptive evidence that the youngest citizens in austria are not particular results which are quite contrary to those reported in the literature (e.g. chan and clayton, 2006). there is therefore so far little evidence in favour of h1b, so that there is little indication that citizens under 18 are less motivated to participate effectively in politics. in sum, citizens under 18 do not differ in terms of their democratic disaffection and their motivation to participate in politics from older age groups. however, they do have relatively little knowledge, indicating that they might be less able to participate. in the next section, we will examine whether age group differences in the motivation and ability to participate effectively in politics can help explain lower turnout levels among citizens under 18. we now turn to examining the causes of lower turnout by following the approach of rubenson. the starting point is a very basic regression model with dummy variables for the four age groups under 31 as well as a series of fundamental socio - demographic controls. in the following steps, a series of independent variables this allows us to test whether different age groups decide not to vote because they lack the ability or motivation to participate effectively in politics ; if this is the case for the youngest cohort, the age coefficient for the under-18s should decrease (or even cease to be significant) when the relevant variables are added to the model. importantly, the cases included in each model are the same, so the most basic model only includes respondents for which all variables in the fullest model are available. due to a heteroscedastic error distribution model 1 includes four age dummy variables for those aged 16 and 17, 1821, 2225 and 2630. the model also includes four socio - demographic controls : education, gender, rural residence and migration background. previous research indicates that education is an important predictor of the propensity to turn out in almost all democracies (e.g. wolfinger and rosenstone, 1980 ; blais., 2004 ; we also include migration background as this may be related to the level of resources and to the level of connectedness one feels to the political system ; we therefore expect having a migration background to have a negative effect on turnout (fieldhouse., 2007). living in a rural area may increase turnout as social capital and thus the pressure to vote may be higher there (e.g. putnam, 2000 ; nevitte., 2009). we can see that turnout intention is indeed predicted to be lower for all younger age groups. the gap between these groups and the reference group of citizens over 30 is quite large : on average, respondents in the youngest groups assess their intention to vote over 1 unit lower than the reference group. the results also underline that there are only small differences between young people under and just over 18. concerning the socio - demographic controls, we find that education is significantly associated with higher levels of voting intention. the coefficients of the other controls are in the expected direction but are not significant. importantly, including basic, mainly socio - demographic controls does not account for the age gap in turnout, so it is not young citizens ' place of residence or their migration background that explains lower rates of electoral participation. model 2 adds two further groups of controls : first, measures of democratic dissatisfaction and alienation ; and second, support for european integration. in general, we expect respondents who trust institutions, believe that they have an important influence and are satisfied with politics to also be more likely to vote. the attitude towards european unification is included as a control as, given that we measure turnout in ep elections, citizens may want to abstain because they disapprove of the eu as a whole. in the model, the only significant effects are for the variables measuring institutional trust and the perceived political impact of the national and european parliaments on the respondent 's life. moreover, these added controls do not substantively affect the interpretation of the key age group dummy for young people under 18. next, in models 36 we add our main independent variables measuring the ability and motivation to engage in politics, first separately and then jointly. we start in model 3 adding our variable measuring political knowledge ; we remove this in model 4 and add non - electoral participation ; model 5 tests only the effect of political interest ; and finally model 6 includes all three controls. in each of these models, our added variables are clearly significant, so they influence people 's stated intention to turn out to vote. as pointed out above, if these variables did explain lower levels of turn out among citizens under 18, then they would cause the coefficients of the dummy variables for the youngest age group to shrink or even cease to be significant. however, we find that the coefficient for the 16-to-17 age group decreases only by a very small amount (0.14 units). this means that the lower turnout levels of citizens under 18 can not satisfactorily be explained by the fact that they are not motivated or able to engage in politics.17 interestingly, it is if anything the age group of young people aged 1821 where we can observe a decrease in the size of this group 's coefficient. thus, citizens just over 18 appear to be substantively influenced by their lacking ability and motivation to vote, but not citizens just under 18. in sum, the results show while turnout among under-18s is indeed lower than among austrians in general, this is not primarily due to a lack of knowledge or interest nor due to democratic dissatisfaction and alienation. h2a and h2b are both rejected. the quality of electoral participation among citizens under 18 goes beyond the reasons that drive abstention : it is the concern that voters under 18 do not choose the party that best represents their views or interests. thus, we analyse whether the quality of the vote decisions taken by voters under 18 once they turn out to vote is any different from those of older voters. we examine vote choice quality directly, though of course this is a concept that is difficult to estimate. we operationalise it as the ideological congruence between voters and the party they want to vote for : the greater the ideological similarity between a voter and the party she chooses, the higher the quality of vote choice. correct voting, which uses measures of voter preferences on a number of different issues by which the competing candidates or parties can be distinguished, as well as on some defensibly objective measure (such as expert judgements) of where the candidates actually stand on those same issues (e.g. lau and redlawsk, 1997 ; lau., 2008).18 although such detailed measures are not available to us, we believe our simplified approach provides a good indication of whether voters choose a party that is ideologically relatively close to them. this dimension can be considered a shortcut or heuristic that voters use in order to simplify ideological competition. the left right dimension has been found to provide an appropriate measure of citizens ' general ideological orientations (fuchs and klingemann, 1990 ; huber, 1989) and to influence vote choice, also in austria (hellwig, 2008). right dimension as the absolute distance of the voter from the party she voted for. voter positions are measured using respondent self - assessments on a 010 left right scale. our first version of the left right congruence measure compares this self - assessment with the voter 's own placement of the party she voted for ; the mean absolute left right distances for our five age groups are shown in the top - left graph of fig. 4.19 second, we compared respondent self - placements with expert survey scores from hooghe. (2010), who asked their respondents to place austrian parties on a general 010 left we can see that there are no significant differences between the different groups of voters for either left while the mean distance is slightly higher among the younger voters, the difference is minimal. as the survey was carried out in the context of ep elections, it might be that voters at that time cared more about congruence on the dimension of european integration rather than on the general left since the survey contains no assessments of party positions on this topic, we again use the hooghe. (2010) scores, who asked experts to score parties on their overall orientation towards european integration using a 17 scale which we rescaled to range from 0 to 10. first, we compare voter 's positions on european unification with rescaled party scores (bottom - left graph). second, we divide voters into two groups sceptical and not sceptical of integration and compare these with hooghe. 's dichotomised measure of party positions (bottom - right graph).20 both measurement approaches show that there are no significant differences between age groups. to the extent that differences even if not statistically distinguishable are present, it is the younger voters whose vote choice is more congruent with party positions on european integration. this is an indication that voters under 18 put some emphasis on the issues that the election, in this case the ep election, is about. in sum, when considering the precise choices made, we have no convincing evidence that the voting decisions of voters under 18 are in any way of lesser quality, that is, less congruent, than that of older groups of voters. critics of lowering the voting age to 16 have argued that such teenage citizens are not able or motivated to participate effectively in politics and that this both drives their turnout decisions and means that their electoral choices are of lower quality. we have tested whether these criticisms have an empirical basis using evidence from austria, the one european country where the voting age has already been lowered for nation - wide elections. first, we do not find that citizens under 18 are particularly unable or unwilling to participate effectively in politics. second, while turnout among this group is relatively low, we find no evidence that this is driven by a lacking ability or motivation to participate. finally, we do not find that the vote choices of citizens under 18 reflect their preferences less well than those of older voters do. in sum, lowering the voting age does not appear to have a negative impact on input legitimacy and the quality of democratic decisions. this means that the potential positive consequences of this reform merit particular consideration and should also be empirically studied. it is not the case that austrian teenagers are particularly unusual in a comparative context. if anything, there are two features of the austrian case that would indicate that young austrians are not particularly interested or engaged in politics. for one, the general educational test scores of austrian school - children are relatively low compared to other oecd countries (oecd, 2011). moreover, there is evidence that it is young voters in austria who are most likely to turn to protest parties such as those on the radical right (e.g. wagner and kritzinger, 2012 ; schwarzer and zeglovits, 2009). thus, we do not think that austrians under 18 are likely to be outliers in their political interest and knowledge compared to teenagers in other countries ; if anything, austria would be a country where we might expect citizens under 18 to be particularly unmotivated to participate in politics. it is also important to note that our study has focused on one point in time. it is therefore impossible for us to distinguish between cohort and age effects. in other words, we can not say with certainty whether citizens under 18 compare favourably with citizens over 18 because of their age or because of their cohort. however, it is unlikely that there will be strong cohort differences between such small differences in ages, so we believe our findings should reflect general age differences rather than time - specific cohort differences. a particularly important question especially in the light of our results of the 1821 age group is the existence of a habit of voting among teenage citizens (franklin, 2004). specifically, it may be easier to instil a habit of voting among those who are still in school and live at home. however, observing a habit requires longer - term data, and citizens under 18 have only had the vote in austria for four years and in one national parliamentary election. we hope that future research will examine whether today 's teenage citizens will be more likely to develop a habit of voting than citizens who were first able to vote at an older age. dalton (2009) has argued that younger generations are engaged in a variety of social and political activities beyond voting, with more direct, action - oriented participation on the increase. several authors have found supporting evidence for this from the uk (henn. dalton 's argument also fits with one our findings, namely that younger people are more likely to say that they would demonstrate in support of their political goals. younger citizens might see voting as less essential and instead turn to non - electoral forms of participation in order to influence political outcomes. for young citizens, norms of engaged citizenship may be changing. while overall turnout rates would suggest a decrease of the bond between citizens and the democratic political system, new participation forms might mean that citizens are actually just as politically active as before, or possibly even more so. future research should explore these other forms of political participation and assess the extent to which they are replacing voting as the primary way of engaging with politics, especially for citizens under 18. to conclude, our findings show that a key criticism of lowering the voting age to 16 does not hold : there is little evidence that these citizens are less able or less motivated to participate effectively in politics. this means that critics of lowering the voting age to 16 need to look again at the arguments they use, and that there are important reasons to consider the potential positive impact of such a reform more closely. | critics of giving citizens under 18 the right to vote argue that such teenagers lack the ability and motivation to participate effectively in elections. if this argument is true, lowering the voting age would have negative consequences for the quality of democracy. we test the argument using survey data from austria, the only european country with a voting age of 16 in nation - wide elections. while the turnout levels of young people under 18 are relatively low, their failure to vote can not be explained by a lower ability or motivation to participate. in addition, the quality of these citizens ' choices is similar to that of older voters, so they do cast votes in ways that enable their interests to be represented equally well. these results are encouraging for supporters of a lower voting age. |
children with special health care needs (cshcn) are defined as those who have or are at increased risk for a chronic physical, developmental, behavioral, or emotional condition and who also require health and related services of a type or amount beyond that required by children generally. the estimated prevalence of cshcn in the united states (us) has increased from 12.8% in 2001 to 13.9% in 2005 and 15.1% (11.2 million children) in 2010. as the needs of cshcn in the us evolved, the federal maternal and child health bureau (mchb) worked with state title v agencies, families, and other stakeholders to develop 6 key system building blocks, referred to as core outcomes or quality indicators, representing the essential elements needed for high - quality systems of health and medical services. in 2011, according to the american community survey, 25 million people in the us had limited english proficiency (lep), with 34% of hispanic / latino and 36.2% of asians reporting not speaking english very well. recognizing the important health care issues facing immigrant and lep communities in the us, the council on community pediatrics of the american academy of pediatrics recently released a policy statement on new insights and recommendations on providing health care for immigrant, migrant and border children. for parents with lep, caring for parental lep has been associated with risks of adverse outcomes in children s health and disparities in medical and oral health, access to care, and use of services.[6 - 8 ] moreover, parents with lep may be unable to read and comprehend important clinical evaluations, prescription instructions, follow - up appointments, referral to specialists and therapy - related documents. in fact, some parents with lep may be unable to understand routinely dispensed written medication instructions. clinical providers and medical entities have reported using untrained interpreters to communicate with families as they lack the capacity or fail systematically to provide effective translation and interpretation services for families with lep.[10 - 12 ] previous research has documented the lack of access to medical home, usual source of care, family - centered care and insurance coverage adequacy for cshcns from non - english primary language (nepl) households.[13 - 15 ] nepl families have also been found to sacrifice more time and employment opportunities to care for their cshcn. with respect to health insurance, parental lep has significantly impacted the enrollment of medicaid - eligible children in publicly funded health insurance programs. moreover, language barriers in accessing health care have been associated with less patient education, worse interpersonal care, and lower patient satisfaction. to our knowledge, no previous study has focused on examining household language use and health care quality as measured by attainment of mchb s six quality indicators of effective implementation of systems of services for cshcn. using data from the 2009 - 2010 national survey of children with special health care needs (ns - cshcn), our study aims to describe the demographic characteristics of cshcn and the prevalence of cshcn meeting six quality indicators by the parent - reported primary household language, and to examine the independent effects of primary household language on the six quality indicators while controlling for confounding variables. the federal mchb within the health resources and services administration (hrsa) of the u.s. the national center for health statistics (nchs) of the centers for disease control and prevention (cdc) oversaw the sampling, data collection, and telephone interviews for the survey as a module of the state and local area integrated telephone survey (slaits). the ns - cshcn is a national telephone survey with independent random samples from 50 states and the district of columbia. the 2009 - 2010 ns - cshcn is the third administration of the ns - cshcn as the previous surveys took place in 2000 - 2001 and 2005 - 2006. the available survey languages included the following : english, spanish, mandarin, cantonese, vietnamese and korean. the topics covered by the ns - cshcn included child health, insurance status, access to health care, preventive care, family - centered care, access to community - based services, impact of child s health on family, transition to adulthood, and child and family demographics. the respondent was the parent or guardian who knew most about the child s health status and health care. the overall interview completion rate was 83.6% for the landline sample, 76.6% for the cell - phone sample, and 80.8% for the combined sample. households were identified as speaking english as the primary language at home (epl) or using another primary language (non - english primary language (nepl)). covariates in the analyses included : child s age (0 - 5, 6 - 11, 12 - 17 years), gender, race / ethnicity (non - hispanic white, non - hispanic black, hispanic, and other), household poverty status measured as a ratio of family income to federal poverty level (fpl) in four categories (< 100%, 100 - 199%, 200 - 399%, 400%), parental education (less than high school, high school graduate, more than high school), family structure (in biological or adopted household, two - parent stepfamily household, mother only household, other), health insurance status (private only, public only, public and private, uninsured), qualification criteria for chscn (functional limitation only or with any other criterion, prescription medications only, service use only, both prescription medication and service use). the quality indicators describe what families should be able to expect from the service system : (1) family partnership in decision - making and satisfaction with care, (2) receipt of care through a medical home, (3) adequate health insurance, (4) early and continuous screening and surveillance, (5) services that are organized for ease of use, and (6) effective transition planning for adult health care. together, these quality indicators represent the essential elements needed for high - quality systems of services. chi - square statistics were used to test socioeconomic and demographic differences between epl and nepl households. bivariate analyses were conducted to examine the association of household language with the six quality indicators. the independent effects of primary household language status were analyzed for all quality indicators in the multivariable logistic regression models while controlling for children s age, gender, race / ethnicity, family poverty level, and family structure. income has been shown to be a better measure of socioeconomic status for immigrants, since education credentials from foreign countries often result in underemployment in the us. adjusted odds ratios (or) and 95% confidence intervals (ci) were computed by using the beta coefficients and standard errors obtained from the multivariable logistic analyses. to account for the complex sample design involving stratification, clustering, and multistage sampling of the ns - cshcn, sas version 9.3 (survey procs) was used to conduct the statistical analyses. the taylor series (linearization) method was used to estimate the covariance matrix of the regression coefficients for complex survey data. households were identified as speaking english as the primary language at home (epl) or using another primary language (non - english primary language (nepl)). covariates in the analyses included : child s age (0 - 5, 6 - 11, 12 - 17 years), gender, race / ethnicity (non - hispanic white, non - hispanic black, hispanic, and other), household poverty status measured as a ratio of family income to federal poverty level (fpl) in four categories (< 100%, 100 - 199%, 200 - 399%, 400%), parental education (less than high school, high school graduate, more than high school), family structure (in biological or adopted household, two - parent stepfamily household, mother only household, other), health insurance status (private only, public only, public and private, uninsured), qualification criteria for chscn (functional limitation only or with any other criterion, prescription medications only, service use only, both prescription medication and service use). the quality indicators describe what families should be able to expect from the service system : (1) family partnership in decision - making and satisfaction with care, (2) receipt of care through a medical home, (3) adequate health insurance, (4) early and continuous screening and surveillance, (5) services that are organized for ease of use, and (6) effective transition planning for adult health care. together, these quality indicators represent the essential elements needed for high - quality systems of services. chi - square statistics were used to test socioeconomic and demographic differences between epl and nepl households. bivariate analyses were conducted to examine the association of household language with the six quality indicators. the independent effects of primary household language status were analyzed for all quality indicators in the multivariable logistic regression models while controlling for children s age, gender, race / ethnicity, family poverty level, and family structure. income has been shown to be a better measure of socioeconomic status for immigrants, since education credentials from foreign countries often result in underemployment in the us. adjusted odds ratios (or) and 95% confidence intervals (ci) were computed by using the beta coefficients and standard errors obtained from the multivariable logistic analyses. to account for the complex sample design involving stratification, clustering, and multistage sampling of the ns - cshcn, sas version 9.3 (survey procs) was used to conduct the statistical analyses. the taylor series (linearization) method was used to estimate the covariance matrix of the regression coefficients for complex survey data. the final analytic sample includes 1,896 cshcn from nepl households and 38,346 children from epl households. children from epl households were predominantly non - hispanic white (63%), while those from nepl households were primarily hispanic (74%).. nearly 40% of nepl parents did not graduate from high school compared to < 10% of epl parents. almost two - thirds of nepl children came from two - parent households, while only 56% of epl children had both parents in their households. one quarter of nepl children, compared to more than half of epl children, had private health insurance. a higher proportion of nepl children qualified for cshcn classification by service use, whereas a higher percentage of epl children qualified by prescription medication use. socioeconomic and demographic characteristics of us children with special health care needs (cshcn) by primary household language use, ages 0 - 17 years source : 2009 - 20010 national survey of children with special health care needs table 2 displays differences in quality indicators attainment by household language type. sixty percent of nepl families, compared to 71% epl families, believed they were partners in decision making for their children s health. only 27% of nepl children received care through a medical home, compared to 44% of epl children. about 49% of nepl families had adequate private and/or public insurance to pay for needed services, compared to 62% of epl families. two - thirds of nepl children were screened early and continuously for special health care needs, in contrast with 80% of epl children receiving early screening. only 58% of nepl children accessed community based services, compared to 66% of epl children. more than 41% of epl youths with special health care needs received the necessary services for transition to adulthood, compared with 22% of nepl youths. weighted percentage of cshcn meeting maternal and children health bureau quality indicators by primary household language use source : the 2009 - 2010 national survey of children with special health care needs table 3 includes the results of the multivariable logistic regression analysis for adjusted odds ratios of not meeting each of the quality indicators, while controlling for child s age, race / ethnicity, gender, insurance and household poverty status, and family structure. nepl families had 31% higher odds of not feeling like partners in decision making (or=1.31, 95% ci=1.16, 1.48). they also had 67% higher odds of lacking care through a medical home (or=1.67, 95% ci=1.46, 1.90) and 42% higher odds of reporting inadequate health insurance (or=1.42, 95% ci=1.26, 1.59). nepl children had 32% higher odds of not receiving early and continuous screening for special health care needs (or=1.32, 95% ci=1.15, 1.51). in addition, nepl youths were more likely to report not receiving necessary services for transition to adulthood (or=1.69, 95% ci=1.3, 2.2). access to community based services was not associated with household language use. adjusted odds of us children with special health care needs (cshcn) not meeting health care quality indicators source : the 2009 - 2010 national survey of children with special health care needs minority race / ethnicity, lower incomes, and families other than two biological or adopted - parents households all conferred additional risks to not meeting quality indicators. publicly insured or uninsured cshcn were also at higher risk of not meeting quality indicators. our study provides compelling evidence that significant disparities exist for cshcn by primary household language status across all quality indicators examined. results of multivariable analyses further substantiate the independent effects of language barriers on the overall wellbeing of cshcn. since the lep population is a subset of nepl households, the risks of not attaining quality indicators for care identified in our study are likely underestimated for the true lep population. cshcn may also be underdiagnosed in lep populations because parts of the case definition such as prescription medication and services use are based on access to health care. compared to reports from the 2005 - 2006 national survey for children with special health care needs, nepl families continue to report access barriers to medical home as well as insurance coverage adequacy. more importantly, cshcn from nepl families are at an even greater disadvantage in receiving services for early and continuous screening for special health care needs and transition to adulthood services compared to the previous survey. the surveillance and clinical evaluation provided through early and continuous screening are pivotal for clinicians and public health professionals to carry out early identification and detection of cshcn for enrollment in early intervention (ei) services who may be at risk for developmental delays. ei services have a substantial, positive impact on the developmental trajectory and long - term outcomes for cshcn with developmental delay and different types of disabilities. during this critical, sensitive period in the life course, it is important that systems of care for cshcn continue to extend outreach efforts to nepl families. in particular, clinicians, allied health professionals, and other providers play critical roles in assisting children from culturally and linguistically diverse early intervention populations to reach developmental milestones and supporting their families in understanding public health system. similarly, cshcn from nepl require improved access to transition services for successful entry into adulthood with respect to post - secondary education, employment, and independent living. clinicians will need to work in collaboration with youth with special health care needs and their nepl families for the development of a medical transition plan focused on seamless connection to clinical adult providers, especially to address needs of parents from immigrant households with regards to assistance with navigation through the often unfamiliar and complex health care delivery system in the us. the fact that cshcn from both epl and nepl households reported comparable experiences on ease of access to community - based services is a reassuring finding. this may have resulted from the creation and expansion of ethnic community - based organizations (cbos) serving specific immigrant populations that built stronger civic ethnic communities and engendered higher levels of political trust and social engagement among their members. as the number of immigrant families in the us increases over time, strategic action plans must address the needs of cshcn from these families, as well as clinicians, and public health professionals who serve them. service providers will need to advance their understanding of the cultural context and life experiences of immigrant families, in particular to leverage cbos that have become important cultural brokers for immigrant families as effective intermediaries to connect families to the complex system of service delivery in the us. first, the 2009 - 2010 ns - cshcn is a cross - sectional survey conducted in english, spanish, and four asian languages, with the screener being in english or spanish. this may bias the non - english respondents towards those who are more educated and fluent in english, resulting in a likely underestimate of risk for the actual us immigrant populations. second, the cross - sectional nature of the survey data collections does not allow causal inferences. thirdly, undocumented families (i.e., those living in the us without a legal status) would likely not have been included in the survey. finally, in addition to identifying a language barrier, household language is also a proxy for immigrant household status and the length of stay of the family in the united states. while asian ethnicity was not disclosed in this public - use dataset, we can infer that nepl future research will need to explore partnerships among cbos, government agencies and immigrant communities with particular emphasis on evidence - based interventions that provide effective and optimal services for cshcn from immigrant families. finally, ongoing surveillance through mchb s six quality indicators for cshcn from immigrant families will provide the necessary monitoring and assurance for this underserved population. among the 25 organisation for economic cooperation and development (oecd) countries, the us ranks poorly in its social and economic inclusion of people with disabilities and in its disability benefit, compensation, and integration policies. the worldwide immigration of children to the us has risen significantly from countries with diverse health systems and cshcn. our study has demonstrated that cshcn including children with disabilities from immigrant families confront challenges regarding inadequate access to high - quality health care that warrants further policy solutions to improve their health care utilization and to reduce health disparities for cshcn from immigrant populations both in the us and abroad. with the continual increase in prevalence of cshcn in the us and aboard, the global burden will likely rise.significant health disparities exist in the timely receipt of early and continuous screening, comprehensive care within a medical home, and insurance adequacy for cshcn from nepl household.strategic action plans must address the needs of cshcn from nepl families through the cultural context and life experiences of immigrant families, in particular to leverage ethnic community - based organizations (cbos) that have become important cultural brokers for immigrant families as effective intermediaries in navigation of the complex health care system of service delivery in the us. with the continual increase in prevalence of cshcn in the us and aboard significant health disparities exist in the timely receipt of early and continuous screening, comprehensive care within a medical home, and insurance adequacy for cshcn from nepl household. strategic action plans must address the needs of cshcn from nepl families through the cultural context and life experiences of immigrant families, in particular to leverage ethnic community - based organizations (cbos) that have become important cultural brokers for immigrant families as effective intermediaries in navigation of the complex health care system of service delivery in the us. | background : lower health care utilization and less favorable health outcomes have been demonstrated in children from non - english primary language households (nepl) in previous studies. this study examines prevalence of health care quality indicators among us children with special health care needs (cshcn) and their association with household language use.methods:we used data from the 2009 - 2010 national survey of children with special health care needs, restricted to an analytic sample of 40,242 children. logistic regression models were used to examine the effects of primary household language on the attainment of the 6 health care quality indicators for cshcn.results:compared to cshcn from english primary language households (epl), cshcn from nepl households had 31% higher odds of not feeling like partners in health care decision - making. they had 67% higher odds of lacking care through a medical home and 42% higher odds of reporting inadequate health insurance. nepl children had 32% higher odds of not receiving early and continuous screening for special health care needs. nepl youths had 69% higher odds of not receiving services for transition to adulthood. minority race / ethnicity, lower income and families other than two biological parents all conferred additional risks to not attaining quality indicators. publicly insured or uninsured cshcn were also at higher risk.conclusions and global health implications : our study provides compelling evidence that significant disparities exist for cshcn by primary household language status across all health care quality indicators. establishment of effective surveillance systems and targeting of outreach programs in both developed and developing countries may lead to improved understanding of health care needs and quality of services and reduction of health disparities for this underserved population. |
aspirin (acetylsalicylic acid) is one of the most frequently used drugs, exploiting its anti - inflammatory and anti - platelet actions for the prevention of cardiovascular disease 1. especially, for this purpose, older people are commonly medicated with this agent 2. the so - called ' aspirin resistance (or aspirin non - responsiveness) syndrome ' is characterized by an incomplete inhibition of platelet function by aspirin 2, 3. it may account for the high variability in effective anti - coagulant doses of aspirin in the population 2, 3. increased hydrolysis of circulating aspirin, pharmacokinetically corresponding to faster elimination of the drug from the circulation by transforming it into salicylate and acetate, is thought to participate as a causal factor in the aspirin resistance 4, 5. although the hepatocyte accounts for most of aspirin catabolism, after the first pass, more than 50 % of the aspirin still gets to the systemic circulation where two distinct aspirin hydrolysis pathways act : a spontaneous ph - dependent hydrolysis and an enzymatic hydrolysis by plasma / serum and erythrocyte esterases 6. the circulating aspirin esterase activities are mainly due to butyrylcholinesterase (bche, pseudocholinesterase) and, in part, to albumin 6. because of higher aspirin esterase activity leading to faster aspirin hydrolysis, the same dose of aspirin in people who metabolize aspirin slowly is more effective than in those who metabolize the drug quickly 4, 5. there is a wide range of rates of aspirin metabolism in the population, and ethnic and individual variations exist 7, 8. even for a given individual, age - related factors may be also contributory to the range of turnover rates 8. however, despite the universal use of aspirin, the underpinnings of the etiology of aspirin resistance remain unclear as is the putative relative role of peripheral (serum) and central (hepatic) metabolism 4 - 6, 8. recently, a cross - sectional study has reported aspirin esterase activity to be increased in type 2 diabetics and has shown the association to be modulated by circulating lipid metabolism 6. in addition, aspirin resistance has been previously reported in patients with diabetes and cardiovascular disease 4, 5. however, research on variations of aspirin esterase activity in pathological and physiological states, including the influences of therapeutic lifestyle modifications, is very scarce. we set out this study to explore preliminarily the effects of cardio - metabolic variables such as circulating cholesterol on serum aspirin esterase activity among older people who participated in an intervention study on physical activity. a total of 18 community - dwelling japanese older volunteers (7 men/11women ; mean age = 67.8 7.7 years [range = 56 - 81 years ]), who were not current smokers, not on any medication and had not been diagnosed to have cardiovascular, renal, kidney, thyroid and nutritional diseases, were recruited into a 3-month interventional program for health promotion. the program, which focused on the health benefits of a mild - to - moderate increase in physical activity, included monthly explanatory and motivational classes to increase physical activity such as walking and to instruct on the appropriate methods. the jichi medical university ethics committee approved the present study and each subject gave informed consent. the body mass index (bmi) was calculated as the weight divided by the square of the height measured in light indoor clothing without shoes. serum aspirin esterase activity was measured kinetically by a modification (optimized in our laboratory) of the procedure described initially by sorensen 9. the reagent buffer contained tris - hcl (0.6 mol / l) and cacl2 (0.4 mol / l) in ph 7.6 - 7.7. mmol / l of acetylsalicylic acid (1 g in 10 ml 100 % ethanol was used as the mother solution). briefly, 15 l of serum sample was pipetted per well (in duplicate) into a 96 well uv flat bottom plate (thermo 8404). substrate was extemporaneously diluted 1 in 50 into reagent buffer, and then 200 l of this final substrate solution was added per well with a multi - channel pipette. the runs were blanked against reagent (to control for spontaneous hydrolysis of acetylsalicylic acid). plates were placed in a temperature - controlled plate reader spectramax uv from molecular devices, and samples were kinetically read at 300 nm, 37c for 15 min, every 57 sec. data were expressed in nmol of acetylsalicylic acid hydrolyzed per minute and per milliliter (nmol / ml / min). the intra - assay cv (coefficient of variation) is 4% and the inter - assay 5%, respectively. data are shown as the mean standard deviation or the median [interquartile range ]. wilcoxon test was used to compare the pre- and post - interventional values of respective measured variables. the level changes were calculated by subtracting the pre - interventional values from the post - intervention values. a single linear regression analysis (pearson 's rank correlation test) and a multiple linear regression analysis adjusted for confounders the log - transformed values of aspirin esterase were used in these correlation analyses because of the skewed distribution. a value of p < 0.05 was considered to be significant. the intervention on physical activity reduced weakly but significantly the bmi levels (23.4 3.3 kg / m at baseline to 23.1 3.0 in post - intervention, p = 0.037). the averaged levels of other measured variables between pre- and post - intervention were unchanged at statistical significant levels : 5.7 1.1 to 5.8 1.1 mmol / l in total cholesterol, 5.6 0.8 to 5.5 1.1 mmol / l in glucose, 41.4 [36.0 - 50.0 ] to 39.8 [34.6 - 48.1 ] nmol / ml / min in aspirin esterase activity. at baseline (pre - intervention), aspirin esterase activity levels were correlated significantly and inversely to age (r = -0.569, p = 0.014). after adjustment for sex, aspirin esterase had a significant positive correlation with age (r = -0.548, p = 0.020), but the significance of this correlation was lost after adjusting for sex and bmi (r = -0.406, p = 0.113). the results were not affected by adjustment for total cholesterol and glucose levels. during the intervention period, the changes in aspirin esterase activity levels were correlated significantly and positively with those of total cholesterol concentrations (table 1). moreover, both variables showed a significantly and independently positive correlation after the adjustments for age (r = 0.534, p = 0.021), age and sex (r = 0.475, p = 0.035) as well as age, sex and the changes in bmi (r = 0.618, p = 0.018). the results on the significant, independent and positive correlation between total cholesterol and aspirin esterase were not affected by adjustment for all the measured variables (table 1). the present study 's main finding is that the changes in serum total cholesterol concentrations are clearly associated with those of aspirin esterase activity during a period of physical activity modification in an older population. this finding suggests cholesterol metabolism alterations may be important in mediation of aspirin metabolism among older people. a recent report has shown that greater aspirin hydrolysis can be associated with decreased cholesterol levels of high - density lipoprotein in type 2 diabetics 6. although our present results are not strictly comparable to those of this previous report since that evidence was obtained in diabetics, both studies potentially seem to bring our attention to the notion that aspirin hydrolysis rates may be linked to cholesterol metabolism 6. whereas the molecular mechanisms underlying the modulation of circulating aspirin esterase activity remain to be elucidated, our data showing its significant correlations with the changes in cholesterol concentrations may open new avenues for exploration of aspirin metabolism. our present results of improvement of aspirin effect with decreased total cholesterol may partly account for non - effectiveness of aspirin in cardiovascular disease among patients with concurrent increases of total cholesterol levels 4, 5 ; thus, emphasizing the clinical importance of control of total cholesterol with a mild - to - moderate increase of physical activity among such patients. while age - related reductions in the activities of several enzymes are known 2, data regarding the correlation of age to aspirin esterase are inconsistent 2, 10 - 12. some studies reported a decrease of aspirin esterase in unhealthy, frail older subjects 10 - 12. our present results at baseline only showed an inverse correlation of age to aspirin esterase activity, but the present study population was not frail, and bmi was also a confounder of this correlation. bmi is a surrogate clinical marker for various common clinical syndromes ; thus, more research is needed to ascertain its role on the effects of aging on aspirin metabolism. this study had the limitation of a small sample size and a short - term interventional period. nevertheless, albumin has a very small contribution to the whole circulating aspirin esterase activity as compared to that of bche 8, and our study population did not include subjects with malnutrition nor there were any with nutritional modifications during the interventional period. more studies with larger sample sizes and longer follow - up as well as including bche measurement and nutritional assessment will be considered in the future. in summary, we found a significant and positive association of changes in circulating total cholesterol with those of serum aspirin esterase activity in an older population that participated in a physical activity modification program. although aspirin metabolism is probably multi - factorial, the association with cholesterol metabolism has merits for further investigations. | objective : metabolism of aspirin (acetylsalicylic acid), commonly used in older people for the prevention of cardiovascular disease, is important to the effectiveness of this drug. whereas part of aspirin hydrolysis occurs in blood, there is a paucity of information in regards to circulating aspirin esterase activity in various physiological and pathological conditions. high aspirin esterase activity, corresponding to faster aspirin hydrolysis (thus aspirin non - responsiveness), may occur in cardiovascular disease - prone states. the objective of this study was to investigate the effects of cardio - metabolic variables such as cholesterol on serum aspirin esterase activity in older people who participated in an intervention study on physical activity.methods : a total of 18 non - medicated subjects (7 men/11 women, mean age 67.8 years, body mass index = 23.4 3.3 kg / m2), who completed a 3-month interventional program for a mild - to - moderate increase in physical activity, were analyzed. the body mass index, plasma glucose, serum total cholesterol and aspirin esterase activity were measured in the pre- and post - interventional phases of the study.results : during the interventional period, the changes in aspirin esterase activity correlated significantly and positively with those of total cholesterol concentrations (r = 0.542, p = 0.020 ; = 0.609, p = 0.035 in a multiple linear regression analysis after adjusting for all the measured variables).conclusion : the results suggest that cholesterol metabolism alterations may be associated with aspirin metabolism in older people. |
acute kidney injury (aki) remains a common and significant problem in the last decade. between 5% and 20% of critically ill patients in the intensive care unit (icu) have an episode of aki, with acute tubular necrosis (atn) accounting for about 75% of cases [13 ]. despite significant advances in both critical care and nephrology, the mortality rate of hospitalized patients with aki has remained relatively unchanged at around 50% over the past few decades. the most common causes of aki are septic shock, ischemia, and nephrotoxins. aki has been defined conceptually as a rapid decline in glomerular filtration rate (gfr) that occurs over hours and days. it propels to a clinical syndrome characterized by a rapid decrease in renal excretory function, with the accumulation of products of nitrogen metabolism such as creatinine and urea clinically unmeasured waste products. the described notions have led to a consensus definitions of aki by the acute dialysis quality initiative. these rifle (risk, injury, failure, loss, end stage) criteria have been broadly supported with minor modifications by the acute kidney injury network (akin) [5, 6 ], and both definitions have now been validated in thousands of patients. the akin group attempted to increase the sensitivity of the rifle criteria by recommending that a smaller change in serum creatinine (0.3 mg / dl) be used as a threshold to define the presence of aki and identify patients with stage 1 aki (rifle - risk). in the akin classifications of aki, however, there still remains much improvements to be made in increasing the sensitivity of current markers of aki. its use in the diagnosis of aki remains a problem ; however, as it often requires as much as a 50% loss in renal function before creatinine levels rise. it is dependent on nonrenal factors independent of kidney function (age, sex, muscle mass, infection). several medications (trimethoprim, cimetidine, and salicylates) alter the tubular secretion of creatinine, leading to changes in serum creatinine independent of gfr [9, 10 ]. these numerous problems with creatinine limit both clinical practice and the development of new therapeutics in aki. clinicians need tools that are not influenced by other clinical parameters or patient characteristics and that can identify losses in gfr soon after occurence. the ideal biomarker of aki would be a substance that the kidney releases immediately in response to injury and that can be detected in the blood or urine without significant metabolism. extensive research efforts over this past decade have been directed at the discovery and validation of novel aki biomarkers to detect injury prior to changes in kidney function and potentially to aid in the differential diagnosis of aki. the purpose of this paper is to review the relevant aki biomarker literature ofwhich the search performed on medline / pubmed using the search terms acute kidney injury, urine and serum new biomarkers and articles were selected from all publication types in english. human ngal is a 25 kda protein firstly identified bound to gelatinase in specific granules of the neutrophil. ngal is a critical component of innate immunity to bacterial infection and is expressed by immune cells, hepatocytes, and renal tubular cells in various disease states [11, 12 ]. it is synthesized and secreted by tubular epithelial cells of the proximal and distal segment. it is freely filtered by the glomerulus, undergoing rapid clearance by the proximal tubule via receptor binding and endocytosis. in healthy kidneys, however, in the setting of acute tubular injury, ngal undergoes rapid and profound upregulation with large increases in both urine and plasma. distinct from traditional markers of function such as creatinine, this rapid response enables ngal to potentially identify injured kidney much earlier than was previously possible. it seems to be involved with iron transportation to and from the proximal tubular epithelial cells, and animal studies demonstrate a renoprotective effect of exogenously administered ngal in the setting of acute ischemic injury [1315 ]. the appearance of ngal in the urine preceded the appearance of the other urinary markers such as the tubular proteins n - acetyl - beta - d - glucosaminidase and beta2- migroglobulin. studies in cultured human proximal tubule cells subjected to in vitro hypoxic injury confirmed the origin of ngal from tubule cells. ngal was also detected in the urine of mice in the early stage of cisplatin - induced nephrotoxicity. these animal studies demonstrated that ngal may represent an early, sensitive, and noninvasive urinary biomarker for ischemic and nephrotoxic kidney injury. urinary and serum ngal were demonstrated to be sensitive, specific, and highly predictive early biomarkers of aki in children after cardiac surgery. seventy - one children undergoing cardiopulmonary bypass were studied and 20 children developed acute renal injury, but diagnosis with serum creatinine was only possible 13 days after cardiopulmonary bypass. microg / l at baseline to 147 microg / l 2 h after cardiopulmonary bypass, and the amount in serum increased from a mean of 3.2 microg / l at baseline to 61 microg / l 2 h after the procedure. ngal demonstrated a near - perfect performance for identifying aki after pediatric cardiac surgery with an area under the receiver operator characteristic curve (auc roc) of 0.99 and 1.0 at 2 and 4 h after cardiopulmonary bypass (cpb), respectively. they were divided into group i, patients who suffered aki grades ii and iii, and group ii, patients who did not develop aki. the results showed that there were highly significant differences in plasma ngal levels between the groups ; this significant difference started as early as 2 h after surgery, which reflects the potential role of plasma ngal as an early biomarker in predicting aki. the another study of 374 children undergoing cpb, plasma, and urine ngal significantly increased in aki patients at 2 h after cpb and remained elevated for at least 48 hours, with the 2 h ngal being the earliest and strongest independent predictor of aki. 2-hour plasma and urine ngal thresholds strongly correlated with length of hospital stay and severity and duration of aki.. the auc for plasma and urine ngal at various time points after cpb ranged from 0.88 to 0.97 indicating that both are excellent predictors of aki. these decision have been confirmed in some studies of adults undergoing cardiac surgery [2229 ]. however, results in adult cardiac surgery have been mixed and the auc for the prediction of aki have ranged widely from 0.61 to 0.96. the auc roc for early diagnosis of aki by urinary ngal has varied from 0.61 at 18 h after cpb to 0.96 at 2 h after cpb. similarly, performance of plasma ngal for the diagnosis of aki has varied from an auc roc of 0.54 within 6 h of cpb to 0.87 at 24 h after cpb. the additional comorbidities in adult populations may be reflective of confounding variables such as age, preexisting kidney disease, bypass time, chronic illness, and diabetes. ngal may also represent early sensitive biomarker of aki after contrast administrations for coronary angiography. ngal was measured in the serum and urine before and at 2, 4, 12, 24, and 48 h after contrast administration. they found a significant rise in serum ngal 2 and 4 h after percutaneous coronary intervention (pci), and a rise in urinary ngal 4 and 12 h after pci. a similar study of 25 patients with normal serum creatinine undergoing pci due to unstable angina. both markers increased significantly after 4 hours and remained elevated up to 48 hours after pci. lastly, a study of 30 patients with normal serum creatinine undergoing coronary angiography were evaluated. there was a significant increase in serum ngal level 4 hours and 24 hours after coronary interventions compared to the baseline value before coronary angiography. ngal is an early predictive biomarker of contrast - induced nephropathy (cin) in children. they studied 91 children with congenital heart disease undergoing elective cardiac catheterization and angiography with contrast administration. cin, defined as a 50% increase in serum creatinine from baseline, was found in 11 subjects (12%), but detection using increase in serum creatinine was only possible 624 h after cardiac catheterization (cc). in contrast, significant elevation of ngal concentrations in urine and plasma was noted within 2 h after cc. by multivariate analysis, the 2 h ngal concentrations in the urine and plasma were found to be powerful independent predictors of cin. with respect to other common nephrotoxins, early urinary ngal measurements may be useful for the prediction of cisplatin, vancomycin, or cyclosporine - associated nephrotoxicity [3537 ]. urine and plasma ngal levels also represent early biomarkers of aki in an intensive care unite (icu) [19, 38 ]. a multicenter study of serum ngal was performed in 143 critically ill children with systemic inflammatory response syndrome (sirs) or septic shock during the first 24 h of admission to the icu. there was a significant difference in serum ngal between healthy children, ill children with sirs, and septic shock. the study was concluded that serum ngal is a highly sensitive but nonspecific predictor of acute kidney injury in critically ill children with septic shock. a 2010 study evaluated 88 icu patient and found that an ngal level of 150 nmol / l predicted aki with 82% sensitivity and 97% specificity. a recent study had 98 patients who were divided in two groups depending on the presence of sepsis. a more study of 65 patients with septic shock were found that urine ngal levels 12 hours before aki diagnosis were a good predictor of aki. ngal has been evaluated as a biomarker of delayed graft function (dgf) in a patient undergoing kidney transplantation. hollmen and colleagues demonstrate that donor urine ngal is similarly useful to help with the preharvest prediction of dgf after renal transplantation from deceased donors. specifically, they studied preharvest serum and urine ngal levels in 99 consecutive, deceased donors, and followed the clinical courses of their 176 kidney recipients. they found that high preharvest urine ngal levels were more common in those who developed prolonged dgf. in receiver operating characteristic (roc) curve analysis, donor urine ngal had poor utility for prediction of dgf (area under the curve 0.595) or prolonged dgf (area under the curve 0.616). however, increased donor urine ngal was a significant risk factor for prolonged dgf. in others prospective studies, urine ngal levels in sample collected on the day of transplant identified those who developed dgf with an auc of 0.8 - 0.9 [50, 51 ]. ngal is an early biomarker of aki in children and adults in the following situations : postcardiopulmonary bypass, after contrast administration, nephropathy, in critically ill icu patients, and delayed graft function in patients undergoing kidney transplantation. kidney injury molecule-1 (kim-1) is a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain. kim-1 mrna and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney [5254 ]. kim-1 has been identified as the first nonmyeloid phosphatidylserine receptor that confers a phagocytic phenotype on injured epithelial cells both in vivo and in vitro. urinary kim-1 has been found to be an early indicator of aki that compares favorably to a number of conventional biomarkers and tubular enzymes [53, 56 ]. kim-1 is also a tissue and urinary biomarker for nephrotoxicant - induced kidney injury. tissue and urinary expressions were measured with different nephrotoxic doses of cisplatin, folic aside, cadmium, gentamycin, mercury, and chromium [53, 5760 ]. as early as 1 day after cisplatin treatment, positive kim-1 immunostaining, observed in the outer medulla of the kidney, and changes in urinary clusterin indicated the onset of proximal tubular injury in the absence of functional effects. tissue kim-1 was the most sensitive biomarker for detection of cisplatin - induced kidney damage. urine samples were collected from 32 patients with various acute and chronic kidney diseases, as well as from eight normal controls. there was extensive expression of kim-1 in proximal tubule cells in kidney biopsies from six patients with biopsy confirmed acute tubular necrosis (atn). urinary kim-1 levels were significantly higher in patients with ischemic atn compared to levels in patients with other forms of acute renal failure or chronic renal disease. adjusted for age, gender, and length of time delay between the initial insult and sampling of the urine, a one - unit increase in normalized kim-1 was associated with a greater than 12-fold (or 12.4, 95% ci 1.2 to 119) risk for the presence of atn. the aucs for kim-1 to predict aki immediately and 3 h after operation were 0.68 and 0.65, 0.61 and 0.63 for nag, and 0.59 and 0.65 for ngal, respectively. combining the three biomarkers enhanced the sensitivity of early detection of postoperative aki compared with individual biomarkers : the aucs for the three biomarkers combined were 0.75 and 0.78. a similar study described that preoperative kim-1 and -gst were able to predict the future development of aki. in hospital patients with aki, urinary levels of kim-1 that higher levels correlated with a higher odds ratio for dialysis requirement or hospital death. this study demonstrated that urinary biomarker of aki such as kim-1 can predict adverse clinical outcomes in patients with aki. renal kim-1 expression is significantly increased in human kidney tissue among patients with a wide range of kidney diseases, including various types of glomerulonephritis, chronic allograft nephropathy, acute rejection, immunoglobulin a nephropathy, systemic lupus erythematosus, diabetic nephropathy, hypertension, and wegener 's granulomatosis. both renal and urinary kim-1 correlate with kidney damage and negatively with renal function, but not with proteinuria. a recent study has explored urinary kim-1 correlated with kidney function in kidney allograft recipients. this study concluded that even a successful kidney transplantation is associated with kidney injury as reflected by elevated urinary kim-1. in a similar study explored urinary biomarkers in 63 renal transplant recipients who require graft biopsy because of progressive worsening of kidney function. they reported that the rate of renal function decline significantly correlated with urinary kim-1 expression after being followed for an average of 39.7 months. in kidney allograft recipients, urinary kim-1 expression provides prognostic information in relation to the rate of renal function decline. it is freely filtered by the glomerulus, completely reabsorbed by the proximal tubules and is not secreted by the renal tubules. in this way, some of the limitations of serum creatinine, effect of muscle mass, diet, gender, and tubular secretion may not be a problem with cystatin c and appear to rise one to two days earlier than serum creatinine. cystatin c is better marker of gfr than serum creatinine as demonstrated in several studies [7077 ]. the ability of serum cystatin c to identify a creatinine rate a schwartz creatinine clearance rate under 80 ml / min/1.73 m was better than creatinine (areas under the roc curve : 0.85 and 0.79 for cystatin c and 0.63 and 0.62 for creatinine). this study concluded that serum cystatin c was better than serum creatinine to detect aki in critically ill children. forty - four patients developed aki and the increase of cystatin c significantly preceded that of creatinine. specifically, serum cystatin c increased already by more than 50% at 1.5 0.6 days earlier compared to creatinine. serum cystatin c demonstrated a high diagnostic value to detect aki as indicated by area under the curve of the roc analysis of 0.82 and 0.97 on the two days before the r - criteria (risk of kidney dysfunction) was fulfilled by creatinine. aki was defined as a 25% or greater increase in plasma creatinine or renal replacement therapy within the first 72 hours following surgery. plasma cystatin c and ngal did not predict the development of aki within the first 6 h following surgery. however, both urinary cystatin c and ngal were increased in the 34 patients who later developed aki. the urinary cystatin c at 6 h after icu admission was the most useful for predicting aki. a recent study compared the sensitivity and rapidity of aki detection by cystatin c level relative to creatinine level after 150 high - risk adult patients in the cardiac surgery. serum creatinine level detected more cases of aki than cystatin c level, 35% developed a 25% increase in serum creatinine level, whereas only 23% had a 25% increase in cystatin c level. this study concluded that cystatin c level was less sensitive for aki detection than creatinine level. another study in 2011 examined presurgical values for cystatin c, creatinine, and creatinine - based estimated glomerular filtration rate (egfr) in 1147 adults undergoing cardiac surgery for high risk aki. presurgical cystatin c is better than creatinine or egfr at prediction risk of aki after cardiac surgery. abnormalities of thyroid function and glucocorticoid therapy may affecte cystatin c independently for kidney function. these situations are limitations to the use of cystatin c as a marker of gfr [8183 ]. early determination of allograft function and prognosis could lead to the development of therapies for kidneys with significant ischemia - reperfusion injury (iri) and more effective recipient management, thereby improving outcomes. a study analyzed urine cystatin c in 91 patients who received deceased - donor kidney transplants to determine its peritransplant excretion pattern, utility for predicting delayed graft function, and association with 3-month graft function. the area under the curve for predicting dgf at 6 h was 0.69 for urine cystatin c and 0.74 for urine cystatin c / urine creatinine. the urine cystatin c / urine creatinine ratio on the postoperative day was associated with 3-month graft function. it is synthesized as an inactive 23 kda precursor by several tissues including monocytes, macrophages, and proximal tubular epithelial cells. animal studies demonstrated that the caspase-1-mediated production of il-18 plays a deleterious role in aki. parikh showed that urinary ngal and il-18 represent early biomarkers for dgf. in patients with dgf, urine ngal and il-18 were elevated in the first day after transplant in patients with dgf. the receiver - operating characteristic curve for prediction of dgf based on urine ngal or il-18 at day 0 showed an area under the curve of 0.9 for both biomarkers. the same author reported that increased levels of il-18 in patients with aki of varying etiology, especially those with delayed renal allograft function and ischemic atn. in kidney transplant recipients, lower urinary il-18 levels were associated with a steeper decline in serum creatinine concentrations postoperative days from 0 to 4. immunohistochemical staining of protocol biopsies showed constitutive il-18 expression in the epithelium of distal tubules with the induction of immunoreactivity in acute rejection patients where also proximal tubules, infiltrating leukocytes, and endothelium were strongly positive. furthermore, serum levels of il-18 were significantly elevated in patients with acute rejection of kidney allograft as compared to patients with uncomplicated outcome of kidney transplantation and subjects with acute tubulointerstitial nephropathy. in a study of critically ill adult patients with acute respiratory distress syndrome, increased il-18 was found to be an early marker of aki and it was an independent predictor of death. the area under the receiver operating characteristic curve for urinary il-18 predicting subsequent aki within 24 hours was 0.62. it was found that urinary il-18 remained independently predictive of composite outcome of death or acute dialysis within 28 days of ascertainment (odds ratio, 1.86).. the predictive ability of urinary il-18 has been demonstrated in one hundred thirty - seven critically ill children. the peak levels of il-18 correlated with the severity of aki. in nonseptic aki patients, urinary il-18 rises to a level higher than control levels 2 days prior to a significant rise in creatinine. urinary il-18 concentration from the first urine specimen was associated with aki development within 48 h (odds ratio = 3.5) independent of the pediatric risk of mortality. this study concluded that urinary il-18 rises prior to serum creatinine in nonseptic critically ill children, predicts severity of aki, and is an independent predictor of mortality. the study tested whether urinary il-18 is a predictive biomarker for aki in patients undergoing cpb. these study results indicated that urinary il-18 is an early, predictive biomarker of aki after cpb. 311 children undergoing surgery for congenital cardiac lesions to evaluate whether early postoperative measures of urine il-18, urine ngal, or plasma ngal could identify which patient would develop aki. elevated urine il-18 and urine ngal levels associated with longer hospital stay, longer intensive care unit stay, and duration of mechanical ventilation. the same author also examined urine il-18, urine, and plasma ngal markers in adults cardiac surgery. they found that urine il-18 and plasma ngal at six hours were strongly associated with risk of aki. l - fabp is a family of carrier proteins for fatty acids and other lipophilic substances such as eicosanoids and retinoids. it was correlated with the degree of tubulointerstitial damage in a folic acid induced nephropathy. fabp expression and urinary excretion have been described in several animal models of aki [100103 ]. in ischemic and cisplatin - induced aki, l - fabp was increased in the urine before the increase in bun. in cisplatin - induced aki, urinary l - fabp levels increased exponentially even in the lowest dose group as early as 2 hours, whereas bun levels increased at 48 hours. in ischemia reperfusion - induced aki, bun levels increased only in the 30-minute ischemia group 24 hours after reperfusion, however, urinary l - fabp levels increased more than 100-fold, even in the 5-minute ischemia group after 1 hour. in both aki model, urinary l - fabp showed a better correlation with histology injury scores and gfr. urine l - fabp was measured in 40 pediatric patients prior to and following cardiopulmonary bypass surgery. urinary l - fabp levels at 4 h after surgery were an independent risk indicator with the area under the receiver - operating characteristic curve 0.810, sensitivity 0.714, and specificity 0.684 for a 24-fold increase in urinary l - fabp. this study demonstrated that urinary l - fabp levels represent a sensitive and predictive early biomarker of aki after cardiac surgery. a recent study was undertaken to evaluate urinary l - fabp and nag for aki diagnosis in adult post - cardiac surgery patients. urinary l - fabp and nag were significantly increased. however, roc analysis revealed that the biomarkers ' performance was statistically significant but limited for clinical translation, auc - roc for l - fabp at 4 hours 0.72 and nag 0.75. urinary l - fabp levels in patients with septic shock were significantly higher than severe sepsis without shock. serum l - fabp levels did not have significant differences between patients with septic shock, severe sepsis, and healthy. urinary l - fabp measured at admission was significantly higher in the nonsurvivors of septic shock with established acute kidney injury than in the survivors and with an auc for mortality prediction of 0.99. l - fabp was evaluated as a biomarker of renal ischemia in both human kidney transplant patients and a mouse model of aki. in 12 living related kidney transplant patients after reperfusion of their transplanted organs, and intravital video analysis of peritubular capillary blood flow was performed. a significant direct correlation was found between urinary l - fabp level and peritubular capillary blood flow, ischemic time of the transplanted kidney as well as hospital stay. a recent study examined the ngal and l - fabp in diagnosis aki in liver transplant recipients. urinary ngal was slightly elevated at 2 h in the non- aki patients while rose and stayed high from 2 to 6 h in the aki patients. however, urinary l - fabp rose transiently in both patients 2120 h following surgery. the level of urinary ngal presented differences at 26 h and urinary l - fabp at 4 h between aki and non - aki patients. this study concluded that urinary ngal rather than l - fabp appeared to be a sensitive and specific marker of aki in liver transplant recipients. there are multiple promising serum and urinary biomarkers, ngal, kim-1, cystatin c, il-18, and l - fabp, which detect aki before the rise in serum creatinine. however, serum creatinine is still the major determinant of kidney function. determination of biomarkers of aki in patients with aki due to different causes, sepsis, ischemia, nephrotoxins, and contrast is important to monitoring. establishing the optimal biomarkers for a clinical situation must require prospective validation in large numbers of patients with aki and needs to be performed in different critically ill populations. these new biomarkers must be worked together to develop better definitions of aki, probably in conjunction with serum creatinine, gfr, and urine output. | acute kidney injury (aki) is a common and strong problem in the diagnosis of which based on measurement of bun and serum creatinine. these traditional methods are not sensitive and specific for the diagnosis of aki. aki is associated with increased morbidity and mortality in critically ill patients and a quick detection is impossible with bun and serum creatinine. a number of serum and urinary proteins have been identified that may messenger aki prior to a rise in bun and serum creatinine. new biomarkers of aki, including ngal, kim-1, cystatin - c, il-18, and l - fabp, are more favourable tests than creatinine which have been identified and studied in several experimental and clinical training. this paper will discuss some of these new biomarkers and their potential as useful signs of aki. we searched the literature using pubmed and medline with acute kidney injury, urine, and serum new biomarkers and the articles were selected only from publication types in english. |
generally, heparin is used to control circuit coagulation during hemodialysis ; however, it does affect the systemic blood coagulation. for patients at high risk of bleeding, alternative modalities, such as saline flushes, regional citrate anticoagulation, prostacyclin, danaparoid, argatroban (direct thrombin inhibitor), and lepirudin (recombinant hirudin), it has been studied for its anticoagulant effect and used clinically in hemodialysis since 1989. nafamostat mesilate inhibits various enzyme systems, such as coagulation and fibrinolytic systems (thrombin, xa, and xiia), the kallikrein kinin system, the complement system, and pancreatic proteases. because it is not absorbed by the anion exchange resin it has a biological half - life of 8 minutes or less, and approximately 40% of the molecule is dialyzed through the dialyzer. because of the characteristics described previously, it has been used in patients who are at high risk of bleeding and those with heparin - induced thrombocytopenia. however, most allergic reactions reported that there were mild symptoms, such as abdominal pain, nausea, vomiting, anorexia, myalgia, and arthralgia. more serious symptoms such as severe hypotension and dyspnea, however, have also been reported in several cases. there have been no previous case reports of allergic reactions caused by nafamostat mesilate in korea. therefore, we would like to present a case of anaphylactic shock caused by nafamostat mesilate. a 65-year - old man was admitted to this hospital in march 2015 to establish new vascular access for dialysis. he was diagnosed with hypertension and diabetes in 2004 and underwent lower anterior resection for rectal cancer followed by radiotherapy in the same year. he was dialyzed through a permanent catheter placed in the right jugular vein at the time of admission. the polyflux 14l (polyarylethersulfone, polyvinylpyrrolidone, polyamide blend, steam sterilized ; gambro, lund, sweden) was used as a dialyzer, and heparin was applied as an anticoagulant. he underwent incision and drainage for treatment of a buttock abscess after having received dialysis 4 times. after the second dialysis after the operation, nafamostat mesilate was used because of bleeding from the incision and drainage site. after initiating dialysis with nafamostat mesilate, the patient complained of bilateral eye congestion and buttock pain ; sudden cardiac arrest occurred after 15 minutes of dialysis. laboratory results at that time were as follows : white blood cells 27.5 10/l (eosinophil 0.1%), hemoglobin 9.9 g / dl, hematocrit 30.5%, platelets 296 10/l, activated partial thromboplastin time 118.3 seconds, prothrombin time 13.2 seconds (international normalized ratio 1.26), serum creatinine 2.58 mg / dl, blood urea nitrogen 26.8 mg / dl, aspartate transaminase 35 u / l, alanine transaminase 20 u / l, total protein 4.8 g / dl, albumin 1.9 g / dl, na 131 mmol / l, k 3.3 mmol / l, cl 100 mmol / l, total co2 15 mmol / l, ca 7.1 mg / dl, phosphate 2.9 mg / dl, glucose 297 mg / dl, c - reactive protein 4.77 mg / dl, and lactic dehydrogenase level of 317 iu / l (normal range, 100450 iu / l). arterial blood gas showed ph 7.263, pco2 48.6 mmhg, po2 153.1 mmhg, hco3 21.5 mmol / l, and o2 saturation 98.7%. echocardiography results were as follows : normal left ventricular systolic function, no regional wall motion abnormality, left ventricular hypertrophy, and relaxation abnormality. norepinephrine was continuously infused for 2 days to maintain blood pressure, and continuous veno - venous hemodiafiltration was started without any anticoagulant because of constant bleeding from the site of the drained abscess on the buttock area. the patient was treated in the intensive care unit for 5 days, and there were no other complications during continuous veno - venous hemodiafiltration. conventional hemodialysis was reinitiated, and nafamostat mesilate was used again because of a small amount of continued bleeding on the buttock area. ten minutes after hemodialysis, the patient complained of a sensation of heat on the buttock area, dyspnea, facial swelling, and congestion of both eyes. blood pressure was 110/60 mmhg just before hemodialysis, but soon was unmeasurable, with only a weak pulse felt over the femoral artery. intramuscular epinephrine, intravenous dexamethasone, and intravenous pheniramin were injected under the suspicion of anaphylactic shock, and the blood pressure recovered to 80/50 mmhg. predialysis laboratory results were as follows : white blood cells 7.4 10/l (eosinophil 9.6%), hemoglobin 10.0 g / dl, hematocrit 30.1%, and platelet count 65 10/l. serum creatinine was 2.18 mg / dl, blood urea nitrogen 23.0 mg / dl, aspartate transaminase 24 u / l, alanine transaminase 8 u / l, total protein 5.3 g / dl, albumin 2.3 g / dl, na 137 mmol / l, k 4.3 mmol / l, cl 102 a skin prick test and total immunoglobulin e (ige) test were performed for evaluation of nafamostat mesilate allergy. nafamostat mesilate allergy was ultimately diagnosed based on a weak positive result on skin prick test and elevation of total ige. nafamostat mesilate is a serine protease inhibitor that inhibits various enzyme systems, such as coagulation and fibrinolytic systems (thrombin, xa, and xiia), the kallikrein kinin system, the complement system, and pancreatic proteases. first, it does not affect systemic coagulation ; second, it inhibits the complement system ; and third, it has no lipolytic activity. in addition, chika reported that nafamostat mesilate inhibited plasma bradykinin elevation and can prevent anaphylactic shock, which is induced by low - density lipoprotein apheresis with heparin. therefore, the use of nafamostat mesilate has advantages over heparin if the patients are at high risk of bleeding. yamazato reported a patient who complained of severe abdominal pain and throat irritation during the sixth hemodialysis run using nafamostat mesilate ; in that case, the drug lymphocyte stimulation test and specific ige were positive. akizawa reported that 3 of 66 patients complained of anorexia, general lassitude, slight myalgia, and arthralgia during hemodialysis using nafamostat mesilate, and symptoms disappeared after the end of dialysis. maruyama described the first case of an anaphylactoid reaction caused by nafamostat mesilate. their patient complained of nausea, vomiting, sweating, chest discomfort, and abdominal pain followed by shock. the drug lymphocyte stimulation test was positive for nafamostat mesilate, whereas specific ige was negative in the reported case. higuchi reported a patient with hypotension that occurred during his ninth hemodialysis run using nafamostat mesilate. in that case, the drug lymphocyte stimulation test for nafamostat mesilate was positive, but specific ige was negative. shunsuke reported a case of acquired allergic reaction to nafamostat mesilate during long - term maintenance of hemodialysis. similar to the case of shunsuke, our patient had a history of nafamostat mesilate exposure at least 1 year before, but there were no previous adverse events. unfortunately, the mechanism of nafamostat mesilate anaphylaxis remains unclear, as in the previously reported cases. nagase reported that the specific ige antibody to nafamostat mesilate has high sensitivity to allergic reaction. we could not conclude the exact mechanism of nafamostat mesilate anaphylaxis because specific ige was not tested in our case. nevertheless, elevated serum total ige and weak positivity to skin prick test indirectly suggested that the main mechanism might be related to ige - mediated allergic reaction. in conclusion, various etiologies, such as cardiovascular disease, sepsis, air embolism, and membrane reaction, must be evaluated as potential causes of cardiac arrest during dialysis, and anaphylactic shock due to drug allergy should also be considered. if patients complain of unusual symptoms repeatedly during dialysis using nafamostat mesilate, it is important to check their allergy histories and to perform a skin prick test for nafamostat mesilate. skin prick test is very useful because it is easy to perform and it provides results within few minutes. if an allergic reaction occurs, typical treatment for anaphylaxis including hydration, epinephrine, antihistamine, and steroid injection should be performed. | a 65-year - old man was transferred from the department of vascular surgery to nephrology because of cardiac arrest during hemodialysis. he underwent incision and drainage for treatment of a buttock abscess. nafamostat mesilate was used as an anticoagulant for hemodialysis to address bleeding from the incision and drainage site. sudden cardiac arrest occurred after 15 minutes of dialysis. the patient was treated in the intensive care unit for 5 days. continuous veno - venous hemodiafiltration was started without any anticoagulant in the intensive care unit. conventional hemodialysis was reinitiated, and nafamostat mesilate was used again because of a small amount of continued bleeding. ten minutes after hemodialysis, the patient complained of anaphylactic signs and symptoms such as dyspnea, hypotension, and facial swelling. epinephrine, dexamethasone, and pheniramin were injected under the suspicion of anaphylactic shock, and the patient recovered. total immunoglobulin e titer was high, and skin prick test revealed weak positivity for nafamostat mesilate. we first report a case of anaphylactic shock caused by nafamostat mesilate in korea. |
malign tumours of the face are generally seen in the periocular area, especially on eyelids [13 ]. malign tumours of periocular area are localized in the inferior eyelid, medial cantus and superior eyelid, respectively. the primary treatment for eyelid malign tumours is the total excision of the lesion with adequate depth and leaving the tissue border intact. some complications accompanying visual loss such as corneal ulcer and keratitis could be seen in defects after the wide excision of superior eyelid tumours. therefore, postoperative defects that are seen in eyelids have to be reconstructed near to normal anatomical structure after careful peroperative planning. tissues similar to normal eyelid structure have to be used in reconstructing superior eyelid full thickness defects. the cutler - beard flap is a good alternative in the reconstruction of superior eyelid defects. the cutler - beard procedure and its consequences were examined in our study after wide excision of superior eyelid malign tumours. the records of patients that had the cutler - beard procedure after superior eyelid malign tumour excision between march 2009 and january 2013 were examined retrospectively in dr. the age and gender of patients and size, localization, and width of defects were recorded. the histopathological diagnosis of the patients was examined. the surgical procedure and complications were noted. patients whose defects occupied less than 50% of the superior eyelid were excluded from the study. the cutler - beard flap was applied to patients with more than 50% of superior eyelid loss after surgical excision of the tumour. surgical excision margins were marked from the tumour border with 5 mm intact skin tissue. tumour excision was done with 15 number lancet and stevens scissor. after having clear surgical margins, the exact size of the defect was determined. the cutler - beard flap was prepared from the inferior eyelid below tars in order to be compatible with superior eyelid defect by doing a horizontal incision. the bridge flap was advanced to the superior eyelid below the eyelash margin by separating the anterior and posterior lamellae. the anterior and posterior lamellae were sutured separately to the levator muscle and the rest of orbicularis muscle by placing the flap on the defective area. the patients ' superior and inferior eyelids were separated from each other at 6 weeks postoperatively. the four patients included in this study had the cutler - beard procedure after malign tumour excision from the superior eyelid at the ankara oncology education and research hospital, ophthalmology clinic, between march 2009 and january 2013. the diagnosis of patients was consistent with basal cell carcinoma (bcc), sebaceous gland carcinoma (sgc), eyelid lymphoma (ell), and squamous cell carcinoma (scc). two patients with scc and bcc diagnosis had secondary operations because of recurrence. after the surgical excision of the tumour, the tumours were excised totally with a 5 mm surgical safety margin in all cases (figure 1). after excision of the lesions, the defects of the superior eyelids were reconstructed with the cutler - beard flap separating the anterior and posterior lamellae and advancing the bridge flap to the superior eyelid below the eyelash margin (figure 2). the patients ' superior and inferior eyelids were separated 1 month postoperatively (figure 3). there was no recurrence but one superior eyelid entropium and dermatochalasis is seen during followup with the patients. eyelids, which have important functions such as protecting the eyeball and providing tear film continuity and lacrimal pump, are composed of skin, mucosa, muscle tissue, and secretory glands. lesions on the eyelids are different from the other parts of body because of the eyelid 's histologic properties and specific surgical treatment procedures. primary treatment of eyelid malign tumours is surgery. lesions have to be excised with a 25 mm safety margin and absolute depth of tumour tissue. the major function of the superior eyelid is to protect the eye from foreign objects and to provide tear film continuity over the cornea. improper reconstruction of eyelid defects causes serious problems such as conjunctivitis, keratitis, and esthetic deformities [79 ]. the complication rate of full thickness defects of superior eyelids is more than inferior eyelids [10, 11 ]. a major aim is to excise a lesion completely for the treatment of eyelid tumours while maintaining anatomical and physiological functions and the cosmetic appearance of the eye. the reconstructed eyelid has to be mobile enough to protect the eyeball from negative outside effects. if the lesion is limited inside the skin, skin grafts and local flaps could be enough but resections of full thickness tumours that have invaded tars and conjunctiva need more complicated surgical procedures for reconstruction. reconstruction procedures of superior eyelid defects change according to defect localization, size, depth, and the patient. tissues used in superior eyelid reconstruction have to consist of well vascularized skin muscle, tarsoligamentous and mucosal membran structures. tumoural defects that occupy less than 25% of superior eyelids could be fixed with primary saturation after transformation to a pentagonal shape, but full thickness defects that occupy 2550% of the eyelid need lateral and medial canthotomy and cantholysis. also, tenzel semicircular flaps could be used in defects that occupy more than 50% of superior eyelids and have 2 mm tars tissue laterally and medially. cutler - beard flaps could be used in defects that occupy more than 50% of superior eyelids. the cutler - beard procedure is a two - stepped full thickness eyelid allocation process that is used in defects with intact levator aponeurosis. the most important advantage of this flap is that it is usable in nearly all defects. complications of this flap include superior eyelid entropium, lid margin irregularity, eyelash loss, retraction because inferior eyelid cicatrization and bridge - flap necrosis [14, 15 ]. entropium and eyelid shrinkage because of the cutler - beard flap could be minimized by using autogen nasal or auricular cartilage tissue instead of tars between skin and mucosa to provide an eyelid [16, 17 ]. the generation of a secondary surgical area and cartilage tissue hardness are disadvantages of this modification. bridge flap necrosis could be prevented by using a 4 - 5 mm thick flap to protect marginal vascular arcade. lid switch and lid bridge flaps prepared from the inferior eyelid are also alternatives to the cutler - beard flap [10, 11 ]. wide resections of the inferior eyelid and long - time exposure of the cornea to the outside are the most important disadvantages of these procedures. in addition, the frick flap and myocutaneous flaps could also be used to reconstruct anterior and posterior lamellae with different grafts and flaps. however, inappropriate lid dynamics in wide defects after these procedures are important disadvantages. as a result, the cutler - beard flap is a good option for adequate results from an anatomical aspect in wide defects that occupy the superior eyelid. | background. to assess cutler - beard procedure results in patients after wide excision of malignant eyelid tumours. materials and methods. the records of two women and two men (four patients) referred to our clinic with eyelid mass complaints and malign eyelid tumour diagnosis according to the histopathological examination were examined retrospectively. results. the patients were 6073 years old and their average age was 66 11.10. the follow - up period of the cases was 16 (625) months. total excisional biopsy was applied to all patients and then cutler - beard full thickness lid reconstruction was done because of the wide localization of the tumour. the patients ' diagnoses were consistent with basal cell carcinoma, sebaceous gland carcinoma, eyelid lymphoma, and squamous cell carcinoma. the patients ' eyelids were separated from each other 1 month postoperatively with a second operation. superior eyelid entropium and blepharochalasis were seen in one patient during followup. conclusions. cutler - beard flap is a successful procedure for superior eyelid tumours accompanied by wide tissue loss. the long - time closure of the eyelids and the need for secondary surgery are the major disadvantages of this procedure. our experience with this procedure will reveal better results with large case series. |
in the last decade, many investigations with several types of iron oxides have been carried out in the field of nanosized magnetic particles (mostly, magnetite (fe3o4) or maghemite (-fe2o3) single domains of about 520 nm in diameter). these iron oxide particles of nanometer size present superparamagnetic property and are ideal for magnetic resonance imaging (mri) contrast agent by enhancement of proton relaxation in the tissue microenvironment [24 ]. for the mri application, these spions must have high magnetization values, stability in physiological environment, and size smaller than 20 nm with overall narrow particle size distribution so that the particles have uniform physical and chemical properties. however, the iron oxide magnetic nanoparticles have several problems such as aggregation in water, chemical instability in air, biodegradability in physiological environment, and toxicity, which limit their use to the medical diagnostic agent. ferrofluids are colloidal suspensions of magnetic nanoparticles, forming magnetizable fluids that remain liquid in the most intense magnetic fields. even though the stability of the ferrofluid is of utmost importance for its biomedical applications, the nanoparticles in colloidal suspension are likely to agglomerate and form large cluster due to hydrophobic interactions between the magnetic iron oxide particles with a large hydrophobic surface area to volume ratio. these problems of naked iron oxide nanoparticles have been overcome by coating the surface of magnetic particles with synthetic polymers such as polyethylene glycol (peg), polyvinyl alchol (pva), polyvinyl pyrrolidone (pvp), and natural polymers like dextran, chitosan, and pullulan. however, the nanoparticles coated with the above - mentioned polymers were nonspecifically accumulated into tissues and organs, resulting in the poor availability in the imaging of specific tissues and organs [13, 14 ]. therefore, targeted delivery of mri contrast agent is a highly desirable strategy for enhancing efficiency and reducing unintended side - effects and toxicity [14, 15 ]. one strategy to realize efficient and specific delivery of spions is to modify the nanoparticle surface with a ligand that is efficiently taken up by target cells via receptor - mediated endocytosis. in this work, our prime aim is to achieve hepatocytes - specific delivery of spions by coating with galactose - carrying polymer for liver imaging as well as to enhance their functions in vivo containing the stabilization of magnetic fluid suspension. pvla, that is, a galactose - carrying polymer, has an amphiphilic structural unit composed of hydrophilic oligosaccharide side chains covalently bound to a hydrophobic polystyrene backbone. the binding profile of the carbohydrating ligands with cell surface receptors has been well characterized for asgp - r on hepatic parenchyma1 cells [1719 ]. the asgp - r recognizes galactose or n - acetylgalactosamine residues of desialylated glycoproteins and brings into the hepatocytes through endocytotic process as a ligand - receptor complex [20, 21 ]. maruyama. reported that the nanoparticles prepared using the plva as an emulsifier were hardly aggregated during storage and centrifugal treatment. therefore, such capabilities of pvla will open the door to the design and synthesis of mri contrast agent that can be accumulated specifically on liver without aggregation in physiological medium. in this study, pvla - coated spions were prepared by traditional coprecipitation method, followed by a thermochemical treatment and postcoating with pvla solution. various characterization techniques have been applied to obtain information about the sizes, structure, and coating of the nanoparticles. intracellular trafficking of the pvla - coated spions was visualized by confocal laser scanning microscopy and their hepatocyte - specific delivery was also investigated through mr images of rat liver. ferric chloride hexahydrate (fecl36h2o > 97%) and ferrous chloride tetrahydrate (fecl24h2o > 99%) were purchased from sigma - aldrich (st. pvla (mw = 5104) was prepared by the same method previously reported. all other chemicals were of analytical reagent grade and were used without further purification. pvla - coated spions were prepared by alkaline coprecipitation of ferric and ferrous chlorides in aqueous solution [24, 25 ]. fecl36h2o (3.255 g, 12.04 mmol) and fecl24h2o (1.197 g, 6.02 mmol) were dissolved in 70 ml of deoxygenated water, respectively. in a typical experimental procedure, the solutions were mixed and precipitated by adding 7 ml of nh4oh solution (2830%) while stirring vigorously. the black precipitate which immediately formed was washed several times with ultrapure water until the ph decreased from 10 to 7. after sedimenting the precipitate with a permanent magnet, the supernatant was removed by decantation. then 20 ml of 2 m hno3 was added to the black sediment and the mixture was stirred for 5 minutes. after adding 30 ml of 0.35 m fe(no3)3 to the mixture, the system was allowed to cool to room temperature, the remaining liquid was discarded, and 50 ml of ultrapure water was added to the slurry which immediately dispersed. the brown suspension was dialyzed for 2 days against 0.01 m nitric acid, and stored at 4c. in a final step, the obtained product was mixed with solution of pvla to obtain spions coated with pvla. for biological investigations, the content of spions in the ferrofluid was determined by dry weight analysis. for use as a control, 2-pyrrolidone - coated spions were prepared through thermal decomposition of ferric triacetylacetonate (fe(acac)3) in hot organic solvent, 2-pyrrolidone, by following previously developed method. however, 2-pyrrolidone not only serves as a media for high - temperature reaction, but also involves surface coordination which renders the magnetic nanoparticles water - soluble and colloidal solution stable. before the sample was withdrawn, the dispersed solution was sonicated for 5 minutes to obtain the better particle dispersion. fourier transform infrared (ft - ir) spectra were recorded in the transmission mode on a nicolet magna 550 series ii spectrometer (midac, atlanta, ga, usa). the transparent films for spions, pvla, and pvla - coated spions were prepared by casting each solution on silicon wafer and followed by drying at room temperature. the size of spions was assessed using an electrophoretic light scattering spectrophotometer (els 8000, otsuka electronics, osaka, japan) with 90 and 20 scattering angles at 25c. the volume of the samples was 4 ml containing a final concentration of 1 mg / ml in distilled water. the average particle size and morphology of spions were observed by transmission electron microscopy (tem) using a jeol model jem 1010 at 80 kv. for sample preparation, a diluted drop of spions suspension was placed on a carbon - coated copper grid. the grid was allowed in air to dry further for 15 minutes and was then examined with the electron microscope. to investigate the nanocrystallinity of spions, x - ray diffraction (xrd) data were collected on an x - ray diffractometer (bruker - axs gmbh d8 advance, karlsruhe, germany) equipped with a rotating anode, sol - x energy dispersive detector, and cu - k radiation source (= 0.1542 nm). hepatocytes were prepared by noncirculation perfusion of male icr mouse liver with a two - step collagenase perfusion technique of seglen. briefly, the male icr mice (57 weeks of age) employed in this study were purchased from jungang lab. the liver was perfused by 0.5 mm of ethylene glycol - bis[-amino ethyl ether]-n, n, n,n-tetraacetic acid (egta) in hanks ' balanced salt solution (hbss) without cacl2 and 5103% (wt./wt.) collagenase in hbss with cacl2 (5 mm) through a disposable needle (25g-1) aligned along the inferior vena cava. the collagenase - perfused liver was dissected, suspended in hbss, and filtered through cheesecloth and 100-m nylon membrane to remove connective tissue debris and cell clumps. hepatocytes were purified by a density - gradient centrifugation (50 g force, 10 minutes) using 45% percoll solution (pharmacia, piscataway, nj, usa) at 4c. isolated hepatocytes were suspended in a serum - free williams ' e (we) medium (gibco brl, ny, usa) containing 50 g / ml penicillin and 50 g / ml streptomycin. the isolated hepatocytes were plated on collagen - coated glass cover slips in 12-well plates (iwaki glass co., tokyo, japan) at 1105 cells per well., the old medium was removed and we medium containing fitc - pvla - coated spions (1 mg / ml) was added to cells. after 15-, 30-, and 60-minute incubation, cells were rinsed twice with 0.1 m pbs. the coverslips were enclosed in 1 ml of glycerol and visualized by confocal laser scanning microscope (micro systems lsm 410, carl zeiss, germany). gallery mode of optical sections was used for checking internalization of complexes into cells [29, 30 ]. for all rats, liver mr images were taken prior and 1 hour after injection of contrast agents. pvla - coated spions and pyrrolidone - coated spions were intravenously injected through the tail vein. rats (sd, female, 6 weeks) were anesthetized with the use of a general inhalation anesthesia (1.5% isoflurane in a 1:2 mixture of o2/n2) during mr examination. mr imagings were performed with a 1.5 t mr scanner (ge signa exite twin - speed, ge health care, milwaukee, wiss, usa) using an animal coil (4.3 cm quadrature volume coil, nova medical system, wilmington, del, usa). for fast spin echo (fse) t2-weighted mr imaging, the following parameters were adopted : repetition time (ms / echo) time milliseconds of 4,800/102, flip angle of 90, echo train length of 8, field of view of 6 cm, section thickness of 2 mm, intersection gap of 0.1 mm, and 256160 matrix. the signal intensity (si) was measured in defined regions of interest (roi) in identical site of liver before (si pre of liver) and after (si post of liver) contrast injection. in addition, the si in the roi of back muscle adjacent to the liver was measured before (si pre of bm) and after (si post of bm). relative signal enhancement of liver was calculated by using the formula [(si post of liver / si post of bm si pre of liver / si pre of bm)/si post of liver / si post of bm ] 100. surface modification of iron oxide nanoparticles with biocompatible polymers is potentially beneficial to prepare mri contrast agents for in vivo applications. in particular, targeted delivering, nonaggregating, and nontoxic properties are required for the nanoparticles to achieve the accumulation in liver. to produce well - dispersed iron oxide colloidal solutions with hepatocytes targeting properties, we prepared spions coated with pvla having galactose residues as hepatocyte - specific ligand and serving as an emulsifier due to its amphiphilic property. the parent nanoparticles were synthesized by coprecipitation of ferrous and ferric ions in an aqueous solution upon addition of ammonium hydroxide. common problems of the naked magnetic nanoparticles are their tendency to agglomerate once formed and their chemical instability with respect to oxidation in air. the final iron oxide compositions are very often intermediate between magnetite (fe3o4) and maghemite (-fe2o3), due to the oxidation of the particles during the synthesis. the problem of oxidation - sensitive magnetite could be overcome by the deliberate introduction of a second oxidation step. stable ferrofluids for liver targeting can be prepared by adsorption of pvla on the surface of magnetic nanoparticles after the second oxidation step. polymer can be adsorbed by electrostatic, covalent, hydrophobic, and hydrogen bonding mechanism. hydrogen bonding is assumed to be the predominant mechanism for the adsorption of nonionic polymer such as pvla on oxide surface. the hydrogen bonding result from the interaction between polar functional groups of pvla and hydroxylated and protonated surface sides of the oxide. the importance of surface hydroxyl functions in hydrogen bonding has been further verified by adsorbing the polymer onto pure gold sol, the surface of which is not oxidized and, therefore, dose not carry any hydroxyl groups ; in this case, no adsorption of the polymer is detected. accordingly, it is believed that the hydrogen bonding may be strengthened by the second oxidation step mentioned above. figure 2 shows a comparison between the ftir spectra of the spions (a) the pure pvla itself (b), and the pvla - coated spions (c). the presence of magnetic iron oxide nanoparticles can be seen by two strong absorption bands at around 631 and 564 cm. these bands result from split of the 1 band at 570 cm, which corresponds to the fe o bond of bulk magnetite. furthermore, an adsorption band was observed at around 434 cm, which corresponds to the shifting of the 2 band of the fe o bond of bulk magnetite (at 375 cm) to a higher wave number. the strong band at 1078 cm as shown in the ftir spectrum of pvla (b) is attributed to the skeletal vibrations and co stretch of oligosaccharide, and the band at 2926 cm and a pair of bands at 1542 and 1423 cm are attributed to the stretch of ch2 and aromatic c = c of polystyrene backbone, respectively [12, 36 ]. in addition, the nh bending and c = o stretching bands are overlapped at 1647 cm. comparing the ftir spectrum of spions (a) and that of pvla - coated spions (c), the characteristic bands resulted from the oligosaccharide and polystyrene containing pvla appeared near 2926 and 1078 cm, respectively, for pvla - coated spions, indicating that pvla was coated at the nanoparticle surface. after the adsorption of pvla, the characteristic bands of fe o bond of bulk magnetite (631 and 564 cm) shifted to the higher wave numbers of about 636 and 588 cm, an indication of the occurrence of hydrogen bonding between hydroxyl groups of pvla and hydroxylated and protonated surface sides of the oxide. we compared the structure, size, and uniformity of the pvla - coated spions with those of the 2-pyrrolidone - coated spions prepared for use as a control. figure 3 shows the xrd patterns for the 2-pyrrolidone - coated and pvla - coated spions. six characteristic peaks (2 = 30.1, 35.5, 43.1, 53.4, 57.0, and 62.6), marked by their indices [(220), (311), (400), (422), (511), and (440) ], were observed for 2-pyrrolidone - coated spion samples. the positions and relative intensities of all diffraction peaks in figure 3(a) 85 - 1436) for magnetite and reveal that the resultant nanoparticles were pure magnetite with spinal structure. as shown in figure 3(b), the xrd pattern of pvla - coated spions also proved its highly crystalline nature and the peaks are consistent with standard maghemite reflections [38, supporting information ], being an indication of the magnetite - maghemite transformation due to second oxidation step. it is already known that magnetite is transformed to maghemite at ph 2 upon addition of ferric ions. jolivet and tronc have reported on the behavior of colloidal magnetite in acidic medium in the presence of iron nitrate and studied the phenomena that induce the magnetite - maghemite transformation [25, 39 ]. further, xrd confirmed the high degree of crystallinity of the both types of particles. the shape, size, and uniformity of the 2-pyrrolidone - coated and pvla - coated spions were observed by means of tem. the tem images of these both particles show ellipsoidal iron oxide particles with an average size less than 10 nm as shown in figure 4. as well known, a representative tem image of naked magnetic nanoparticles showed that the particles strongly agglomerated [6, 25 ]. on the other hand, our samples that were obtained after monomer or polymer coating especially, in pvla - coated particles (b), the particle distribution is more homogeneous than the 2-pyrrolidone - coated particles (a). figure 5 shows number average sizes and size distribution of synthesized magnetite and pvla - coated spions by els. it was clear that size distributions of both the 2-pyrrolidone - coated and pvla - coated spions were unimodal and particles were uniformly prepared. the average particle sizes were 20.84.4 and 25.86.1 nm for 2-pyrrolidone - coated and pvla - coated spions, respectively. the average diameters obtained by els were larger than the sizes determined from the tem image for corresponding samples. this may presumably be because els gives a mean hydrodynamic diameter of magnetic nanoparticles surrounded by pvla layer in aqueous solution whereas tem gives the diameter of magnetic nanoparticles alone in dry state. the fitc on the nanoparticles allowed direct visualization of the nanoparticle uptakes into cells. figure 6 shows fluorescence (a) and phasecontrast (b) micrographs of hepatocytes cultured in medium containing spions coated with fitc - labeled pvla according to treatment time. the fluorescence micrograph demonstrates a time - dependent uptake of fitc - pvla - coated spions into hepatocytes as shown in figure 6(a). a significant uptake of the nanoparticles was clearly observed after 1 hour of culture. we performed a quantitative evaluation of uptake in the in vivo although only qualitative evaluation was performed in the in vitro, because the specific interaction between galactose moieties of pvla and asialoglycoprotein receptors in the hepatocytes has been already reported. it was also checked whether the pvla - coated spions existed in the cytosol or was only attached to the plasma membrane of hepatocyte. the cell was sectioned at various depths from cell surface by confocal laser scanning microscopy, and each fluorescence distribution was observed (see figure 7). fluorescence of the magnetic nanoparticles was observed more intensively inside the cell membrane and uniformly distributed in the cytosol. these results suggest that the many nanoparticles were internalized in the cytosol through a receptor - mediated endocytosis. the affinity of asgpr to natural and synthetic oligosaccharides having nonreducing galactose residues had been reported by lee.. we examined an applicability of the pvla - coated spions for in vivo liver imaging based on a t2weighted fse echo imaging, which is a useful way to the liver accumulation of the magnetic nanoparticles. however, we did not calculate the statistical analysis due to small number of animals. at 1 hour postinjection of the pvla - coated spio, signal intensity of liver was hypointense compared to si of back muscle fse t2-weighted mr image (data not shown). the relative signal enhancement on the fse t2-weighted mr image was observed in the liver with a t2 signal drop of 70.9% for pvla - coated spio, indication of the accumulation of nanoparticle in liver. figure 8 shows the t2-weighted mr images of middle part of liver before and after injection of spions through tail vein. after injection of the pvla - coated spions, the si of liver clearly dropped on t2-weighted mr image. the relative signal enhancement of the t2-weighted mr image was observed in the liver with a t2 signal drop of 75.4% for pvla - coated spions and si of liver on t2-weighted mr image after injection of pvla - coated spions was darker than that after pyrrolidone - coated spions (see figures 8(b) and 8(d)). thus, pvla - coated spions were more successfully targeted the liver than pyrrolidone - coated spions. pvla - coated spions can be used as the hepatocyte targeted contrast agent such as mangafodipir trisodium, formerly known as mn - dpdp. additionally, we observed accumulation in kidneys after injection of pvla - coated spions (data not shown). renal excretion of nanoparticles is beneficial to develop nontoxic mr nanoparticles in the clinical use. we have demonstrated that pvla can serve as the coating material for spions to achieve the stabilization and liver - specific delivery of ferrofluid. the pvla - coated spions of about 10 nm diameter having a core shell structure with magnetic core and polymeric shell have been successfully prepared. the ftir experimental results proved that the pvla is adsorbed onto the surface of spions through the hydrogen bonding between polar functional alcohol groups of pvla and hydroxylated and protonated surface sides of the oxide. hence the resultant nanoparticles possess an excellent solubility and stability in ferrofluid. in vivo experimental result indicated that the pvla - coated spions were accumulated in liver appreciably. therefore, pvla as a coating material not only prevented the aggregation between spions in physiological medium but also provided a capacity to be delivered in liver specifically, which suggests the potential utility of pvla - coated spions as a contrast agent for liver diagnosis. | our goal is to develop the functionalized superparamagnetic iron oxide nanoparticles (spions) demonstrating the capacities to be delivered in liver specifically and to be dispersed in physiological environment stably. for this purpose, spions were coated with polyvinylbenzyl - o--d - galactopyranosyl - d - gluconamide (pvla) having galactose moieties to be recognized by asialoglycoprotein receptors (asgp - r) on hepatocytes. for use as a control, we also prepared spions coordinated with 2-pyrrolidone. the sizes, size distribution, structure, and coating of the nanoparticles were characterized by transmission electron microscopy (tem), electrophoretic light scattering spectrophotometer (els), x - ray diffractometer (xrd), and fourier transform infrared (ft - ir), respectively. intracellular uptake of the pvla - coated spions was visualized by confocal laser scanning microscopy, and their hepatocyte - specific delivery was also investigated through magnetic resonance (mr) images of rat liver. mri experimental results indicated that the pvla - coated spions possess the more specific accumulation property in liver compared with control, which suggests their potential utility as liver - targeting mri contrast agent. |
collaborative studies were performed to develop a functional assay for fish - killing activity produced by pfiesteria piscicida. eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2 - 4)]-diphenyltetrazolium bromide cytotoxicity assay. diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (gh(4)c(1)) cells. residual water as well as preextracted culture water containing p. piscicida cells induced c - fos - luciferase expressed in gh(4)c(1) cells with a rapid time course of induction and sensitive detection. the reporter gene assay detected activity in toxic isolates of p. piscicida from several north carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum.imagesfigure 1figure 2figure 3figure 4 |
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mucosal damage produced by the abnormal reflux of gastric contents into the esophagus is observed in some cases of gerd. male gender, hiatal hernia, helicobacter pylori infection, smoking, and alcohol consumption can increase the risk of erosive esophagitis (ee) [3, 4 ]. in addition, common features of metabolic syndrome are risk factors for ee [5, 6 ]. obesity, a central component of metabolic syndrome, is known to promote gastroesophageal reflux. of note, the increase in intra - abdominal pressure due to obesity contributes to ee. some studies have suggested that the risk of gerd or ee increases with increasing body mass index (bmi) [79 ]. however, bmi is an imperfect estimate of adiposity, particularly in men, owing to the greater muscle mass in this group. moreover, the association between increased bmi and ee is inconsistent and varies with sex, ethnic origin, and other confounding factors [1113 ]. therefore, the pattern of obesity may be a better predictor of gerd than bmi. a previous study indicated that gerd symptoms were positively associated with the abdominal diameter in caucasian men independently of bmi. moreover, abdominal adipose tissue consists of subcutaneous fat (sf) and visceral fat (vf), and recent studies revealed the association between abdominal vf and ee [17, 18 ]. a recent study reported that abdominal vf volume measured by cross - sectional computerized tomography (ct) is a more important risk factor for ee than other factors such as bmi and abdominal circumference in both sexes. however, because of individual differences in the absolute amount of vf, this parameter may not indicate obesity directly ; therefore, the relative ratio of vf to sf may indicate obesity more accurately. we hypothesized that the ratio of visceral fat area (vfa) to subcutaneous fat area (sfa), measured by ct, rather than bmi, might significantly correlate with the risk of ee. therefore, the aim of this study was to investigate the association between abdominal fat distribution, represented by the vfa / sfa ratio, and the incidence of ee in the general population. subjects older than 40 years were enrolled in a routine checkup program at the health care center of chung - ang university hospital from january 2007 to december 2012. a total of 728 participants underwent a physical examination (height, body weight, waist circumference, and blood pressure), blood tests [glucose, triglycerides (tg), and high - density lipoprotein (hdl) cholesterol ], esophagogastroduodenoscopy (egd), and abdominal ct scan. height and body weight measurements were automated (gl-150 ; g - tech international co., uijungbu city, korea, inbody 720 ; biospace co., chun - an city, korea), and bmi was calculated as weight divided by height squared (kg / m). waist circumference was measured by trained nurses at the midpoint between the lower borders of the rib cage and upper pole of the iliac crest. after fasting for 12 h metabolic syndrome was defined on the basis of the criteria established by the national cholesterol education program (ncep). the ncep criteria for metabolic syndrome require that at least three of the following conditions be met : waist circumference > 40 in. in men and > 35 in. in women, plasma tg > 150 mg / dl, hdl cholesterol 40 in. in men and > 35 in. in women, plasma tg > 150 mg / dl, hdl cholesterol < 40 mg / dl in men and < 50 mg / dl in women, blood pressure 130/85 mmhg, and fasting plasma glucose 110 mg / dl. egd was performed using a flexible endoscope (cv-260sl ; olympus, tokyo, japan) after overnight fasting. we inspected the gastroesophageal junction at the start of the endoscopic examination before inflation of the stomach or at the end of the examination after deflation of the stomach. the severity of ee was graded from a to d according to the los angeles (la) classification system. hiatal hernia was considered present if the gastroesophageal junction extended at least 2 cm above the diaphragmatic hiatus impression during quiet respiration. the abdominal adipose tissue area was quantified using 64-multidetector ct (brilliance ; philips medical systems, cleveland, oh, usa). the fat area was determined by measuring the mean value of the pixels within the range between 175 and 25 hounsfield units. the total abdominal fat area (tfa), vfa, and sfa were measured by the acquisition of a 10-cm ct slice scan image of the third and fourth lumbar vertebrae during suspended respiration. the area (cm) was calculated using extended brilliance workspace software (version 1 - 4.5.2, philips healthcare, best, netherland). vfa was calculated by delineating the intra - abdominal cavity bound by the parietal peritoneum or transversalis fascia, and excluding the vertebral column and paraspinal muscles. continuous variables were expressed as mean sd. a p value of < 0.05 was considered statistically significant. the mean age of the patients was 47.15 years ; 629 (86.4%) patients were male, and 99 (13.6%) were female. the mean bmi was 24.66 kg / m, and 65 (8.9%) patients had metabolic syndrome. on egd, 65 (8.9%) patients had ee, and 11 (1.5%) patients had hiatal hernias. on ct, the mean vfa, sfa, and vfa / sfa ratio were 2886.97, 3627.27, and 1.02 cm, respectively (table 1).table 1baseline demographic and clinical characteristicsvariablesnumber of subjects728age (mean sd, years)47.15 8.35sex male629 (86.4) female99 (13.6)bmi (kg / m) (mean sd)24.66 2.85metabolic syndrome yes65 (8.9) no663 (91.1)blood pressure (mean sd, mmhg)124.30 14.72hdl cholesterol (mg / dl)49.09 9.21triglyceride (mg / dl)41.78 36.08fasting glucose (mg / dl)101.02 19.39erosive esophagitis yes65 (8.9) no663 (91.1)hiatal hernia yes11 (1.5) no717 (98.5)vf area (cm)2886.97 5260.61sf area (cm)3627.27 7764.85vf / sf ratio (mean sd)1.02 0.49 bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, sf subcutaneous fat, vf visceral fat baseline demographic and clinical characteristics bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, sf subcutaneous fat, vf visceral fat among the 728 subjects, 65 (8.9%) patients had ee. the mean bmi was higher in the ee group than in the non - ee group (25.80 vs. 24.40, p = 0.002). metabolic syndrome was more prevalent in the ee group than in the non - ee group (30.8 vs. 13.7%, p < 0.001). moreover, tg and blood pressure levels were higher in patients with ee. on egd, hiatal hernia was more prevalent in the ee group than in the non - ee group (9.2 vs. 0.8%, p < 0.001). however, vfa was not significantly different between the two groups (1595.00 vs. 1426.00 cm, p = 0.927). conversely, sfa was higher in the non - ee group than in the ee group (1226.92 vs. 1514.46 cm, p = 0.010). moreover, the mean vfs / sfa ratio was higher in the ee group than in the non - ee group (1.30 vs. 0.92, p < 0.001).table 2comparison of clinical characteristics between subjects with reflux esophagitis and those withoutsubjects with reflux esophagitis (n = 65)subjects without reflux esophagitis (n = 663) p valueage (range, years)46 (4250)48 (4253)0.260sex0.003 male (%) 1 (1.5)98 (14.8) female (%) 64 (98.5)565 (85.2)bmi (kg / m)25.80 (23.5527.65)24.40 (22.7026.20)0.002metabolic syndrome<0.001 yes (%) 20 (30.8)91 (13.7) no (%) 45 (69.2)572 (86.3)hdl cholesterol (mg / dl)46 (4052)48 (4354)0.066triglyceride (mg / dl)45 (2665)29 (2049)<0.001high blood pressure0.006 yes (%) 35 (53.8)242 (36.6) no (%) 30 (46.2)420 (63.4)fasting glucose (mg / dl)98.00 (93.50107.00)97.00 (91.00105.00)0.307hiatal hernia6 (9.2)5 (0.8)<0.001vf area (cm)1595.00 (841.501936.50)1426.00 (998.001950.00)0.927sf area (cm)1226.92 (707.582054.67)1514.46 (945.682772.09)0.010vf / sf1.30 (0.871.63)0.92 (0.671.29)<0.001 bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, ns nonspecific, sf subcutaneous fat, vf visceral fat comparison of clinical characteristics between subjects with reflux esophagitis and those without bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, ns nonspecific, sf subcutaneous fat, vf visceral fat the results of the multivariate logistic regression analysis demonstrated that hiatal hernia (p < 0.001), vfa / sfa ratio 1.165 (p = 0.010), and high tg level (p = 0.015) were independent risk factors for ee. hiatal hernia was strongly associated with an increased risk of ee [adjusted odds ratio (or), 12.90 ; 95% confidence interval (ci), 3.5746.65 ]. similarly, a vfa / sfa ratio 1.165 was a significant risk factor for ee (or 2.04 ; 95% ci 1.183.51 ; table 3).table 3logistic regression analysis of covariables for erosive esophagitisvariableor (95% ci)univariate analysis (p)or (95% ci)multivariate analysis (p)age (years) < 65 (n = 714)1 65 (n = 14)1.88 (0.418.68)0.326sex female (n = 99)1 male (n = 629)0.09 (0.010.66)0.0030.19 (0.031.40)0.191bmi (kg / m) < 25 (n = 665)1 25 (n = 63)2.25 (1.333.81)0.0021.64 (0.932.87)0.086metabolic syndrome no (n = 663)1 yes (n = 65)2.79 (1.584.95)<0.0010.82 (0.332.01)0.819hdl cholesterol (mg / dl) < 40 (n = 631)1 40 (n = 97)1.37 (0.692.72)0.371triglyceride (mg / dl) < 150 (n = 714)1 150 (n = 14)6.06 (1.9718.65)0.0054.74 (1.3516.62)0.015high blood pressure no (n = 452)1 yes (n = 276)2.04 (1.223.41)0.0061.72 (0.973.07)0.065fasting glucose (mg / dl) < 110 (n = 609)1 110 (n = 119)0.93 (0.461.87)0.826hiatal hernia no (n = 717)1 yes (n = 11)13.38 (3.9745.17)0.00112.90 (3.5746.65)<0.001vf / sf ratio < 1.165 (n = 477)1 1.165 (n = 251)2.77 (1.654.65)<0.0012.04 (1.183.51)0.010 bmi body mass index, ci confidence interval ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, vf visceral fat, sf subcutaneous fat logistic regression analysis of covariables for erosive esophagitis bmi body mass index, ci confidence interval ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, vf visceral fat, sf subcutaneous fat the analysis of the correlation between ee and the vfa / sfa ratio indicated that the incidence of ee was higher in the group with the higher vfa / sfa ratio (p < 0.001 ; fig. 1). in addition, the receiver - operating characteristic (roc) curve indicated that a vfa / sfa ratio 1.165 had good accuracy to predict ee (area under the roc curve = 0.643 ; fig. the incidence of erosive esophagitis was higher in the group with the higher vfa / sfa ratio (p for trend < 0.001) fig. receiver - operating characteristic (roc) curve indicated that a vfa / sfa ratio 1.165 had a good accuracy to predict erosive esophagitis (auroc = 0.643). auroc area under the receiver operator characteristic curve association between erosive esophagitis and vf / sf ratio. the incidence of erosive esophagitis was higher in the group with the higher vfa / sfa ratio (p for trend < 0.001) auroc of vf / sf ratio in the identification of erosive esophagitis. receiver - operating characteristic (roc) curve indicated that a vfa / sfa ratio 1.165 had a good accuracy to predict erosive esophagitis (auroc = 0.643). auroc area under the receiver operator characteristic curve the severity of ee was positively correlated with the vfa / sfa ratio and vf volume (p = 0.002 ; table 4). the risk of ee types la - a, la - b, and la - c / lac - d increased 1.23-fold, 1.27-fold, and 1.56-fold, respectively. of note, a vfa / sfa ratio 1.165 was strongly correlated with the severity of ee (p < 0.001 ; table 5).table 4association between visceral fat / subcutaneous fat ratio and severity of erosive esophagitisnormalla - ala - bla - c, dno. of subjects (%) 663 (91.1)50 (6.9)14 (1.9)1 (0.1)vf / sf (mean sd)1.00 0.491.23 0.531.27 0.471.56 p value0.002 vf visceral fat, sf subcutaneous fat table 5severity of erosive esophagitis according to visceral fat / subcutaneous fat rationormalla - ala - bla - c, dno of subjects (%) 663 (91.1)50 (6.9)14 (1.9)1 (0.1)vf / sf < 1.165449 (94.1)24 (5.0)4 (0.9)0 (0.0)vf / sf 1.165214 (85.3)26 (10.3)10 (4.0)1 (0.4) p for trend<0.001 p value<0.001 vf visceral fat, sf subcutaneous fat association between visceral fat / subcutaneous fat ratio and severity of erosive esophagitis vf visceral fat, sf subcutaneous fat severity of erosive esophagitis according to visceral fat / subcutaneous fat ratio vf visceral fat, sf subcutaneous fat the mean age of the patients was 47.15 years ; 629 (86.4%) patients were male, and 99 (13.6%) were female. the mean bmi was 24.66 kg / m, and 65 (8.9%) patients had metabolic syndrome. on egd, 65 (8.9%) patients had ee, and 11 (1.5%) patients had hiatal hernias. on ct, the mean vfa, sfa, and vfa / sfa ratio were 2886.97, 3627.27, and 1.02 cm, respectively (table 1).table 1baseline demographic and clinical characteristicsvariablesnumber of subjects728age (mean sd, years)47.15 8.35sex male629 (86.4) female99 (13.6)bmi (kg / m) (mean sd)24.66 2.85metabolic syndrome yes65 (8.9) no663 (91.1)blood pressure (mean sd, mmhg)124.30 14.72hdl cholesterol (mg / dl)49.09 9.21triglyceride (mg / dl)41.78 36.08fasting glucose (mg / dl)101.02 19.39erosive esophagitis yes65 (8.9) no663 (91.1)hiatal hernia yes11 (1.5) no717 (98.5)vf area (cm)2886.97 5260.61sf area (cm)3627.27 7764.85vf / sf ratio (mean sd)1.02 0.49 bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, sf subcutaneous fat, vf visceral fat baseline demographic and clinical characteristics bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, sf subcutaneous fat, vf visceral fat the mean bmi was higher in the ee group than in the non - ee group (25.80 vs. 24.40, p = 0.002). metabolic syndrome was more prevalent in the ee group than in the non - ee group (30.8 vs. 13.7%, p < 0.001). moreover, tg and blood pressure levels were higher in patients with ee. on egd, hiatal hernia was more prevalent in the ee group than in the non - ee group (9.2 vs. 0.8%, p < 0.001). however, vfa was not significantly different between the two groups (1595.00 vs. 1426.00 cm, p = 0.927). conversely, sfa was higher in the non - ee group than in the ee group (1226.92 vs. 1514.46 cm, p = 0.010). moreover, the mean vfs / sfa ratio was higher in the ee group than in the non - ee group (1.30 vs. 0.92, p < 0.001).table 2comparison of clinical characteristics between subjects with reflux esophagitis and those withoutsubjects with reflux esophagitis (n = 65)subjects without reflux esophagitis (n = 663) p valueage (range, years)46 (4250)48 (4253)0.260sex0.003 male (%) 1 (1.5)98 (14.8) female (%) 64 (98.5)565 (85.2)bmi (kg / m)25.80 (23.5527.65)24.40 (22.7026.20)0.002metabolic syndrome<0.001 yes (%) 20 (30.8)91 (13.7) no (%) 45 (69.2)572 (86.3)hdl cholesterol (mg / dl)46 (4052)48 (4354)0.066triglyceride (mg / dl)45 (2665)29 (2049)<0.001high blood pressure0.006 yes (%) 35 (53.8)242 (36.6) no (%) 30 (46.2)420 (63.4)fasting glucose (mg / dl)98.00 (93.50107.00)97.00 (91.00105.00)0.307hiatal hernia6 (9.2)5 (0.8)<0.001vf area (cm)1595.00 (841.501936.50)1426.00 (998.001950.00)0.927sf area (cm)1226.92 (707.582054.67)1514.46 (945.682772.09)0.010vf / sf1.30 (0.871.63)0.92 (0.671.29)<0.001 bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, ns nonspecific, sf subcutaneous fat, vf visceral fat comparison of clinical characteristics between subjects with reflux esophagitis and those without bmi body mass index ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, ns nonspecific, sf subcutaneous fat, vf visceral fat the results of the multivariate logistic regression analysis demonstrated that hiatal hernia (p < 0.001), vfa / sfa ratio 1.165 (p = 0.010), and high tg level (p = 0.015) were independent risk factors for ee. hiatal hernia was strongly associated with an increased risk of ee [adjusted odds ratio (or), 12.90 ; 95% confidence interval (ci), 3.5746.65 ]. similarly, a vfa / sfa ratio 1.165 was a significant risk factor for ee (or 2.04 ; 95% ci 1.183.51 ; table 3).table 3logistic regression analysis of covariables for erosive esophagitisvariableor (95% ci)univariate analysis (p)or (95% ci)multivariate analysis (p)age (years) < 65 (n = 714)1 65 (n = 14)1.88 (0.418.68)0.326sex female (n = 99)1 male (n = 629)0.09 (0.010.66)0.0030.19 (0.031.40)0.191bmi (kg / m) < 25 (n = 665)1 25 (n = 63)2.25 (1.333.81)0.0021.64 (0.932.87)0.086metabolic syndrome no (n = 663)1 yes (n = 65)2.79 (1.584.95)<0.0010.82 (0.332.01)0.819hdl cholesterol (mg / dl) < 40 (n = 631)1 40 (n = 97)1.37 (0.692.72)0.371triglyceride (mg / dl) < 150 (n = 714)1 150 (n = 14)6.06 (1.9718.65)0.0054.74 (1.3516.62)0.015high blood pressure no (n = 452)1 yes (n = 276)2.04 (1.223.41)0.0061.72 (0.973.07)0.065fasting glucose (mg / dl) < 110 (n = 609)1 110 (n = 119)0.93 (0.461.87)0.826hiatal hernia no (n = 717)1 yes (n = 11)13.38 (3.9745.17)0.00112.90 (3.5746.65)<0.001vf / sf ratio < 1.165 (n = 477)1 1.165 (n = 251)2.77 (1.654.65)<0.0012.04 (1.183.51)0.010 bmi body mass index, ci confidence interval ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, vf visceral fat, sf subcutaneous fat logistic regression analysis of covariables for erosive esophagitis bmi body mass index, ci confidence interval ; high blood pressure, 130/85 mmhg or documented use of antihypertensive therapy, ldl low - density lipoprotein, hdl high - density lipoprotein, vf visceral fat, sf subcutaneous fat the analysis of the correlation between ee and the vfa / sfa ratio indicated that the incidence of ee was higher in the group with the higher vfa / sfa ratio (p < 0.001 ; fig. 1). in addition, the receiver - operating characteristic (roc) curve indicated that a vfa / sfa ratio 1.165 had good accuracy to predict ee (area under the roc curve = 0.643 ; fig. the incidence of erosive esophagitis was higher in the group with the higher vfa / sfa ratio (p for trend < 0.001) fig. receiver - operating characteristic (roc) curve indicated that a vfa / sfa ratio 1.165 had a good accuracy to predict erosive esophagitis (auroc = 0.643). auroc area under the receiver operator characteristic curve association between erosive esophagitis and vf / sf ratio. the incidence of erosive esophagitis was higher in the group with the higher vfa / sfa ratio (p for trend < 0.001) auroc of vf / sf ratio in the identification of erosive esophagitis. receiver - operating characteristic (roc) curve indicated that a vfa / sfa ratio 1.165 had a good accuracy to predict erosive esophagitis (auroc = 0.643). the severity of ee was positively correlated with the vfa / sfa ratio and vf volume (p = 0.002 ; table 4). the risk of ee types la - a, la - b, and la - c / lac - d increased 1.23-fold, 1.27-fold, and 1.56-fold, respectively. of note, a vfa / sfa ratio 1.165 was strongly correlated with the severity of ee (p < 0.001 ; table 5).table 4association between visceral fat / subcutaneous fat ratio and severity of erosive esophagitisnormalla - ala - bla - c, dno. of subjects (%) 663 (91.1)50 (6.9)14 (1.9)1 (0.1)vf / sf (mean sd)1.00 0.491.23 0.531.27 0.471.56 p value0.002 vf visceral fat, sf subcutaneous fat table 5severity of erosive esophagitis according to visceral fat / subcutaneous fat rationormalla - ala - bla - c, dno. of subjects (%) 663 (91.1)50 (6.9)14 (1.9)1 (0.1)vf / sf < 1.165449 (94.1)24 (5.0)4 (0.9)0 (0.0)vf / sf 1.165214 (85.3)26 (10.3)10 (4.0)1 (0.4) p for trend<0.001 p value<0.001 vf visceral fat, sf subcutaneous fat association between visceral fat / subcutaneous fat ratio and severity of erosive esophagitis vf visceral fat, sf subcutaneous fat severity of erosive esophagitis according to visceral fat / subcutaneous fat ratio vf visceral fat, sf subcutaneous fat our study identified a positive correlation between ee and each of the following individual factors : high bmi, metabolic syndrome, high vfa / sfa ratio, hiatal hernia, high tg level, high blood pressure, and female sex. however, multivariate analysis revealed that only high vfa / sfa ratio, hiatal hernia, and high tg level were associated with ee. in a previous study, previous studies demonstrated that women have lower vf and higher sf than men [17, 23 ]. in our study, however, vfa did not show a significant association with ee, as a higher proportion of women might have resulted in a lower vfa than expected in the ee group. in other studies, multivariate analysis found that high bmi, waist circumference, and waist - to - hip ratio were associated with ee [17, 18 ]. although these parameters are easily obtainable, these are imperfect measures of adiposity owing to confounding factors such as sex and ethnic origin. while in some asian populations the prevalence of obesity is lower than that in western countries, health risks associated with obesity occur at a lower bmi in asian populations, and these populations are predisposed to abdominal obesity. several hypotheses have been formulated to explain how obesity can cause gerd [2527 ]. previous studies have indicated that obesity can lead to ee via dietary, mechanical, and humoral factors [15, 26 ]. a previous study has shown that the amount and type of dietary intake are responsible for gerd. moreover, an increase in abdominal adipose tissue leads to increased intra - abdominal and intragastric pressure, increased rate of transit, lower esophageal sphincter relaxation, formation of hiatal hernia, and subsequent esophageal acid reflux. a large cohort study found that compared to men with obesity, women with obesity had a greater risk of gerd symptoms, with the risk being highest in premenopausal women and postmenopausal women undergoing estrogen therapy. furthermore, vf is strongly associated with elevated serum levels of several proinflammatory cytokines, including interleukin-6 and tumor necrosis factor - alpha, which are overexpressed in patients with ee [30, 31 ]. waist circumference and bmi are significantly correlated with intragastric pressure, gastroesophageal pressure gradient, and separation of the gastroesophageal junction pressure components [33, 34 ]. however, the association between bmi and gerd is affected by many confounding factors [7, 12, 13, 16 ]. moreover, anthropometric measurements are less accurate and less reproducible in obesity studies. abdominal visceral adipose tissue, which is associated with increased intra - abdominal pressure, may be a better predictor of gerd than other obesity parameters. the most important mechanism involved in the correlation between obesity with gerd is intra - abdominal pressure, which plays a significant role in acid reflux. moreover, abdominal ct is more accurate and more reproducible than anthropometry because ct allows for the direct assessment of sf and vf compartments, whereas anthropometric measurements do not. vfa is an indicator of abdominal fat and a strong predictor of insulin resistance and coronary artery disease [33, 36 ]. however, the high cost and radiation exposure should be considered. in our study, the vfa / sfa ratio was a more significant risk factor for ee than bmi, waist circumference, waist - to - hip ratio, and vfa. moreover, we found that a vfa / sfa ratio 1.165 might be a useful indicator for predicting ee. in addition, the severity of ee was positively correlated with the vfa / sfa ratio. furthermore, ee was objectively evaluated via egd and categorized using the los angeles classification, and minor changes were excluded to increase specificity. second, abdominal adipose tissue was measured using 64-multidetector ct, which has a high degree of validity and reproducibility. first, the study population was based on subjective screening ; therefore, there could have been a selection bias. second, we did not evaluate individual diet or lifestyle factors such as alcohol consumption and smoking status. the pattern of obesity was more important than bmi, and the vfa / sfa ratio was used in the diagnosis of vf - type abdominal obesity. we suggest that a high vfa / sfa ratio is a useful clinical predictor of ee. therefore, individuals with a high vfa / sfa ratio may need endoscopic evaluation for ee. further studies are needed to determine the treatment success of ee based on the vfa / sfa ratios. the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. | backgroundto investigate the association between abdominal fat distribution represented by the visceral fat area (vfa) to subcutaneous fat area (sfa) ratio, and erosive esophagitis (ee). methodsseven hundred and twenty - eight participants aged > 40 years underwent physical examination, blood tests, esophagogastroduodenoscopy, and abdominal computer tomography at chung - ang university hospital from 2007 to 2012.resultsof 728 subjects, 65 (8.9%) had ee. the ee patients had higher body mass index, metabolic syndrome prevalence, triglyceride levels, and blood pressure (p < 0.05). the mean vfa / sfa ratio was higher in the ee group than in the non - ee group (1.30 vs. 0.92, p < 0.001). the predominance of ee in the group with higher vfa / sfa ratio was higher than in the group with lower vfa / sfa ratio (p < 0.001). a vfa / sfa ratio 1.165 had good accuracy to predict ee (area under the receiver - operating characteristic curve, 0.643). the vfa / sfa ratio and visceral fat volume were positively correlated with the severity of ee (p = 0.002), and a vfa / sfa ratio 1.165 was strongly correlated with the severity of ee (p < 0.001).conclusionthe high vfa / sfa ratio can be a useful clinical predictor of ee. |
the subjects of the present study were 40 patients with cataracts and 30 normal persons who visited the department of ophthalmology. the contents of the survey and examination were explained to all of the subjects, and written consent was obtained. the questionnaire assessed gender, age, and life habit variables such as smoking status, drinking status, and daily life, in addition to survey items about personal history. the medical examinations included measurements of height, weight, blood pressure, fasting plasma glucose, and total cholesterol and genetic tests for abl1 and tdrd7. for the genetic and clinical examinations involving survey, we obtained approval from the institutional review board of chosun university hospital. thirty healthy, age- and sex - matched subjects were also included for the controls. blood for testing was obtained before noon the next day after instructing the subjects to keep their stomachs empty for 12 hours or longer after eating dinner. for serum cholesterol, plasma glucose and gene analysis, peripheral blood (4 - 5 ml) was taken into vacuum tubes treated with edta (ethylene diamine tetraacetic acid), and transported on ice from the time it was obtained until centrifugation. the blood that arrived at the test room was then centrifuged for 10 minutes at 3,000 rpm at 4 prior to testing. total cholesterol and plasma glucose were measured using a commercial analytical system (architect ci16200 analyser ; abbott laboratories, abbott park, il, usa). total rna was extracted from each venous blood sample with the easyred rna extraction kit (intronbio, seoul, korea) in accordance with the manufacturer 's protocol. the cdna synthesis was conducted using a superscript vilo cdna synthesis kit (invitrogen, carlsbad, ca, usa) in accordance with the manufacturer 's protocol. for quantification of the tdrd7 transcript relative to the abl1 control gene, we used the primers specific for tdrd7 (forward : 5'-acgagtagagatcacaaatg-3 ' ; reverse : 5'-tctgctaaacagcacttaat-3 ') and abl1 (forward 5'-ctgctagagaaggactacc-3 ' ; reverse : 5'-tcgtctgagatactggattc-3 '). real - time polymerase chain reaction (pcr) reactions with the above primers were conducted in a total volume of 20 l using phusion flash (thermo scientific, wilmington, de, usa) with evagreen (biotium, hayward, ca, usa) in accordance with the manufacturer 's recommendations. thermocycling was carried out using a cfx96 real - time pcr detection system (bio - rad, hercules, ca, usa) with the following conditions : 1 cycle of 98 for 10 seconds, 40 cycles of 98 for 1 second, and 55 for 15 seconds. the amplification curves of 2 genes, tdrd7 and abl1 mrna, are shown in fig. 1. amplification of an endogenous control may be conducted to standardize the amount of tdrd7. for the quantification of gene expression the relative expression levels of tdrd7 mrna, normalized to abl1 mrna, were calculated using the 2 value. in order to apply the 2 method, the results of real - time reverse transcription - pcrs were represented as cycle threshold (ct) values. the ct value was defined as the cycle at which a sample crosses a threshold that is significantly above the background fluorescence and within the exponential phase of the amplification. the average of three ct measurements was calculated for both the tdrd7 and abl1 mrna. ct was determined as the mean of the triplicate ct values for the tdrd7 genes minus the mean of the triplicate ct values for the abl1 gene. the ct represented the difference between the two groups for a given target gene, namely ct = ct (cataract group) - ct (the mean of normal group). the -fold higher expression of a given target gene in the cataract group compared to the normal group was calculated as 2. a past history of ophthalmologic disease such as ocular trauma and cataract surgery was checked, as well as whether the subjects had systemic disease such as hypertension, diabetes, or cardiovascular disease, along with family history. in addition to uncorrected visual acuity, corrected visual acuity was examined in the case of subjects that wore corrective lenses, and the best corrected visual acuity was measured after checking the refractive state via a manifest refraction test. the state of the lens was classified by lens opacities classification system (locs) iii through an examination using a slit lamp microscope after mydriasis. if, in one eye or two eyes, there was nuclear opacity of second stage or higher in locs iii classification, cortical opacity of second stage or higher, or posterior subcapsular opacity of first stage or higher, those subjects were classified as cataract patients, and were otherwise categorized as control group members. cataract patients were classified into nuclear, cortical, posterior subcapsular, or mixed types. data analysis was conducted using spss ver. 17.0 (chicago, il, usa) and the actual numbers, percentage, average, and standard deviation were obtained as descriptive statistics in order to study the general characteristics of the patient group and control group. the comparison of the general characteristics of the patient group and the control group was conducted via t test and test, and, in comparison of the genetic values of the patient group and the control group, cases in which the p - value was found to be below 0.05 by an independent sample t test were determined to be statistically significant. in order to compare the tdrd7 gene values of different types of cataracts, one - way anova was used ; to compare the tdrd7 gene values of normal eyes of different age objects, correlation analysis was conducted. cases in which the p - value was below 0.05 were determined to be statistically significant. the subjects of the present study were 40 patients with cataracts and 30 normal persons who visited the department of ophthalmology. the contents of the survey and examination were explained to all of the subjects, and written consent was obtained. the questionnaire assessed gender, age, and life habit variables such as smoking status, drinking status, and daily life, in addition to survey items about personal history. the medical examinations included measurements of height, weight, blood pressure, fasting plasma glucose, and total cholesterol and genetic tests for abl1 and tdrd7. for the genetic and clinical examinations involving survey, we obtained approval from the institutional review board of chosun university hospital. thirty healthy, age- and sex - matched subjects were also included for the controls. blood for testing was obtained before noon the next day after instructing the subjects to keep their stomachs empty for 12 hours or longer after eating dinner. for serum cholesterol, plasma glucose and gene analysis, peripheral blood (4 - 5 ml) was taken into vacuum tubes treated with edta (ethylene diamine tetraacetic acid), and transported on ice from the time it was obtained until centrifugation. the blood that arrived at the test room was then centrifuged for 10 minutes at 3,000 rpm at 4 prior to testing. total cholesterol and plasma glucose were measured using a commercial analytical system (architect ci16200 analyser ; abbott laboratories, abbott park, il, usa). total rna was extracted from each venous blood sample with the easyred rna extraction kit (intronbio, seoul, korea) in accordance with the manufacturer 's protocol. the cdna synthesis was conducted using a superscript vilo cdna synthesis kit (invitrogen, carlsbad, ca, usa) in accordance with the manufacturer 's protocol. for quantification of the tdrd7 transcript relative to the abl1 control gene, we used the primers specific for tdrd7 (forward : 5'-acgagtagagatcacaaatg-3 ' ; reverse : 5'-tctgctaaacagcacttaat-3 ') and abl1 (forward 5'-ctgctagagaaggactacc-3 ' ; reverse : 5'-tcgtctgagatactggattc-3 '). real - time polymerase chain reaction (pcr) reactions with the above primers were conducted in a total volume of 20 l using phusion flash (thermo scientific, wilmington, de, usa) with evagreen (biotium, hayward, ca, usa) in accordance with the manufacturer 's recommendations. thermocycling was carried out using a cfx96 real - time pcr detection system (bio - rad, hercules, ca, usa) with the following conditions : 1 cycle of 98 for 10 seconds, 40 cycles of 98 for 1 second, and 55 for 15 seconds. the amplification curves of 2 genes, tdrd7 and abl1 mrna, are shown in fig. 1. amplification of an endogenous control may be conducted to standardize the amount of tdrd7. for the quantification of gene expression the relative expression levels of tdrd7 mrna, normalized to abl1 mrna, were calculated using the 2 value. in order to apply the 2 method, the results of real - time reverse transcription - pcrs were represented as cycle threshold (ct) values. the ct value was defined as the cycle at which a sample crosses a threshold that is significantly above the background fluorescence and within the exponential phase of the amplification. the average of three ct measurements was calculated for both the tdrd7 and abl1 mrna. ct was determined as the mean of the triplicate ct values for the tdrd7 genes minus the mean of the triplicate ct values for the abl1 gene. the ct represented the difference between the two groups for a given target gene, namely ct = ct (cataract group) - ct (the mean of normal group). the -fold higher expression of a given target gene in the cataract group compared to the normal group was calculated as 2. a past history of ophthalmologic disease such as ocular trauma and cataract surgery was checked, as well as whether the subjects had systemic disease such as hypertension, diabetes, or cardiovascular disease, along with family history. in addition to uncorrected visual acuity, corrected visual acuity was examined in the case of subjects that wore corrective lenses, and the best corrected visual acuity was measured after checking the refractive state via a manifest refraction test. the state of the lens was classified by lens opacities classification system (locs) iii through an examination using a slit lamp microscope after mydriasis. if, in one eye or two eyes, there was nuclear opacity of second stage or higher in locs iii classification, cortical opacity of second stage or higher, or posterior subcapsular opacity of first stage or higher, those subjects were classified as cataract patients, and were otherwise categorized as control group members. cataract patients were classified into nuclear, cortical, posterior subcapsular, or mixed types. data analysis was conducted using spss ver. 17.0 (chicago, il, usa) and the actual numbers, percentage, average, and standard deviation were obtained as descriptive statistics in order to study the general characteristics of the patient group and control group. the comparison of the general characteristics of the patient group and the control group was conducted via t test and test, and, in comparison of the genetic values of the patient group and the control group, cases in which the p - value was found to be below 0.05 by an independent sample t test were determined to be statistically significant. in order to compare the tdrd7 gene values of different types of cataracts, one - way anova was used ; to compare the tdrd7 gene values of normal eyes of different age objects, correlation analysis was conducted. cases in which the p - value was below 0.05 were determined to be statistically significant. among the total 70 subjects, 40 (57.1%) were cataract patients while 30 (42.9%) belonged to the control group ; 33 (47.1%) were males and 37 (52.9%) were females. the average age of the whole subject population was 52.67 12.96 years and the age range of the subjects was between 10 and 89 years. the age of the cataract patients was significantly higher, with an average of 62.68 13.40 years, whereas the average age of the control group was 38.60 23.99 years (p 0.05). although the serum glucose level of the cataract patients was higher than that of the control group with a value of 109.63 32.72 mg / dl, while that of the control group was 95.31 15.45 mg / dl, the difference was not statistically significant (p > 0.05) (table 1). in the results of our classification of the cataracts of 40 patients, in accordance with the positions of the opacity, the cataracts were identified as 19 nuclear types, 10 cortical, 6 posterior subcapsular, and 5 mixed types. the abl1 and tdrd7 values of the control group were 26.95 0.91 and 29.68 1.07, respectively, and those of the cataract patients were 26.17 1.25 and 29.11 1.34, respectively. ct was determined as the mean of the triplicate ct values for the tdrd7 genes minus the mean of the triplicated ct values for the abl1 gene. ct represented the difference between the two groups for a tdrd7 gene, more precisely ct = ct (cataract group) - ct (normal group). the mean and standard deviations for all ct and ct values (cataract versus normal group) are provided in table 2. the results, including standard deviations, are listed in table 2. as the result of adjusting the tdrd7 value in reference to abl1 mrna, the tdrd7 expression level of the cataract patients was found to be significantly lower than that of the control group, with a value of 1.05 0.34, whereas that of the control group was 1.52 0.63 (p = 0.048) (table 2). to allow for a better overview and to show the broad spectrum of expression rates, all 2 values (with standard deviations) the results of our comparisons of the genetic values of different types of cataracts demonstrated that the tdrd7 expression levels of the cortical type and mixed type were statistically significantly lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017) (table 3). to allow for a better overview and to demonstrate the wide spectrum of expression rates, all 2 values (with standard deviations) 3). the results of measuring the tdrd7 gene expression levels of the normal eyes of different age objects demonstrated that there was no statistically significant correlation between the two variables (p = 0.785) (fig. the abl1 and tdrd7 values of the control group were 26.95 0.91 and 29.68 1.07, respectively, and those of the cataract patients were 26.17 1.25 and 29.11 1.34, respectively. ct was determined as the mean of the triplicate ct values for the tdrd7 genes minus the mean of the triplicated ct values for the abl1 gene. ct represented the difference between the two groups for a tdrd7 gene, more precisely ct = ct (cataract group) - ct (normal group). the mean and standard deviations for all ct and ct values (cataract versus normal group) are provided in table 2. the results, including standard deviations, are listed in table 2. as the result of adjusting the tdrd7 value in reference to abl1 mrna, the tdrd7 expression level of the cataract patients was found to be significantly lower than that of the control group, with a value of 1.05 0.34, whereas that of the control group was 1.52 0.63 (p = 0.048) (table 2). to allow for a better overview and to show the broad spectrum of expression rates, all 2 values (with standard deviations) the results of our comparisons of the genetic values of different types of cataracts demonstrated that the tdrd7 expression levels of the cortical type and mixed type were statistically significantly lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017) (table 3). to allow for a better overview and to demonstrate the wide spectrum of expression rates, all 2 values (with standard deviations) 3). the results of measuring the tdrd7 gene expression levels of the normal eyes of different age objects demonstrated that there was no statistically significant correlation between the two variables (p = 0.785) (fig. this study focused on the loss - of - function mutation of tdrd7, which has been shown to affect its expression level and the clinical presentation of cataracts. cataracts account for approximately 50% of blindness globally, and the only way to treat cataracts is to remove the lens through surgery. understanding the causes of cataracts and risk factors can be helpful in the search for non - operative methods to delay the occurrence of or prevent cataracts. most known gene mutations that lead to congenital cataracts derive from genes that encode for structural proteins of the lens. the gene mutations involved in the generation of transcription factors that regulate the expression of structural genes account for the subset of mutations that cause cataracts. there are currently several reported mutations in congenital and hereditary cataracts involving genes that encode proteins with structural, metabolic, and transport functions. it is possible that some of these genes may be involved in adult cataract with late phenotype expression due to the interaction with several harmful factors along with the aging process. suggested that rna - containing granules can regulate the subcellular localization of mrna as well as the processing which plays an important role in the transparency of the lens. cytoplasmic rna granules play a role in determining whether mrnas undergo degradation, stabilization, or intracellular localization. the structure of the lens in vertebrates requires a very high concentration of specialized cytoplasmic proteins : the crystalline, a specialized intermediate filament cytoskeleton to stabilize the structure of the lens ; cellular membranes with high concentrations of both the lens - specific water channel, aquaporin 0 ; and lens - preferred connexins, which allow the lens to transport nutrients and waste products despite its lack of blood vessels. a number of the genes encoding for these proteins are abundantly expressed in the lens, resulting in a highly biased transcriptome. have reported the identification of tudor domain - containing 7 protein, or tdrd7, as an rna granule component with a highly enriched and conserved pattern of developmental expression in the ocular lens. they suggested that human tdrd7 mutations result in cataract formation via the misregulation of specific, developmentally critical lens transcripts. the official name of the tdrd7 gene is " tudor domain containing 7 " ; it is also referred to by other names such as catc4, kiaa1529, pctaire2bp, rp11 - 508d10.1, and trap. more accurately, it is located from base pair 100,174,301 to base pair 100,258,406 on chromosome 9. tdrd7 is a member of a large family of tudor domain - containing proteins that mutually interact with methylated arginine residues on other proteins. many members of the tdrd family, including tdrd7, are found in rna granules - cytoplasmic complexes of protein and rna that regulate gene expression by influencing rna degradation, stabilization, and the intracellular localization of rna. although tdrd7 transcripts and proteins are found in a variety of cells, especially of the male germ line, tdrd7 expression is very high in lens fiber cells and may control the production of extremely high amounts of structural proteins of the lens. tdrd7 is a tudor domain rna binding protein which is expressed in lens fiber cells, and is required for the posttranscriptional control of mrnas that are essential for normal lens development and rna granule function. demonstrated that tdrd7 is found in a granular distribution in lens fiber cells and colocalizes with concentrations of rna found in the cytoplasm of these cells. they demonstrated that human organogenesis defects can result from the perturbation of a distinct, tissue - specific rna granule component that regulates the posttranscriptional steps of developmentally critical mrnas. simple rna distribution within lens fibers would presumably be inefficient because of the relatively long distance between the lens fiber cell nucleus and the fiber cell tips, as well as the high concentration of proteins in the fiber cell cytoplasm. accordingly, when we look into the matter retrospectively, the requirement for a lens - preferred rna transport mechanism may not be entirely surprising. most notably, the lens epithelial cells express mrnas for lens structural proteins that are not translated into proteins until the onset of fiber cell differentiation, which is also when the onset of tdrd7 expression occurs. additionally, another possible role for tdrd7-containing rna granules would be to regulate the translation of fiber cell structural proteins. determined that reduced tdrd7 expression in a lens epithelial cell line induced statistically significant changes in the expression levels of 6% of the genes expressed in those cells, including some crystallins. overall, lachke. demonstrated that the vertebrate lens contains several classes of rna granules and that tdrd7-containing granules are critically important for the normal functioning of the lens. these findings open up a new field of investigation that may ultimately help to elucidate both the physiological and biochemical functions that regulate rna granules in the lens. when we look into different risk factors of cataract, there are some risk factors which clearly affect specific types of cataracts. for example, smoking increases the prevalence of nuclear cataracts, and steroids are related to the occurrence of posterior subcapsular cataract. the results of comparing the gene values of different types of cataracts in this study demonstrated that the tdrd7 expression ratios of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular type ; this implies that, while many senile cataracts are of nuclear type, many cataracts caused by genetic loss - of - function mutations may be of the cortical type. although blood cholesterol is not known as a risk factor for cataracts, a case was recently reported wherein posterior subcapsular cataract occurred in two eyes of a lathosterolosis patient showing a defect of cholesterol biosynthesis ; klein. suggested that the ingestion of statin, which is widely used for the purpose of preventing cardiovascular disease by lowing the blood cholesterol value lowers the incidence rate of nuclear cataracts. in this study, although the total cholesterol value of cataract patients was higher than that of the control group, this was not statistically significant. the result of comparing the age and the tdrd7 gene expression ratios of the patients with normal eyes demonstrated that there is no statistically significant correlation between the two variables (fig. although this study is significant in that it is the first study regarding the relationship between the tdrd7 gene and cataracts using koreans as subjects, further proactive studies with a greater number of subjects selected from a larger number of regions are needed to more clearly establish the relationship between the tdrd7 gene and cataracts. in conclusion, as the tdrd7 gene expression levels of the cataract patients found in a test conducted with 70 subjects living in a local region in the present study were statistically significantly lower than those of the control group, we can assert that there is a causal relationship between the occurrence of human cataracts and the loss- of - function mutations of the tdrd7 gene. lachke. suggested that rna - containing granules can regulate the subcellular localization of mrna as well as the processing which plays an important role in the transparency of the lens. cytoplasmic rna granules play a role in determining whether mrnas undergo degradation, stabilization, or intracellular localization. the structure of the lens in vertebrates requires a very high concentration of specialized cytoplasmic proteins : the crystalline, a specialized intermediate filament cytoskeleton to stabilize the structure of the lens ; cellular membranes with high concentrations of both the lens - specific water channel, aquaporin 0 ; and lens - preferred connexins, which allow the lens to transport nutrients and waste products despite its lack of blood vessels. a number of the genes encoding for these proteins are abundantly expressed in the lens, resulting in a highly biased transcriptome. have reported the identification of tudor domain - containing 7 protein, or tdrd7, as an rna granule component with a highly enriched and conserved pattern of developmental expression in the ocular lens. they suggested that human tdrd7 mutations result in cataract formation via the misregulation of specific, developmentally critical lens transcripts. the official name of the tdrd7 gene is " tudor domain containing 7 " ; it is also referred to by other names such as catc4, kiaa1529, pctaire2bp, rp11 - 508d10.1, and trap. more accurately, it is located from base pair 100,174,301 to base pair 100,258,406 on chromosome 9. tdrd7 is a member of a large family of tudor domain - containing proteins that mutually interact with methylated arginine residues on other proteins. many members of the tdrd family, including tdrd7, are found in rna granules - cytoplasmic complexes of protein and rna that regulate gene expression by influencing rna degradation, stabilization, and the intracellular localization of rna. although tdrd7 transcripts and proteins are found in a variety of cells, especially of the male germ line, tdrd7 expression is very high in lens fiber cells and may control the production of extremely high amounts of structural proteins of the lens. tdrd7 is a tudor domain rna binding protein which is expressed in lens fiber cells, and is required for the posttranscriptional control of mrnas that are essential for normal lens development and rna granule function.. demonstrated that tdrd7 is found in a granular distribution in lens fiber cells and colocalizes with concentrations of rna found in the cytoplasm of these cells. they demonstrated that human organogenesis defects can result from the perturbation of a distinct, tissue - specific rna granule component that regulates the posttranscriptional steps of developmentally critical mrnas. simple rna distribution within lens fibers would presumably be inefficient because of the relatively long distance between the lens fiber cell nucleus and the fiber cell tips, as well as the high concentration of proteins in the fiber cell cytoplasm. accordingly, when we look into the matter retrospectively, the requirement for a lens - preferred rna transport mechanism may not be entirely surprising. most notably, the lens epithelial cells express mrnas for lens structural proteins that are not translated into proteins until the onset of fiber cell differentiation, which is also when the onset of tdrd7 expression occurs. additionally, another possible role for tdrd7-containing rna granules would be to regulate the translation of fiber cell structural proteins. determined that reduced tdrd7 expression in a lens epithelial cell line induced statistically significant changes in the expression levels of 6% of the genes expressed in those cells, including some crystallins. overall, lachke. demonstrated that the vertebrate lens contains several classes of rna granules and that tdrd7-containing granules are critically important for the normal functioning of the lens. these findings open up a new field of investigation that may ultimately help to elucidate both the physiological and biochemical functions that regulate rna granules in the lens. when we look into different risk factors of cataract, there are some risk factors which clearly affect specific types of cataracts. for example, smoking increases the prevalence of nuclear cataracts, and steroids are related to the occurrence of posterior subcapsular cataract. the results of comparing the gene values of different types of cataracts in this study demonstrated that the tdrd7 expression ratios of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular type ; this implies that, while many senile cataracts are of nuclear type, many cataracts caused by genetic loss - of - function mutations may be of the cortical type. although blood cholesterol is not known as a risk factor for cataracts, a case was recently reported wherein posterior subcapsular cataract occurred in two eyes of a lathosterolosis patient showing a defect of cholesterol biosynthesis ; klein. suggested that the ingestion of statin, which is widely used for the purpose of preventing cardiovascular disease by lowing the blood cholesterol value lowers the incidence rate of nuclear cataracts. in this study, although the total cholesterol value of cataract patients was higher than that of the control group, this was not statistically significant. the result of comparing the age and the tdrd7 gene expression ratios of the patients with normal eyes demonstrated that there is no statistically significant correlation between the two variables (fig. although this study is significant in that it is the first study regarding the relationship between the tdrd7 gene and cataracts using koreans as subjects, further proactive studies with a greater number of subjects selected from a larger number of regions are needed to more clearly establish the relationship between the tdrd7 gene and cataracts. in conclusion, as the tdrd7 gene expression levels of the cataract patients found in a test conducted with 70 subjects living in a local region in the present study were statistically significantly lower than those of the control group, we can assert that there is a causal relationship between the occurrence of human cataracts and the loss- of - function mutations of the tdrd7 gene. | purposeto investigate the relationship between plasma tdrd7 mrna and lens transparency, and to evaluate plasma tdrd7 mrna as a potential marker for cataracts and its sub - type by quantitatively analyzing human peripheral blood.methodsplasma rna was extracted from 40 patients with cataracts, and 30 normal controls of matched age and gender. blood cholesterol and fasting glucose were measured, and the rna extracted from the sample was synthesized into cdna. after polymerase chain reaction, the results were compared after quantifying the tdrd7 mrna using abl1 mrna for normalization. we analyzed the relative gene expression data via the ct method.resultsthe normalized 2-ct of plasma tdrd7 mrna based on abl1 mrna was 1.52 0.63 in the case of the control group and 1.05 0.34 in the case of the cataract patients, and the tdrd7 expression level of the cataract patients was lower than that of the control group (p = 0.048). the comparison of the genetic values of different types of cataracts demonstrated that the tdrd7 expression level of the cortical type and mixed type were lower than those of the nuclear type and posterior subcapsular opacity type (p = 0.017).conclusionshuman cataracts and the tdrd7 gene loss - of - function mutations are strongly causally related, as the expression level of plasma tdrd7 mrna in patients with cataracts was statistically significantly lower than in the normal control group. |
nowadays, overweight and obesity are global problems, as the marketing of the energetic and junk food is one of the principal reasons (1). different studies have confirmed that many commercial advertisements are contrary to the healthy nutrition (2). the consumption of the advertised foods is more than the non - advertised foods (3). despite the growing power of social media, tv advertising is still the most influential medium in buying decisions of people (4). advertising of unhealthy food means that television advertisements encourage consumption of the products with high fat, sugar, and/ or sodium. according to previous studies, most of the television food advertisements promote unhealthy food products or reveal unhealthy nutritional information that causes more preferences for and buying unhealthy food products. also, some studies have shown that food advertising affects consumption of foods with high total calorie (5). various studies have shown that exposure to television food advertisements is one of the main factors influencing the diet and eating behaviors and preferences of people, which finally affects the outcomes such as obesity and overweight (4,6). according to a study in the usa, the american children watch 20000 - 40000 advertisements per year (7). a similar study in australia showed that watching more ads increases interest and intention to harmful foods such as sweet, meat, fast food and sweet drinks (8,9). in other research on adults, similar findings were reported (10). for analysis of commercial advertisements in swiss media, six prominent swiss tv channels, they mainly advertised the consumption of sweet and fatty foods, without any healthy advertisements for fruit and vegetables (11). in an identical research in canada, four canadian tv and three famous british channels were monitored for one week in 2006. after analysis of advertisements according to the scientific criteria of healthy/ unhealthy foodstuff, it was concluded that 52 - 61% of the advertisements were related to the unhealthy foodstuff (12). according to the findings of an iranian study in 2008, television s food advertisements were identified as the factors influencing healthy lifestyle among the senior citizens (13). a study in 2009 showed that children exposed to food advertising consumed 45% more food. adults also had more consumption of both healthy and unhealthy snack foods in similar condition (14). advertising plays a significant role in habits and preferences of people. mass media, especially tv, has a major effect on adopting a healthy lifestyle by people. this mass media associated with social support of friends, family members, and health professionals and other organizations can be effective while concentrating their programs on the promotion of healthy lifestyles. messages provided in this area should be attractive, clear, proportionate, understandable and in accordance with people culture. it is clear that making the messages consistent with age and change stage of people is one of the duties of health professionals (13). advertisements of harmful services and products have been declared forbidden for all media which could be followed by fine according to iranian 5-year development program law passed by islamic legislative assembly in schedule 2011. since there may be differences between the law and execution, so the executive organizations shall enforce and control the regulation to influence on their performance. in any cases, there is a close relationship between consuming high - quality healthy foodstuff and the standard matters with public health, and if it is ignored, it may cause serious danger to public health of society in the future.all health authorities have to notice the role of advertising programs on health goals in order to prevent the related diseases. moreover, the majority of studies on the status of health - related television and radio advertising have been conducted in europe and the usa. in iran an extensive study has not been done yet to compare the advertisements of hazardous or healthy products and to determine their congruence with current health recommendations provided by the ministry of health. therefore, this research was conducted to study the health - related advertisements in islamic republic of iran broadcasting (irib), and specifying the kind of the advertisements was not healthy. so the findings may also help policymakers and authorities to plan and make reasonable and constructive decisions on health - related program of advertisements broadcasting to promote public health. this study was a cross - sectional research conducted to explore the status of health - related advertisements in 2012. different channels of irib were considered as the research population, and the list of which was prepared by internet websites. because of a variety of irib channels, the researchers decided to monitor tv and radio programs using a sampling method. the sample channels were chosen for monitoring according to the list of all local networks and based on the priority of advertisements and geographical distribution on the map. among 16 nationwide tv networks, 17 provincial tv networks and 14 radio networks, the following channels were selected to study : nationwide networks : channel 1, channel 2, channel 3 ; provincial networks : a) tehran, b) gilan, c) khouzestan, d) isfahan ; radio networks : 1) nationwide radio, 2) payam radio, and 3) youth radio. to have different audiences of tv and radio channels, recording the advertising information was on a rotating basis throughout the week. since some popular programs include movies, serials, entertainments, and sport programs such as football leagues were broadcasted in special week days, mid - week days were selected in rotation to cover all programs and their audience. also, because the audience may have much more time for watching irib programs at weekends, tv and radio programs were recorded in thursdays and fridays for all the weeks. the selected weekdays of the first week were included in : saturday, sunday, thursday, and friday, and the second week were also involved : monday, tuesday, thursday and friday, and the third week took in : wednesday, thursday and friday, which was repeated the same from the fourth weeks as explained. all of the advertisements broadcasted in irib (such as commercial, educational, etc.) except two types of tv advertisements (blipverts : a blipvert is a very brief television advertisement that lasts just one or several seconds) and subtitle advertising) were included in this study. the study data was collected using the observational method and a researcher - made checklist which its validity was attested using an expert panel method and carrying out a pilot study. the basic items of the data collection checklist were extracted based on the literature review, and the checklist draft was reviewed by nine experts who were specialists in community medicine, nutrition, health education, media, health economics and health care management. then according to the experts feedbacks, the corrections were made to the initial draft, and the final checklist was composed and approved. the checklist items were included : number, date, broadcasting time, product type, advertisements duration and channel name/ code. for the pilot study, all observers completed some checklists according to a sample of recorded tv and radio advertisements, and then their forms were compared with each other, and some minor revisions were made in the final checklist. the observers were chosen among volunteer experts working at iran university of medical sciences. because all observers were trained to complete the checklists and all advertising information were extracted from the recorded advertisements, the study checklist was fully completed with no missing data in this study. after checking and analysis of checklists, data processing was started using excel software. the encoding method was used to separate and classify the advertised products according to the criteria based on the guidelines of iran ministry of health. to control and restrict the advertising of unhealthy products and services, ministry of health in collaboration with other departments (industry and mines, welfare and social security, trade and economic affairs and finance ministries) enacted the regulations concerning health - related advertisements broadcasting. therefore, adequate supervision and monitoring should be done in this area, and television networks and radio stations have to pay fines in the cases of the rules violation. according to ministry of health guidelines, the classifications of unhealthy products and services are listed below : 1) less healthy : bulked cereals (puff paste, crunchy, etc.), beverages (such as cola, energized drinks), chips, etc. 2) harmful to health : cosmetic services (such as solarium tanning, tattoos, hair and nail implant, etc.) spraying poison services without supervision of ministry of health nutritional counseling and medications for weight loss and obesity, the intervention measures to increase the height, and bodybuilding clubs without supervision of ministry of health pools and recreation water centers(water parks) without supervision of ministry of health environment and water decontamination services without supervision of ministry of health 3) harmful to health and probability of abuse drugs : all kinds of medicines without the authorization of ministry of health products or actions representative of high - risk behaviors (such as consuming fast food and industrial food products, tobacco use including cigarette and hookah, etc). statistical analysis of the study was performed using spss 18. data was analyzed in the descriptive method, and statistical diagrams and tables specify the status of irib advertisements generally regarding average time in seconds, and healthy products in percentage among which the harmful products and services were determined and classified according to the related criteria. however the advertisements broadcasted by irib and monitored during this study in 2012 was as follows : a) advertisements unrelated to health (such as civil, communication services, banking, education, private institutes, culture, trading, home appliances, cars, insurance, industry, etc) ; b) harmless advertisements (e.g., detergent, health products, dairy, groceries, rice, spaghetti, fish can, foodstuff, restaurants, swimming pools, sports equipment, etc) ; c) less healthy advertisements (such as beverage, types of chips, puff paste, crunchy, etc) ; d) harmful advertisements (just hair implant was broadcasted) ; e) harmful advertisements with a probability of abuse include (just the fast food was broadcasted). the results of analyzing tv and radio advertisements are shown in the statistical diagrams and tables. the overall time in seconds spent for all the advertisements in ten tv and radio channels were as follows : total recorded time : 638811 seconds (100% non - harmful to health advertisements : 612339 seconds (95.86% harmful to health ads : 2334 seconds (0.37% harmful to health with the probability of abuse : 5495seconds (0.86% less healthy : 18643seconds (2.91%) (table 1) because of lack of scientific criteria, it is impossible to determine harmfulness of health services advertised by irib in national and local levels in 2012. nevertheless, advertisement of health services includes the following items : clinicians complex, clinics, swimming pools and water parks, etc. table 2 indicates the frequency and time spent (in seconds) for total advertisements of the ten tv and radio channels according to types of health products (table 2). furthermore, 26.1% of the time spent (in seconds) for total advertisements out of ten tv and radio channels of concerned to health. the time percentage of advertisements of harmful products and services compared to total irib advertisements in national and local levels during the period of study in 2012 are as follows : less healthy advertisements (2.9%), harmful advertisements (0.4%), harmful advertisements with a probability of abuse (0.9%) (table 3, diagram 1). percentage of time spent for advertisements related and unrelated to health in ten tv and radio channels according to the findings, 80.9% of advertisements were broadcasted between different programs, 0.8% of total advertisements during children programs, 1.38% ad during sport programs, 13.66% ad during entertainment programs, 0.3% ad during educational programs, 2.5% ad during family programs, and 0.49% ad during documentary programs, interviews, and scientific meetings (diagram 2). the status of advertisements of ten tv and radio channels in terms of advertising within or between the programs the main findings of this study revealed that the majority of advertisements of islamic republic of iran broadcasting in 2012 were unrelated to health, and a tiny number of advertisements were harmful to health. it showed that 4.14% of the advertisements were related to the harmful, less healthy foodstuff and harmful services and products, such as beverage, types of chips, puff paste, crunchy, hair implant, and fast food, etc. the advertisements of the ten iranian tv and radio channels in the special days for two successive months were monitored. the results showed non - harmful to health advertisements involved more than 95% of total advertisements of the ten tv and radio channels in the study ; and the remained advertisements were related to the harmful, less healthy foodstuff and detrimental services and products. also, near one percent of the advertisements were shown during children programs. in 2014, in a similar study which was performed in ireland, 322 tv advertisements of foodstuff and drinks were recorded, of which 66.3% were related to the food, and the more recorded advertisements concerned fast foods (27.3%). the findings also indicated that new laws shall be passed for controlling advertisements of unhealthy foodstuff broadcasted during children programs (15). it seems that the levels of fast foods advertising in irib broadcasting are less than the other country. an article published in 2013 indicated that, in spite of the reduction of tv advertisement costs by 19.4%, the children and teenagers still watch 12 - 16 advertisements a day which included the foods with high saturated fats, sugar or sodium (16). in a study carried out in singapore, 33% of analyzed advertisements were related to the foodstuff, 38% of foodstuff advertisements were recognized healthy and 57% unhealthy (2). these results are not in compliant with the findings of the current study which may show irib more commitment to the regulations related to foodstuff advertising. in a research carried out in mexico, 22% of advertisements were concerned to the foodstuff and 50% of the advertisements were related to the children (17). it was confirmed in an analysis of three australian commercial channels that 25.5% of advertisements were related to the foodstuff, and a large number of advertisements were broadcasted in the hours children were watching tv (18). in this study, the advertisements broadcasted during children programs were less than the ending hours of the night, which does nt match to the results of two mentioned studies in this case. the results of analyzing six national tv commercial channels in malaysia during a period of six months in 2008 indicated that the highest rate of advertisements was related to snack (34.5%) mostly broadcasted during the hours children were watching tv (19). furthermore, the findings of studying foodstuff advertisements in three australian tv channels showed that 31% of advertisements were concerned to foodstuff, and 81% focused on the unhealthy foods (1). whereas the findings of the current study revealed that tv and radio advertisements may be more supervised by health authorities. commercial advertisements of six prominent swiss tv channels, a german and an italian channel were recorded and analyzed for six months in 2011. the most commercial messages advertised sweet and fatty foods, without any healthy advertisement of fruits and vegetables (11). in a similar research in 2009, four tv canadian and three famous british channels were studied for one week. after advertisement analysis according to the scientific criteria of healthy and unhealthy foodstuff, it was confirmed that 52 - 61% of advertisements were related to unhealthy foodstuff (12). it may be due to neglecting or non - compliance with regulations and rules. in two third of countries, tv advertisements in 13 countries (australia, denmark, finland, belgium, france, germany, austria, greece, netherlands, norway, sweden, england and usa) were studied for 20 hours of children programs ; the results showed that australia, usa and england had the highest rate of foodstuff advertisements and the lowest ones were for sweden (7). while, in this research, the health - related advertisements consist of only 27.3% of total advertisements, in this regard, the foodstuff advertisements in irib have a better status than the pointed countries. according to the results of this study, near to one - third of tv advertisements is related to the health of which few amount is concerned to the advertisements of foodstuff without nutritional value, or unhealthy and harmful ones. the findings of this research showed that healthy, anti - obesity, and mixed food advertising decreased consuming total calories, fat, sodium, and carbohydrates (5). it is found that exposure to food advertisements can change eating preferences and behaviors (20). therefore, practitioners and decision makers should be aware of the spread of food advertising and the possibility of the impact on knowledge and behavior. they should make an effort to encourage producers to create and promote weight - friendly foods (3). according to the results of this study, it is recommended that besides keeping the current status of tv and radio advertising, the healthier foodstuff advertisements should be presented, and ministry of health should impose more supervision based on the legal responsibilities, and consider the contribution of the appropriate stakeholders. moreover, the government would be obliged to pay subsidiary to the industries owners for movement of food industries for producing healthier foods. concerning the relation between health and food, and due to the effect of advertising on health, the media especially radio and tv should provide the necessary knowledge to the society in this case, with broadcasting healthy advertisement. as one of the study limitations, due to differences in the nature and type of advertisements broadcasting in iran and its related regulations, the generalizing of the results to other countries may not be possible. it was not possible to determine harmfulness of health services in this study because there had not been performed any particular scientific study yet and the scientific criteria were not available. also, recording tv and radio programs including advertisements needed lots of time and energy, and saving and getting a backup of the recorded tv and radio programs was challenging. | background : media advertisements especially radio and tv are one of the most important and effective ways for health promotion and consumption of healthy productions worldwide. ministry of health and some other ministries in iran agreed to control and restrict the advertising of unhealthy products and services. therefore, adequate supervision and monitoring should be done in this field. a content analysis of health - related advertisements was done in islamic republic of iran broadcasting (irib) methods : this study was a cross - sectional research and collecting of data was carried out in 2012. ten selected tv and radio channels were recorded from 6 a.m. to 12 p.m. for two successive months in the special weekdays. broadcasted advertisements data were extracted by the trained observers according to a checklist and analyzed using spss 18 software and described with descriptive statistics. results : the percentage of different types of advertising were including 73.9% unrelated to health, 21.9% harmless health related, 2.9% less healthy, 1.3% harmful or harmful with a probability of abuse. non - harmful to health advertisements included 95.86% of total advertisements out of ten tv and radio channels ; and the remained advertisements (4.14%) were related to the harmful, less healthy foodstuff and detrimental services and products. also, 0.8% of the advertisements were shown during children programs. conclusion : the main findings of the current study revealed that majority of the advertisements of islamic republic broadcasting were unrelated to health. it seems advertising of harmful for health in irib was less than 5%, and the levels of these type ads were less than the other countries. even so, the policymakers need to pass and enforce some executive and governing law for the prevention of broadcasting unhealthy advertisements to increase the society health level and prevent the diseases resulted from unhealthy products causing the considerable damages in a long time. |
sushruta recognized as the father of cosmetic surgery, centuries earlier quoted a surgeon, by his own experience and intelligence, may invent and add new instruments to facilitate the surgical procedures. he pointed out the hand as the most important and the best instrument but for which the operation of other instruments cease. thereafter it has been the hands and the conventional instruments that have stayed on. however, it was the advent of laparoscopy that marked the modern day minimally invasive surgery. after having being initially utilized in gynaecology, the turn of the present century and the last decade has seen its widespread application in various surgical fields. specifically pertaining to urology nevertheless the rise of laparoscopy had seen many eyebrows of the critics before it has reached its present day status. however, the latter has its own set of concerns in the form of limited degrees of freedom, working with two - dimensional system, transmission of physiologic tremors, the fulcrum effect and so forth. in an attempt to overcome these limitations and at the same time maximizing the benefits of minimally invasive surgery, there came the application of robotics in surgery. the earlier versions such as puma-560, probot, robodoc have given way to the present day comprehensive master - slave surgical robot the da vinci system. of the different ramifications in surgery presently, urology and gynaecology are performing the major loads of robot - assisted surgeries, although the proportions being performed in general surgery, cardiovascular, oncology and even transplants off late, are on the rise. the first indian urological surgical program was started at aiims, new delhi in 2006. ever since then there has been an increase in the surgical robotic systems in india. at present, there are about 19 surgical robotic systems operative in the country. indian cities with robotic surgical platforms include new delhi, gurgaon, mumbai, chennai, nadiad, bengaluru, hyderabad. however, with an ever expanding population which currently stands about 1.2 billion, the number presently still stands skimpy. nevertheless the pertinent question remains : is it the need of the hour in present day indian scenario ? being a combination of computer technology with surgical handicrafts, it is envisaged as a revolution in itself, marking probably, just a new horizon of surgical innovation. the dictum holds true that what you see better you do better. the accompanying 10 - 15 magnification offered by the robotic platform provides an unmatched meticulous anatomy of the surgical field of interest, making chances of inadvertent injury an uncommon event. the three - dimensional vision makes working more ergonomic as compared to that in conventional laparoscopy. in contrast to the latter, there remains no need for the main surgeon to stand and operate, in fact, he sits comfortably on the console site and can perform even marathon surgeries. likewise, the patient side assistant as well as the nursing assistant can remain comfortably seated for the major part of the procedure, with the prime work of just exchanging the robotic instruments and providing sutures and suctioning intermittently. needless to say, this translates into much less exertion at the end of the day for both the operating surgeon and the assistants alike. the endowrist technology, provides a whole range of movements and 7 of freedom making this an ideal platform for suturing and reconstructive procedures such as prostatectomy, pyeloplasty, partial nephrectomy and other surgeries alike. motion scaling feature of the robotic system dramatically reduces the unavoidable physiological tremors, again, making suturing and fine dissection tasks as being ideally suited to be performed by this technology and at same time being more precise. the latter two features in particular improved working in deeper body cavities such as pelvis, again is an advantage as compared to the traditional open surgery. for the existence of any surgical technology or its innovation, it is prudent that the same has surgeon friendly profile and more importantly so, is advantageous to the patient as well. being minimally invasive in nature, it is associated with smaller incisions, improved cosmesis, less post - operative discomfort, less blood loss owing to the meticulous display of anatomy as mentioned, early recovery and discharge. with these bundled advantages, the robotic technology is being applied to virtually every surgery being performed by its open surgical and laparoscopic counterparts. as there are two sides of the coin, there are concerns that need to be addressed. yes, it is tacit that the major hurdle for the robotic technology to become cosmopolitan is the cost. one of the reasons could be a single large manufacturer presently being the dominant market supplier that is, the intuitive surgical of the da vinci system. notwithstanding the fact that robotic surgical platforms have a multi - million market, there are other contenders on the fence to join the business - the university of washington 's raven ii, titan medical 's amadeus systems, sofar 's telelap alf - x, and the araknes project to mention a few. if these are out with cheaper alternatives, probably it would nt be surprising to see these sophisticated systems outreaching to the common man as well. some of these could also come up with reusable instruments, further lowering down the finances involved. there is no doubt that presently very few centres are involved in rendering robotic surgery services to the community. the reason being acquisition of robotic surgical skills involves undergoing either a fellowship program or working under a mentor with necessary skills, over a period of time. the outcome of either of these is a very small fraction of super specialists reaching out to the billion population. however, this can be overcome with time as more surgical platforms become installed including the teaching institutes, so that a larger section of the budding surgeons can serve the community. however, it has been seen that though having a learning curve of its own, the same is not as difficult as is learning laparoscopy. the brighter side is even the surgeons of the open era, who are not facile with the typical laparoscopy, may still learn and perform on a robotic platform, although the ones who are well versed with laparoscopy have an obvious advantage. the most overt advantage is specifically the suturing task which seems much easier as compared to the laparoscopy counterpart. to further shorten the learning curve the number of robotic platforms being established are on the rise so is the number of cases being performed, not only abroad, but in india as well. as with any other technology the outcomes continue to improve with an increase in experience. in a country like ours this coupled with an ever increasing patient load and upcoming medical insurance schemes, renders our country a fertile soil for such platforms to grow and prosper. in the past 3 - 4 years there has been a steep increase in the installation of these robotic systems across the country. all the major cities are experiencing an increase in the number of large corporate sector hospitals which are readily establishing robotic surgical platforms. with the multi - specialty utilization and increase in the number of cases performed each day, the cost - effectiveness can be markedly improved. specifically in india, the cost comes out to be cheaper as compared to similar robotic surgeries performed in the western world. in fact, this can be utilized as a means for promoting medical tourism, which in turn could bring the country to a new frontier. finally, there have been allegations by the critics that the technology is primarily an industry driven. however, there is enough literature support for the utility and advantages of the newer modality in question. there is no doubt that the best piece of evidence comes from the meta - analysis, systematic reviews and well - conducted randomized controlled trials, that is, level 1 evidence. in a meta - analysis of four randomized controlled trials of robotic - assisted versus laparoscopic colorectal surgery to compare the safety and efficacy, liao. concluded lesser blood loss, recovery time and conversion rates with robotic - assisted procedures as compared to laparoscopic ones. in a systematic review and meta - analysis of robotic radical cystectomy versus open radical cystectomy, the former was found to have lower high - grade complication rates and mortality compared to the open approach. with reference to robotic prostatectomy suggests that though being more expensive in terms of capital and maintenance costs, these could be brought down by if the capital cost of equipment is minimized and by maintaining a large patient turnover of at least 100 - 150 procedures per year per robotic system. a comparative direct cost analysis of paediatric urologic robot - assisted laparoscopic surgery versus open surgery by rowe. the authors found the direct incurred costs of robotic surgery to be significantly lower compared to the open counterpart. however, for this to take effect it is important to have a dedicated and consistent robotic surgery team, meticulous patient and procedure selection as well as high patient turnover. a systematic review by gala. comparing robotic gynaecologic surgery with laparoscopic and open counterparts, came out with the fact that robotic surgery consistently conferred shorter hospital stay. the learning curve for robotic surgery was found to be lower than for conventional laparoscopy as well as the technique to be advantageous as compared to its open counterpart for management of endometrial cancer. renal transplantation, a procedure performed by open means, is now no more an exception to the robotic realms and a feat that has been successfully accomplished recently. robotic surgery with its bundled advantages is still in its burgeoning phase, the best of which is yet to come. the unrivalled suturing ease and motion scaling features, transforming into greater precision, has led to its widespread application in different surgical ramifications. these, coupled with the aforementioned advantages, has led to an increasing number of procedures being performed and that too with improved patient outcomes. it seems that the progressing india is readily accepting this robotic surgical innovation, the use of which is on a continuous rise, with the number of robotic platforms coming up in increasing numbers in many tertiary care indian centres and a corresponding increase in demand of the same by the patients as well ; thereby aptly fulfilling the economics of demand and supply. last, but not the least, it would nt be unfair to say the fiction of yesterday, is translating into a vividly seen reality of today and a horizon of unmatched surgical excellence of tomorrow. | robotic surgery with its bundled advantages is still in its burgeoning phase, the best of which is yet to come. the unrivalled suturing ease and motion scaling features, transforming into greater precision, has led to its widespread application in different surgical ramifications. these, coupled with the aforementioned advantages, has led to an increasing number of procedures being performed and that too with improved patient outcomes. it seems that the progressing india is readily accepting this robotic surgical innovation, the use of which is on a continuous rise, with the number of robotic platforms coming up in increasing numbers in many tertiary care indian centres and a corresponding increase in demand of the same by the patients as well ; thereby aptly fulfilling the economics of demand and supply. |
the danish neuro - oncology registry (dnor) is a national clinical database collecting data on diagnostics and treatments in adult patients with primary brain tumors in denmark.1 it was established with the purpose of supporting clinical development and research in this patient group. the danish neuro - oncology group (dnog) is developing and implementing national guidelines on treating adult brain tumor patients in denmark.2 by the supply of data from dnor, the discussions in dnog are facilitated and performed on a more evidence - based background. a few key clinical quality indicators are selected in dnor, giving each clinical department the opportunity to compare its own results to predefined standards and to reported national levels, and to follow - up on time trends of its own results. in denmark (5.5 million inhabitants), approximately 1,550 patients are diagnosed with brain tumor each year and the incidence is increasing. brain tumor diagnoses are classified by the international classification of diseases (icd) codes and the systematized nomenclature of medicine morphology codes.3,4 the icd-10 codes may be grouped into the following entities (table 1) : cerebral tumors, meningioma, sellae tumors, pineal tumors, neurinoma, and spinal tumors. patients in each group are handled in different ways clinically, and accordingly, different data need to be registered. there is an ongoing work to include patients from all groups, but until now, the primary focus has been on glioma patients who make up the major part of adult brain tumor patients. these patients are identified by the m - codes given in the first row of table 1. dnor has included all patients diagnosed with glioma (excluding ependymoma) since january 1, 2009, which numbers approximately 400 patients each year. data are registered in four different forms linked in a hierarchical data model (figure 1). the forms including data on diagnostic and surgery are entered by the department of neurosurgery and the forms including data on chemotherapy and radiotherapy are entered by the department of oncology. the data are transferred to an analytical database which can describe the clinical course of the patients and administrative issues. using the unique ten - digit code given to all danish citizens, the analytical database is further linked to the danish civil registration system5 to obtain residence and vital status. data contain information about symptoms, presurgical magnetic resonance imaging (mri) characteristics, performance status, surgical procedures, residual tumor on postsurgical mri, postsurgical complications, icd-10 codes, systematized nomenclature of medicine morphology codes, radiotherapy, and chemotherapy. there are two process and five results indicators describing survival, early postoperative mri, surgical qualification, extent of surgical resection, postsurgical mortality, radiotherapy, and chemotherapy. from 2009 to 2014, clinical data on 2,238 patients have been reported in dnor, representing 92% of all cases identified in the central registries using the relevant diagnostic codes presented in the first row of table 1. in approximately 5% of the included patients, only oncological data were reported, while basic diagnostic and surgical data were missing. from 2015, the dnor has been using algorithms from the standardized platform for the danish clinical cancer registries,6 which will optimize the completeness of the identification of patients with brain tumors in dnor. there is a trend that postoperative mri is performed in a higher proportion of patients (indicator ii) and another trend that a higher proportion of patients have no residual tumor after surgical resection of the primary tumor (indicator iv). hence, there is an ongoing research project with the purpose of evaluating the completeness of data as well as data validity. furthermore, several epidemiological studies based on data from the initial 6 years from 2009 to 2014 are in progress. generally, dnor provides the opportunity for clinical research in glioma patients in a population - based setting. the possibility to link to other danish national registries such as the danish civil registration system,5 the danish national patient registry,8 and the danish dnor was established under the auspices of dnog, which is the danish multidisciplinary cancer group on primary brain tumors in adult patients. dnog members are appointed by the relevant member society under the organization of danish medical societies including neurosurgery, medical and radiation oncology, neurology, neuroradiology, pathology, and nuclear medicine. dnor is financially supported by the danish health care authorities through a national quality program organized in the danish clinical registries.10 also, there is technical support from the registry support centre of epidemiology and biostatistics, south. the establishment of dnor has optimized the quality in handling primary brain tumor patients in denmark by introducing clinical quality indicators and facilitating a better multidisciplinary collaboration at a national level. dnor variables and indicators are selected by clinicians for the discussion of quality on a more reliable background on selected topics. this discussion takes place in dnog with the possibility of simultaneous adjustment in national guidelines to optimize the implementation of procedures in clinical practice. | aim of databasethe danish neuro - oncology registry (dnor) was established by the danish neuro - oncology group as a national clinical database. it was established for the purpose of supporting research and development in adult patients with primary brain tumors in denmark.study populationdnor has registered clinical data on diagnostics and treatment of all adult patients diagnosed with glioma since january 1, 2009, which numbers approximately 400 patients each year.main variablesthe database contains information about symptoms, presurgical magnetic resonance imaging (mri) characteristics, performance status, surgical procedures, residual tumor on postsurgical mri, postsurgical complications, diagnostic and histology codes, radiotherapy, and chemotherapy.descriptive datadnor publishes annual reports on descriptive data. during the period of registration, postoperative mri is performed in a higher proportion of the patients (indicator ii), and a higher proportion of patients have no residual tumor after surgical resection of the primary tumor (indicator iv). further data are available in the annual reports. the indicators reflect only minor elements of handling brain tumor patients. another advantage of reporting indicators is the related multidisciplinary discussions giving a better understanding of what actually is going on, thereby facilitating the work on adjusting the national guidelines in the danish neuro - oncology group.conclusionthe establishment of dnor has optimized the quality in handling primary brain tumor patients in denmark by reporting indicators and facilitating a better multidisciplinary collaboration at a national level. dnor provides a valuable resource for research. |
wound healing is a complex and dynamic process that includes an immediate sequence of cell migration leading to repair and closure. vacuum - assisted closure (vac, kci international, san antonio, tx, usa) therapy was first reported in 1997. it has become a common modality for treating complex wounds, characterized by a controlled and localized negative pressure applied on polyurethane absorbent foams. these pore sizes are the most effective at transmitting mechanical forces across the wound and provide an even distribution of negative pressure over the entire wound bed to aid in wound healing. vac therapy has shown promising results in several studies, in terms of the wound - healing process and for the treatment of infection after cardiothoracic surgery. however, this method can have some disadvantages like pain, bleeding, heart rupture, death, deterioration in quality of life, loss of protein, fistula formation and secondary infection. lymphatic complications such as lymphocele and lymphatic leakage are common after vascular interventions and surgeries, and vac therapy is used as the primary method of treatment. in this report, we present a case of a patient with thoracic wound infection after rib stabilization that was complicated by a diffuse lymphatic leakage and lymphedema of the breast after continuous vac therapy. this very rare complication of the vac therapy did not promote healing, but further hindered it. a 47-year - old female visited our clinic with dislocated rib serial fractures with flail chest and hematothorax (fig. the medical history was positive for obesity (body mass index 32.2 kg / m). 1b) was performed with stratos (medxpert gmbh, heitersheim, germany) with 3d rib clips and connecting bars for overbridging fragment fixation via an anterolateral approach after diagnostic thoracoscopy and hematothorax evacuation. a broad - spectrum antibiotic therapy was administered, and a surgical revision with large debridement was performed. the osteosynthetic material was not removed. to close the infra - mammary wound temporarily and to accelerate wound healing, vac therapy the site was sealed with an adhesive drape covering the polyurethane foam and tubing, including 45 cm of surrounding healthy tissue to ensure a seal. a controlled continuous pressure of 75125 mmhg was uniformly applied to all tissue on the inner surface of the wound. this system was subsequently renewed every 35 days under general anesthesia for a total of 6 times. necrotic tissue was removed during surgical debridement that was performed when necessary, and adequate hemostasis was achieved. over the course, the patient developed a diffuse lymphatic leakage and congestion of the left breast with partial necrosis (fig. 2a) of the lower breast quadrants, requiring repeated surgical debridement and necrosectomy (fig. 2b). figure 1:(a) computed tomography scan of the chest with multiple displaced anterolateral rib fractures and hematothorax (arrow). (b) osteosynthesis of four ribs with stratos (medxpert gmbh, heitersheim, germany). figure 2:(a) wound infection with partial necrosis and lymphatic leakage with lymphedema of the left breast. (c) result after breast reconstruction with pedicled myocutaneous latissimus dorsi island flap. (a) computed tomography scan of the chest with multiple displaced anterolateral rib fractures and hematothorax (arrow). (b) osteosynthesis of four ribs with stratos (medxpert gmbh, heitersheim, germany). (a) wound infection with partial necrosis and lymphatic leakage with lymphedema of the left breast. (b) (c) result after breast reconstruction with pedicled myocutaneous latissimus dorsi island flap. for reconstruction of the skin and soft tissue defects of the breast, a pedicled myocutaneous latissimus dorsi island flap was performed (fig. no other treatments were necessary, the patient was asymptomatic and the follow - up was uneventful. vac therapy of complex acute- and chronic - infected wounds has increased in popularity among various surgical specialties. there are few reports of vac therapy for treating open thoracic wounds after rib stabilization, and we experienced an interesting case of lymphatic leakage after introduction of the vac therapy. vac therapy was applied in our patient on day 10 after rib stabilization during wound revision due to a local infection of the infra - mammary wound. in general, the adverse events of vac therapy are seldom but can be life - threatening. our case illustrates a first - described complication of exacerbated diffuse lymphatic leakage with lymphedema and necrosis of the breast in relation to the vac therapy, and the following reconstructive treatment resulted in prolongation of the hospital stay. we suggest that this complication occurred due to a too high negative pressure application of the vac system. the level of applied pressure causes a change in microvascular blood flow, and the high negative pressure can result in an impairment of the lymphatic drainage of the breast. this causes lymphedema with congestion of protein - rich fluid, an increase in the size and the number of lymphatic channels and a reduction in the availability of oxygen. this milieu provides a rich medium for bacterial growth resulting in infection, e.g. cellulitis, lymphangitis, lymphadenitis and necrosis of the breast with secondary damage of the lymphatic system. vac has been used successfully for difficult breast wounds such as infections after mammoplasty, before reconstruction of the breast after removal of a giant cystosarcoma phyllodes, necrotizing fasciitis of the breast, thoracic wall recurrence after breast cancer and mastitis. stoeckel. presented 18 cases of complicated breast wounds requiring vac. in 2 of these 18 cases, vac therapy was unsuccessful as the wounds were further complicated by tissue ischemia and infection requiring operative debridement. do not give further information regarding these two cases that appear to have a similar etiology as the study subject in our case report. the wound vasculature of these two patients was significantly compromised as is the case with our study subject. since the vac pressure used is not mentioned, it is difficult to compare the various studies [6, 810 ]. we recommend using vac therapy at a lower negative pressure of, for example, 50 mmhg, to avoid this rare complication. future research should focus on improvement of the device to avoid complications and expand clinical indications. furthermore, high - quality randomized clinical trials with standardized controls and protocols are needed to compare outcomes in specific types of patients and wounds, at different levels of pressure, and using different pressure models adapted to wound composition. | vacuum - assisted closure (vac) therapy is a useful tool in the management of a wide spectrum of complex wounds in cardiothoracic surgery. it promotes healing through the application of a controlled and localized negative pressure on porous polyurethane absorbent foams. known advantages of the vac therapy are the acceleration of wound healing, stimulation of granulation tissue and reduced tissue edema. despite its excellent properties, some related complications after and during the therapy have been reported. we report the case of a 47-year - old female with a thoracic wound infection after rib stabilization, managed with open surgery and vac therapy, which was complicated by a diffuse lymphatic leakage. this is the first case described of diffuse lymphatic leakage followed by partial necrosis of the breast after continuous vac therapy. we recommend the application of a lower pressure level of this device for complex wounds of the chest wall near the breast. |
oral potentially malignant disorder (pmd) is the preferred who term to describe a number of mucosal lesions which demonstrate an increased risk of squamous cell carcinoma (scc) development compared with apparently normal oral mucosa. the list of mucosal pathology considered potentially malignant includes discrete lesions such as leukoplakia and erythroplakia, as well as more widespread conditions such as proliferative verrucous leukoplakia, immunodeficiency, oral submucous fibrosis, and perhaps more controversially oral lichenoid lesions. whilst a vast literature exists describing the aetiology, clinical appearance, and the identifiable histopathological features of dysplasia seen in pmd, there remain no universally agreed clinical management protocols. we have described previously, however, both the diagnostic accuracy of obtaining definitive histopathology specimens and the treatment efficacy of the entire lesion removal by interventional laser surgery, and it is now generally accepted that pmd excision is probably the optimal management option [13 ]. it remains impossible, unfortunately, to predict either the behaviour of individual pmd lesions or the progress of disease in a particular patient, and some authors raise concerns that formal pmd excision is not proven to prevent scc development, although it remains a not unreasonable hypothesis [2, 3 ]. of perhaps more significance is the lack of clarity regarding overall clinical outcome following pmd treatment and a need to both rationalise terminology and define a more structured patient follow - up regime. the aim of this paper, therefore, is to report on the detailed clinical outcome and followup of a cohort of 100 pmd patients who all underwent a standardised interventional laser surgery treatment to excise dysplastic single lesion disease and whose postoperative progress was documented for up to 10 years following the first presentation. following ethical committee approval and informed patient consent, 100 consecutive pmd patients attending the maxillofacial oncology / dysplasia clinics at newcastle upon tyne in northern england over a 3-year period and who underwent co2 laser excision of dysplastic lesions were recruited to the study. all were new patients, with no prior history of oral cancer or precancer and no previous surgical or radiotherapy treatment, and all presented with distinct, single - site pmd lesions proven on incisional biopsy to exhibit dysplasia. laser surgery was carried out by the same operator (p. j. thomason) working to a standardised protocol, which has been previously documented, and which comprised formal excision of mucosal lesions and widespread ablation of mucosal margins [2, 3 ]. the influence of risk factor behaviour such as smoking and alcohol use was identified and appropriate cessation advice was given prior to treatment. all patients were reviewed on a regular basis postlaser intervention, at varying intervals between 1 and 12 months based upon the severity of individual clinical and pathological features, to monitor the clinical course of disease and patients ' outcome. the identification of new mucosal disease, biopsy for histopathological diagnosis, and further interventional treatment was carried out in accordance with defined management protocols [2, 3 ]. all excision biopsy specimens underwent standardised histopathology examination by two experienced oral pathologists (c. m. robinson and p. sloan) working to agreed diagnostic criteria. lesions were graded using both the 2005 world health organisation (who) classification and a binary grading system (high grade versus low grade) that benefits from increased levels of interobserver agreement and improved predictive value. the two pathologists independently assessed the biopsy material, and discordant grading was resolved by review and consensus. the size of dysplastic lesions was assessed by multiplying the length by width of laser excised specimens as recorded in histopathology reports. clinical outcome for each patient was defined at the time of their most recent followup appointment using one of the following terms : clinical resolution, a patient clinically free of pmd disease following treatment, persistent disease, whereby the pmd lesion persisted at the same site despite interventional treatment, recurrent disease, when a pmd lesion recurred at the same site following previously successful excision, further disease, distinguishing pmd lesion development at new oral sites following previously successful excision, malignant transformation, whereby invasive scc arose at the same site of a clinically recognised oral precursor lesion, and oral cancer development, in which invasive scc development occurred but at new oral sites distant from previously recognised or treated precursor lesions. statistical analyses were performed using spss, version 19.0 (statistical package for the social sciences, chicago, il, usa). categorical variables for clinical outcome data were summarized and presented descriptively using frequencies and percentages with the chi - square test or fisher 's exact test used to evaluate relationship between variables. continuous variables were expressed as mean standard deviation and were compared using independent student 's t - test for pairwise comparison ; kruskal - wallis test was used for group comparison. the kaplan - meier survival analysis method with log - rank test was used to assess the differences between outcome groups and to calculate cumulative disease - free survival rates. for all tests, p values sixty - eight male patients (age range 3081 years ; mean 58 years) and 32 female patients (age range 3394 years ; mean 59 years) comprised the study cohort. eighty - six patients were either current or ex - smokers, whilst 83 regularly consumed alcohol. one hundred lesions were formally excised by laser, the majority (76) appearing clinically as leukoplakias (67 homogeneous and 9 nonhomogeneous), and the remaining were classified as erythroleukoplakias (16) and erythroplakias (8). most lesions (79) presented on the floor of mouth and ventrolateral tongue, as summarised in table 1. following consensus who grading of the excision specimens, 42 of the lesions were classified as mild dysplasia, whilst the remainder showed moderate dysplasia (26), severe dysplasia (21), or carcinoma in situ (11) (kappa value = 0.644, p 600 mm (p = 0.010, chi - square test). a higher mean size of presenting lesion was seen in patients developing recurrent disease (393.63 mm) compared to recurrence - free (281.70 mm), albeit nonsignificant (p = 0.356, independent t - test). further (new site) dysplastic disease was significantly more common following intermediate sized precursor lesion excision (p = 0.049, chi - square test). although (same site) malignant transformation was more common following intermediate sized lesion excision, this was not statistically significant (p = 0.593, chi - square test). however, risk estimate showed that if initial dysplasia size exceeded or equalled 425 mm (equivalent to the third quartile), the odds ratio for transformation was 2 times higher than that of smaller sized lesions (95% ci, 0.36511.582). scc development distant from primary lesion sites was only seen in intermediate or major sized lesions, and although nonsignificant (p = 0.104, fisher 's exact test), there was a definite trend for lesions > 200 mm to exhibit further disease, malignant transformation, and scc development following treatment. the vast majority of patients in this study were either current or ex - smokers, and a significant relation was found between smoking status and clinical outcome (p = 0.014, chi - square test), whilst the incidence of both recurrent and further disease was the highest in patients exposed to tobacco, there was a trend for nonsmokers to risk malignant transformation and particularly scc development, whereby new site carcinomas were seen exclusively in nonsmokers. nonsmoking patients also presented with significantly larger lesions (mean size 473.20 mm), compared with both ex - smokers (354.25 mm) and current smokers (241.49 mm), p = 0.026, kruskal - wallis test. also, spearman correlation revealed a significant positive correlation between the degree of histopathological grading and lesion size (r = 0.272 ; n = 96 ; p < 0.01), whereby increased pmd size was associated with increased dysplasia severity. chi - square testing, however, showed no significant relation between clinical outcome and the number of cigarettes smoked per day (p = 0.139). there were no statistically significant relationships seen between alcohol intake and outcome (p = 0.267, chi - square test), although patients consuming regular alcohol posttreatment risked both recurrent and further disease development, whilst all 3 patients who ceased alcohol consumption remained disease - free. the who system showed a significant relationship with defined outcome categories (p = 0.003, chi - square test) ; patients exhibiting malignant transformation or new site scc development displayed were those seen with either severe dysplasia or carcinoma - in - situ in presenting pmds. recategorising clinical outcome as either clinical resolution (disease free) or further disease (encompassing recurrent / further pmds, malignant transformation, or scc development) emphasised that lesions with severe dysplasia and carcinoma - in - situ were statistically more likely to develop further disease (p = 0.010, chi - square test). the degree of dysplasia also had a significant effect on unfavourable outcome, with severe dysplasia / carcinoma - in - situ having a shorter mean time to develop further disease (40 months) compared with moderate (78.8 months) or mild dysplasia (87.83 months). also, 2- and 5-year disease - free survival rates were much lower for severe dysplasia / carcinoma - in - situ than for either moderate or mild dysplasia (63%, 76%, and 85% and 14%, 59%, and 62%, resp.), p = 0.006, log - rank test., there were demonstrably more high - grade lesions in the outcome groups of recurrent, further disease, malignant transformation, and scc development, but this was statistically significant only for recurrent disease (p = 0.025, fisher 's exact test). increased statistical significance was seen, however, by recategorising outcome as either clinical resolution or further disease, confirming an increased risk of further disease with high - grade lesions (p = 0.021, chi - square test). patients with high grade dysplasia also had a significantly shorter mean time (64 months) to develop an unfavourable outcome compared to low - grade lesions (88.7 months), and lower 2- and 5-year disease - free survival rates were seen for high - grade compared to low - grade dysplasia (68% versus 83% and 29% versus 63%, resp.), p = 0.013, log - rank test. fourty eight pmd excision specimens had clear margins on histopathological examination with no discernible dysplastic features, whilst in 23 cases foci of mild dysplasia were identified ; less commonly, moderate (14) and severe dysplasia (12) or rarely foci of carcinoma - in - situ (3) were reported. additional intervention for dysplasia positive margin cases was not usually required due to active ablation of all margins at the time of laser excision, although all cases underwent careful clinical surveillance. whilst the presence of dysplasia in excision margins did not significantly influence overall postlaser surgery clinical outcome (p = 0.053, fisher 's exact test), the majority of patients free from either recurrent (same site) disease or further (new site) disease had clear resection margins, whilst those developing further disease primarily exhibited severe dysplasia in excision margins (p = 0.004 and p = 0.050, resp. clinical outcome in relation to length of followup was determined by plotting pmd - free survival via kaplan - meier survival analysis, and this showed a clear relationship with time. figure 3 confirms that whilst 88 patients exhibited clinical resolution and were disease - free 1 year after surgery, there was a progressive rise in recurrent and further disease through successive years so that disease - free rates fell to 75 at 2 years, 68 at 3 years, and 47 at 5 years, with only 42 patients pmd - free 10 years after surgery. figure 4 shows that recurrent (same site) pmds most commonly presented during the first 2 years following laser surgery (11 out of 17 cases), p = 0.0001, log - rank test. in contradistinction to recurrence, figure 5 illustrates that further (new site) pmd disease could arise at any time during the first five years of followup, but with particularly significant risk at 1 and 3 years after surgery, p = 0.0001, log - rank test. five malignant transformation cases (same site) occurred during the first 15 months of followup (p = 0.0001, log - rank test), figure 6. scc development (new site cancer) only occurred in 2 cases, both nearly 5 years following severe dysplasia excision from the ventro - lateral tongue. the clinico - pathological profile of pmd cases observed in each clinical outcome category is summarised in table 2. further statistical analysis was performed using univariate and multivariate logistic regression analysis to predict the role of patient age, sex, lesion size, type, histopathology, anatomical site, and resection margin status upon unfavourable clinical outcome (disease active state including recurrent or further dysplasia, malignant transformation, and oscc development), table 3. whilst patients ' age and sex showed no significant effects, non - homogenous leukoplakia was a significant predictor of active disease (p = 0.023), increasing risk by nearly 3 times compared to homogenous lesions. tongue lesions showed a 3.4 increased risk compared with floor of mouth (p = 0.013). the presence of severe dysplasia was a highly significant predictor of active disease status (p = 0.007) ; severe dysplasia and carcinoma - in - situ showed a 4.6 and a 4.8 times increased risk, respectively, for active disease compared to mild dysplasia. high - grade dysplasia was also a significant predictor for disease active state (p = 0.020), increasing the risk to approximately 3 times that of low - grade dysplasia, table 3. the presence of dysplasia in surgical resection margins was also a significant prognostic factor for disease active status (p = 0.035), increasing risk by nearly 3 times compared with clear margins. major sized lesions displayed a 4.5 times increased risk compared to minor sized ones (p = 0.045), table 3. the ability to predict clinical outcome for pmds remains elusive in clinical practice, probably due to lack of understanding of the natural history of the disease, confusion over terminology, limited agreement on therapeutic interventions, and uncertainty regarding patient followup. this paper is unique in presenting detailed, long - term clinical outcome data for a 100 patient cohort presenting with dysplasia - proven pmds, all excised by laser to a standardised treatment protocol. it is notable upon reviewing the literature that many previous authors have not found pmd clinical appearance, anatomical site, histopathological assessment, or features related to patient age, gender, or risk factor behaviour to reliably predict clinical outcome [1, 610 ]. the ultimate goal of pmd diagnosis and management must, of course, is the prevention of scc. malignant transformation rates varying widely between 0.1 and 40% have been quoted in the literature, which is extremely unhelpful in individual patient management, although the highest risk of cancer development is believed to occur in the most dysplastic precursor lesions ; larger mucosal lesions and nonsmoking patients also appear to be at enhanced risk of malignancy [7, 8, 11, 12 ]. a number of observational, anecdotal, and retrospective papers have reported clinical outcome and malignant transformation data through the years, but these are significantly weakened by the heterogeneous clinical and histopathological nature of the precursor lesions studied and by a lack of any agreed treatment protocols and uncoordinated follow - up regimes. table 4 lists the malignant transformation rates seen in the dysplastic lesions reported in these studies ; none of the studies distinguished between same site and new site cancer, but overall malignant transformation rates in excess of 36% (with a mean of 16%) were reported, which are significantly higher than either the 2 new site scc cases or the 5 same site malignant transformations seen during this study [9, 1320 ]. more recently, mehanna. quoted a 12% cancer rate over a mean transformation time of 4.3 years using a systematic review and meta - analysis which included a total of 992 cases although interestingly, because of limitations in the published literature, they only felt able to include 14 papers out of a possible 2837 identified oral precancer publications. the authors reported a lack of high quality evidence to date which limited the scope of their study. to date, there has been a paucity of prospective, randomised controlled trials in oral precancer research, and those that do exist have not fundamentally resolved treatment and clinical outcome dilemmas. this 100 patient cohort study, although not a controlled trial population, is a unique data set facilitating analysis of a defined oral precancer population with shared risk factor behaviour presenting with proven dysplastic pmds, standardised diagnostic and treatment protocols, consistent clinical decision making, and longitudinal patient observation with documented clinical followup. whilst interventional laser excision of mucosal dysplastic lesions appears to reduce the risk of same site malignant transformation, scc development at new sites remains a risk reflecting field change in pmd disease [2, 3, 10 ]. active mucosal surveillance and regular clinic follow up remain mandatory for all pmd patients, and interventional management strategies are best regarded as cyclical, passing from active surgical excision through to surveillance and then returning to surgical intervention for early targeting of further pmd disease. a particular advantage of laser surgery is the ability to repeat excisions or ablations at the same site on a number of occasions, without compromising oral healing or function [2, 10 ]. in this study, 62% of pmd patients were disease - free following laser excision of dysplastic mucosal lesions, and the risk of malignant transformation remained low at 2 to 5%. the incidence of further disease, however, increased with the length of patient followup, and was higher for non - homogeneous leukoplakias, large mucosal lesions, more severe dysplasias, floor of mouth and ventral tongue sites, and in nonsmoking patients. long - term patient followup and active clinical surveillance thus remains mandatory for all pmd patients. multicentre, randomised controlled clinical trials for pmd treatment are now urgently required to determine treatment efficacy. | oral potentially malignant disorders (pmds) are at risk of transforming to invasive squamous cell carcinoma (scc), but controversy exists over their management and the precise role of interventional treatment. in this study, a cohort of 100 patients presenting with new, single oral dysplastic pmd lesions were followed for up to 10 years following laser excision. pmds presented primarily as homogeneous leukoplakias on floor of mouth and ventrolateral tongue sites and showed mainly high - grade dysplasia following analysis of excision specimens. sixty - two patients were disease - free at the time of the most recent followup, whilst 17 experienced same site pmd recurrence, 14 developed further pmds at new sites, 5 underwent same site malignant transformation, and 2 developed scc at new oral sites. whilst laser excision is an effective therapeutic tool in pmd management, prolonged patient followup and active mucosal surveillance together with clear definitions of clinical outcomes are all essential prerequisites for successful interventional management. multicentre, prospective, and randomised trials of pmd treatment intervention are urgently required to determine optimal management strategies. |
persistent pulmonary hypertension of the newborn (pphn) is a serious neonatal illness, which in the past was associated with high mortality and morbidity. it results from failure of the neonate to make a postnatal transition from a high resistance fetal pulmonary circulatory state to a low resistance pulmonary circulation. this increased pulmonary vascular resistance and decreased pulmonary blood flow prevents adequate gas exchange in the lungs resulting in severe respiratory distress and hypoxemia in the neonate. the affected newborn usually term or late - preterm infant was not associated congenital anomalies and develop within hours after birth with severe respiratory distress that requires the mechanical ventilation. the mortality rate about 10 - 20% of affected infants despite of treatments such as nitric oxide, extracorporeal membrane oxygenation (ecmo) and advanced modes of mechanical ventilation. in addition, infants who survive pphn at increased risks for long - term sequelae including chronic lung disease, seizures, and neurological developmental problems as a result of hypoxemia and/or the aggressive treatments that pphn often requires. the main goals of treatment of pphn are to decrease pulmonary vascular resistance and increase pulmonary blood flow. this is carried out by correcting the underlying disease, good supportive care, and selective pulmonary vasodilators such as inhaled nitric oxide (ino), magnesium sulphate (mgso4) and oral sildenafil. the aim of this study was to estimate the incidence of pphn at nicu of al - minya university pediatric hospital, to identify the risk factors and to assess the outcome of these cases. this data were prospectively collected from nicu of al - minya university hospital (a tertiary referral hospital) in cooperation between the departments of neonatology and cardiology. a total of 640 neonates greater than 36 weeks of gestation and admitted to the nicu, from january 2009 to april 2012 were studied a written informed consent was obtained from the legal guardians of each patient the study was approved by the scientific ethical committee of al - minya university hospital. patients with a major congenital anomalies, congenital diaphragmatic hernia, congenital heart disease and pre - term were excluded. all neonates in the study were subjected to complete history, and the physical examination was performed and blood drawn to obtain the baseline laboratory data including a complete blood count, arterial blood gases, blood glucose, serum electrolytes and blood culture to exclude sepsis. chest x - ray and echocardiogarphy were carried out to verify shunt and exclude structural congenital heart disease. a neonate with acidosis was given sodium bicarbonate slow bolus infusion : 2 - 3 meq / kg iv for neonates with acidosis and guided by improvement of arterial blood gases. drugs that decrease pulmonary vascular resistance and increase pulmonary blood flow were given for selected neonates 1 group (16 neonates) was given oral sildenafil citrate 1 mg / kg every 6 h and the dose could be doubled if the oxygenation did not improve and blood pressure remained stable and the treatment was discontinued if there was no significant change in oxygenation after 36 h, or patient has received eight doses. this strategy according to previous study and 2 group (10 neonates) was given mgso4, a loading dose of 250 mg / kg of mgso4 iv over 10 - 15 min. a clinical response is obtained once the serum magnesium level exceeds 3 - 5 mmol / l give between 20 and 75 mg / kg / h for 2 - 5 days while maintaining a blood level of between (3.5 and 5.5 mmol / l). mechanical ventilation was used in 12 neonates with extreme acidosis (ph<7.10) resistance to treatment, severe neonatal pneumonia, and massive lung collapse. other drugs ; anti - infective, positive inotropic drugs (dopamine), sedatives and skeletal muscle relaxants according to the clinical condition of the infant. follow - up of these neonates for 6 months for evaluation of neurological injury by performing complete neurological examination by a pediatric neurologist and in some of them by doing cranial ultrasound, electroencephalography, brain computed tomography and or magnetic resonance imaging hearing test and fundus examination were also performed for neonates suspected with complications. all neonates in the study were subjected to complete history, and the physical examination was performed and blood drawn to obtain the baseline laboratory data including a complete blood count, arterial blood gases, blood glucose, serum electrolytes and blood culture to exclude sepsis. chest x - ray and echocardiogarphy were carried out to verify shunt and exclude structural congenital heart disease. a neonate with acidosis was given sodium bicarbonate slow bolus infusion : 2 - 3 meq / kg iv for neonates with acidosis and guided by improvement of arterial blood gases. drugs that decrease pulmonary vascular resistance and increase pulmonary blood flow were given for selected neonates 1 group (16 neonates) was given oral sildenafil citrate 1 mg / kg every 6 h and the dose could be doubled if the oxygenation did not improve and blood pressure remained stable and the treatment was discontinued if there was no significant change in oxygenation after 36 h, or patient has received eight doses. this strategy according to previous study and 2 group (10 neonates) was given mgso4, a loading dose of 250 mg / kg of mgso4 iv over 10 - 15 min. a clinical response is obtained once the serum magnesium level exceeds 3 - 5 mmol / l give between 20 and 75 mg / kg / h for 2 - 5 days while maintaining a blood level of between (3.5 and 5.5 mmol / l). mechanical ventilation was used in 12 neonates with extreme acidosis (ph<7.10) resistance to treatment, severe neonatal pneumonia, and massive lung collapse. other drugs ; anti - infective, positive inotropic drugs (dopamine), sedatives and skeletal muscle relaxants according to the clinical condition of the infant. follow - up of these neonates for 6 months for evaluation of neurological injury by performing complete neurological examination by a pediatric neurologist and in some of them by doing cranial ultrasound, electroencephalography, brain computed tomography and or magnetic resonance imaging hearing test and fundus examination were also performed for neonates suspected with complications. the study population included 640 neonates during the duration from january 2009 to april 2012. only 32 (5%) were diagnosed as pphn. it was higher for those who were born by cesarean section, male, post - term, large for gestational age, meconium stained amniotic fluid, perinatal asphyxia, sepsis, hyaline membrane disease, pneumonia, and down - syndrome. maternal diseases including, diabetes mellitus, hypertension, and anemia [tables 1 and 2 ]. demographic and clinical data of cases with pphn risk factors and causes of pphn table 3, showed the different therapeutic modalities, which were available in nicu units for management of pphn included oxygen administration to all neonates for short duration (6 - 9 days). oral sildenafil was administered to 16 neonates with pphn and the outcome was satisfactory and there was a significant difference between responsive (10 cases) and unresponsive neonates (6 cases) ; (p = 0.001) [table 4 ]. infusion of magnesium sulfate was administered to 10 neonates with pphn the outcome was satisfactory and there was a significant difference between responsive and unresponsive neonates. 12 (37.5%) neonates with extreme acidosis (ph<7.10) resistance to treatment, severe neonatal pneumonia, and massive lung collapse were treated by mechanical ventilation. 8 neonates (25%) therapy used for treatment of newborns with pphn comparison of improvement of cases received oral sildenafil and mgso4 the outcome of the neonates with pphn was mentioned in [table 5 ], it showed that after 6 months of closed follow - up, 12 neonates (37.54%) improved without sequelae, 4 neonates (12.5%) developed some neurodevelopmental impairment, 3 neonates (9.3%) developed chronic lungs diseases, 2 neonates (6.2%) developed hearing (deficits) and another 3 neonates (9.3%) missed follow - up. persistence of pulmonary hypertension leading to respiratory failure in the neonate has been recognized for 40 years since its original description by gersony. in 1969. the mortality rate of infants with pphn was estimated to be around 10 - 20% even with the use of high - frequency ventilation, surfactant, ino, and ecmo but is much higher when these therapies are not available. a total of 32 neonates (18 male and 14 females) were diagnosed as having persistent neonatal pulmonary hypertension prospectively evaluated. pphn represented nearly 5% (32/640 cases) of total neonatal care units admissions and it was considered the sixth leading cause of death in our nicu units in year 2010. the diagnosis of our cases depends on high clinical suspicion, echocardiography, and pulse oximetry and blood gas analysis. the most common cause of pphn in this study was mainly meconium aspiration syndrome, representing 50% of pphn in this series. meconium cause mechanical obstruction to the airways, resulting in air trapping, hyperinflation, and increased risk for pneumothorax. meconium components also inactivate surfactant, trigger an inflammatory response with the release of cytokines, and increase the production of the vasoconstrictors endothelin and thromboxane. bacterial endotoxin causes pulmonary hypertension from several mechanisms, including the release of thromboxane, endothelin, and several cytokines. in this study, pphn occurred as a complication of hyaline membrane disease and transient tachypnea of the newborn in five cases (16.6%) delivered by c - section. the increasing reactivity of pulmonary arteries at this gestation period predisposes these neonates to pulmonary hypertension when gas exchange is impaired because of surfactant deficiency. four cases (12.5%) of pphn of unknown etiology in this series were transient forms with a good outcome, suggesting transient maladaptation to extrauterine life. the majority of neonates were delivered by cesarean section (62.5% of cases) due to prenatal problems in agreement with previous studies had reported cesarean section delivery was associated with a high incidence of respiratory distress syndrome and pphn. maternal diseases such as uncontrolled diabetes mellitus, hypertension, and anemia represented maternal risk factors in our neonates. uncontrolled diabetes mellitus associated is with high incidence of hyaline membrane disease, hypoglycemia, marcosmia, and fetal distress. this was in agreement with many previous studies that maternal diseases play a role in occurrence of pphn especially dm, hypertension and maternal anemia. ino has been one of the latest measures to successfully treat pphn and significantly reduce the need for ecmo, but both were not available in our nicu. hence we used other modalities as pulmonary vasodilator drugs, which are available like oral sildenafil and mgso4 infusion. mgso4 was tried in 10 cases (31.25%), 6 (60%) of cases showed improvement of po2, decrease in total oxygen depending time, and infants survive without sequelae. we discontinued infusion in 2 cases due to hypotension and unresponsiveness to therapy, and shifted to mechanical ventilation. showed that intravenous mgso4 has been used extensively in developing countries for the treatment of pphn. it is cheap and easily available and has a potential neuroprotective effect ; its main disadvantage is that it can cause systemic hypotension. oral sildenatil was used in 16 cases (50%) who were randomly selected, all of them were full - term with moderate respiratory distress syndrome, 10 of them (62.5%) neonates showed improvement and 6 (37.5%) cases discontinued medication due to unresponsiveness and shifted to another line of available therapeutic modalities. stated that oral sildenafil was administered easily and was tolerated as well as placebo and improved oxygenation index in infants with severe pphn, which suggests that oral sildenafil, may be effective in the treatment of pphn and underscores the need for a large, controlled trial. khorana. in a retrospective study concluded that results confirm that sildenafil may be a useful adjuvant therapy for term infants with pulmonary hypertension in centers lacking ino and ecmo, mechanical ventilation was performed in 12 (37.5%) neonates with ppht, extreme acidosis (ph<7.1) severe neonatal pneumonia, massive lung collapse, and whom unresponsiveness to other therapeutic modalities was recorded. three of our cases were subjected to high - frequency oscillatory ventilation mode due to severe condition from the start and one of them after failure of other alternative therapy. after meticulous follow - up (6 months) of these neonates, the outcome was : 12 cases (37.5%) improved and showed normal development and were followed - up in pediatric neurology, ophthalmology, otolaryngology clinics without sequelae 8 cases (25%) died (four of them from severe sequelae of birth asphyxia and myocardial failure, three from severe neonatal septicemia, one from massive air leak syndrome). three cases (9.3%) developed chronic lung disease, two (6.2%) develop hearing defects and another 3 (9.3%) missed follow - up. the mortality and morbidity rates in our neonate were high and may be attributed to late presentation, delayed diagnosis and unavailability of other highly effective therapeutic modalities such as ino, ecmo and more recent vasodilators agents like tolazoline. pphn was diagnosed in 5% of the studied neonates in our nicu, meconium aspiration, birth asphyxia, hyaline membrane diseases, neonatal septicemia, post - term, cesarean section, maternal hypertension, and diabetes mellitus were the most encountered risk factors for pphn. this study is limited by the fact of being a single center analysis, prospective studies with the objective of evaluating the different therapies including a significant number of patients, will give much more information regarding therapeutic efficacy and survival. | background : persistent pulmonary hypertension of the newborn (pphn) result from the failure of the normal fetal - to - neonatal circulatory transition is associated with substantial infant mortality and morbidity.objective:to estimate the possible risk factors and assess the outcome of these cases.materials and methods : prospective study was performed enrolling all full - term and post - term newborn admitted to the nicu from january 2009 to april 2012, all neonates were subjected to complete history and physical examination, laboratory data including a complete blood count, arterial blood gases, blood glucose, serum electrolytes, and blood culture to exclude sepsis. cases with pphn had a continuous pulse oximeter, blood pressure and electrocardiography monitoring. chest x - ray and echocardiogarphy were carried out to verify shunt and exclude structural congenital heart disease.results:out of the studied 640 infants, 32 infants (5%) developed pphn, meconium aspiration, birth asphyxia, hyaline membrane diseases, neonatal septicemia, post - term birth being large for gestational age, cesarean section, maternal overweight, and diabetes mellitus were associated with an elevated risk for pphn. all neonates treated with o2, 10 neonates with mg sulphate, 16 with oral sildenafil and 12 with mechanical ventilation. after 6 months follow - up, 12 (37.54%) improved and followed - up without sequelae, 4 (12.5%) developed some neurodevelopmental impairment, 8 (25%) died, 3 (9.3%) developed chronic lungs diseases, 2 (6.2%) developed hearing defects and another 3 (9.3%) missed follow-up.conclusion:pphn was found in 5% of the studied population. meconium aspiration, birth asphyxia, neonatal septicemia, post - term were associated with an elevated risk for pphn. as this is a unit based study, a comprehensive countrywide survey on pphn in egypt is recommended to determine any regional differences in disease incidence. |
ribonucleotide reductase (rr) catalyzes the conversion of ribonucleoside diphosphates into deoxyribonucleoside diphosphates, playing essential roles in dna synthesis and repair in humans and influencing vital cellular mechanisms [15 ]. the molecular regulations of the three known rr subunits, rrm1, rrm2, and rrm2b (also called p53r2), have long been studied and reported by our and other groups [615 ]. previous research has established that rrm2b, a p53-inducible rr subunit, plays vital roles in dna repair, cell cycle modulation, mitochondrial dna (mtdna) synthesis, metastasis suppression, and oxidative stress resistance [1, 6, 7, 9, 1619 ]. mutation or absence of rrm2b in humans results in defective mtdna, and severe mtdna depletion has been observed in rrm2b/ animals [9, 16 ]. in cells a study on a rrm2b - knockout animal model indicated that the intactness of the rrm2b subunit is critical for maintaining chromosomal stability and that the loss of rrm2b results in plasmacytic neoplasms. furthermore, rrm2b expression is correlated with improved survival in some cancers, whereas a more progressive phenotype of certain other cancers complicates the rrm2b regulatory pathway [2224 ]. several reports have suggested that rrm2b regulates critical cellular mechanisms irrespective of the p53 status. first, although the p53-mediated rrm2b induction is inhibited in the p53-deficient mouse embryonic fibroblasts cells, it has been observed that rrm2b is expressed at basal levels. furthermore, a high rrm2b expression has been observed in various p53-deficient cancer cells, and it continues to influence mitochondrial functions irrespective of the p53 status, suggesting that rrm2b - mediated mitochondrial homeostasis is independent of functional p53, and other factors are involved in rrm2b regulation [25, 26 ]. the findings of our recent study are consistent with those of the aforementioned studies, in which the tumor suppressor foxo3 binds to the rrm2b promoter and activates rrm2b transcription in a p53-independent manner under physiological conditions. rrm2b is a unique member among the rr subunits that can resist reactive oxygen species (ros) [8, 19, 25, 28, 29 ]. however, little is known about the detailed mechanisms and the functional regulations of rrm2b under oxidative stress. we studied the functional regulations of rrm2b in cancer cells under oxidative stress and observed that rrm2b plays a crucial role in the regulation of mitochondrial and inflammation pathways in a p53-independent manner. cells from atcc were cultured in dmem medium containing fetal bovine serum (10%) and penicillin / streptomycin (1%) incubated at 37c with 5% co2., h1299 cells were seeded on coverslips, fixed, permeabilized, stained with primary antibodies, anti - rrm2b (rockland) and -h2ax (active motif), and secondary antibodies (invitrogen), washed, and then mounted with dapi (invitrogen) onto the slides. samples were analyzed by immunofluorescence microscope system, and the scale bars indicate 10 m. antibodies used in this assay were rrm2b (rockland), -h2ax (active motif), gapdh (santa cruz and genetex), vdac1, cox4, p - nfb, p - p38, and p - ib (genetex). the cytoplasmic / nuclear extraction kit was applied to separate cytoplasmic and nuclear fractions as described in the manufactory protocol (tools) before processing to the western on phospho antibodies. cells were treated with h2o2, harvested, washed with pbs, and stained with mitotracker green probes (invitrogen) of 50 nm in serum - free medium for 30 mins. total rna was isolated by trizol, and then rna was reverse transcribed (quanta) to obtain cdna for pcr. cdna was subjected to real - time pcr using the sybr green pcr reagents kit (stratagene) as described. for quantitation of mtdna copy number, nd1 gene level was quantified by q - pcr and normalized to -actin. due to the space limitation the statistical analysis was done as described. in brief, student 's t - test was used for p value calculation, and the star stands for p < 0.05. although it is a known fact that rrm2b can resist ros, little is known about the detailed regulatory mechanisms. stable h1299 cell lines without the functional p53 protein containing overexpressed rrm2b or rrm2bshrna and their respective controls were included in this study, as described previously, and rrm2b expression was evaluated using western blot analysis (figure 1(a)) and an immunofluorescence assay (figure 1(b)). for investigating rrm2b regulation under oxidative stress, hydrogen peroxide (h2o2), a type of oxidative stress - inducing ros, was used as the source of oxidative stress in this study. cells were treated with h2o2 for 2 hours and fixed for immunofluorescence staining, in which -h2ax foci induction indicated dna damage. the results suggested that stronger -h2ax signals were induced by cells expressing rrm2bshrna than by the control cells (figure 2(a)), suggesting that greater dna damage was associated with lower rrm2b expression in cells. for the western blot analysis, we performed the experiment at different time intervals, and the -h2ax signals on the blots were quantified relative to the gapdh activity for quantitative estimation and analysis. consistent rrm2b depletion was observed, which was correlated with the induction of -h2ax signals under h2o2 treatment (figure 2(b)). similarly, we observed low -h2ax foci signals under h2o2 treatment in rrm2b overexpressed cells (figure 2(c)). in summary, the results suggested that rrm2b plays a protective role in securing cells against oxidative stress - induced dna damage in a p53-independent manner. next, we examined the role of rrm2b in regulating mitochondrial proteins under oxidative stress in p53-deficient h1299 cells. only stable cells expressing rrm2bshrna or those acting as control that were subjected to or excluded from h2o2 treatment were included in this study. the cells were then harvested for western blot analysis, in which the expression of mitochondrial proteins, voltage - dependent anion channel 1 (vdac1), and cytochrome c oxidase subunit iv (cox4) [3436 ] were examined along with rrm2b expression and gapdh activity (figure 3(a)(i)). in addition, the relative expression levels of vdac1 and cox4 were analyzed and are shown in figure 3(a)(ii). the results suggested that the expression levels of both proteins, particularly vdac1, were more pronounced under h2o2 treatment, and the expression levels decreased on rrm2b downregulation, confirming that rrm2b regulated the mitochondrial content (figure 3(a)). to further understand the impact of rrm2b on the mitochondria, the mitochondrial mass was measured. facs was performed using a mitotracker green probe for detecting the mitochondrial mass in stable h1299 cells under h2o2 treatment. under oxidative stress, a significant decrease was observed in the relative mitochondrial mass of the cells expressing rrm2bshrna, indicating the protective role of rrm2b in mitochondrial homeostasis (figure 3(b)(i)), which is in agreement with our previous finding. the representative examples of the intensity of mitotracker on facs are shown in figure 3(b)(ii). moreover, the relative copy number of the nadh dehydrogenase subunit 1 (nd1), a mitochondrial gene, under h2o2 treatment was measured using quantitative pcr. low rrm2b expression resulted in a low nd1 copy number (figure 3(c)), suggesting that, under oxidative stress, rrm2b exerts protective effects on the mtdna content. the results establish that the rrm2b pathway affects mitochondrial homeostasis under oxidative stress, which is independent of the action of functional p53. oxidative stress mediated by h2o2 triggers the inflammation pathway [38, 39 ], and human inflammatory diseases have long been associated with nf-b or p38 signaling or both [4042 ]. however, the role of rrm2b in the regulation of inflammation has not been investigated. nf-b and p38 are complex pathways regulating various cellular mechanisms [41, 4346 ]. the nf-b pathway can be activated by various proinflammatory cytokines and is therefore considered a proinflammatory signaling pathway. in addition, extracellular stimuli such as uv light, growth factors, and inflammatory cytokines result in p38 activation. in this study, we investigated the functional role of rrm2b in oxidative stress - mediated nf-b and p38 signaling. stable h1299 control or rrm2bshrna cells were treated with h2o2 and harvested 2 hours later, and the lysates were separated into cytoplasmic and nucleolus fractions. western blot analysis was performed using phosphorylated nf-b, phosphorylated ib, and phosphorylated p38 antibodies for identifying nf-b and p38 activation in cells (figure 4). the results showed that nuclear nf-b and p38 signaling on h2o2 treatments were more pronounced in the cells expressing rrm2bshrna than in the control cells (figure 4), suggesting that a stronger inflammation signal was induced by rrm2b depletion. in summary, the findings suggest for the first time that rrm2b can functionally regulate the mitochondrial and inflammatory pathway and that these rrm2b - mediated regulations are independent of p53. figure 5 illustrates a model summarizing the current findings of rrm2b regulation, suggesting that, under oxidative stress, rrm2b plays critical roles in the upregulation of genes involved in the mitochondrial and inflammation pathways. rrm2b is a unique member of the rr enzyme that exhibits anti - ros potential. it was demonstrated that rrm2b suppressed ros activation mediated by oxidative stress and is highly induced in a p53-dependent manner during senescence. in our recent study, the critical role of rr2 mb in regulation of mitochondrial and inflammation pathways under oxidative stress in a p53-independent manner was reported for the first time. rrm2b plays critical roles in vital cellular mechanisms such as dna replication, and low rrm2b expression sensitizes cancer cells under various stresses, and therefore studies have suggested that low rrm2b expression can potentially be considered a chemosensitizer for cancer treatment [2, 47, 48 ]. in this study, h1299 cells were subjected to oxidative stress, and the induced h2ax foci signals were stronger in rrm2bshrna cells, indicating that the chemosensitivity may generate equal outcomes in p53-deficient cancer cells. additional therapeutical applications remain to be uncovered. as mentioned, rrm2b mutation results in severe mtdna depletion. in this study mitochondrial mass was damaged under low rrm2b expression and was further destroyed under oxidative stress. findings of previous studies and the data in the present study indicate that the intactness of rrm2b is critical for complete functioning of the mitochondria, despite the presence of functional p53., our previous study using rrm2b - knockout animal models suggested that rrm2b is critical in maintaining chromosomal stability and preventing chronic inflammation - associated tumorigenesis. in this study, we demonstrated that the nf-b and p38 signaling pathways were upregulated by oxidative stress, particularly under low rrm2b conditions, which is in agreement with our previous finding, suggesting that rrm2b is crucial in preventing chronic inflammation and acts by inhibiting the nf-b and p38 pathways. both nf-b and p38 signaling pathways affect vital cellular regulatory mechanisms that include inflammation and apoptosis [46, 49 ]. therefore, accompany with our data, the rrm2bshrna cells could potentially trigger stronger inflammation and apoptosis signals under dna damage conditions. recently, more complex roles of nf-b have been suggested in nf-b activation in pro- and anti - inflammation processes and pro- and antiapoptosis [49, 50 ], which may depend on the nature of the model systems. our current study provides new insights into the role of rr in inflammatory diseases, and the intriguing regulation mechanisms of rrm2b under oxidative stress in inflammation remain to be explored. | rrm2b is a critical ribonucleotide reductase (rr) subunit that exists as p53-inducible and p53-dependent molecule. the p53-independent regulation of rrm2b has been recently studied, and foxo3 was identified as a novel regulator of rrm2b. however, the p53-independent regulation of rrm2b, particularly under oxidative stress, remains largely unknown. in this study, we investigated the role of rrm2b underoxidative stress - induced dna damage and further examined the regulation of mitochondrial and inflammatory genes by rrm2b. our study is the first to report the critical role of rrm2b in mitochondrial homeostasis and the inflammation signaling pathway in a p53-independent manner. furthermore, our study provides novel insights into the role of the rr in inflammatory diseases. |
quality of diabetes care was assessed in a cross - sectional survey of 33 practices in 1995 (2). all practices (n = 35) with electronic medical records in two representative areas of norway all patients with type 2 diabetes in 1995 (n = 1,667) and 2005 (n = 3,013) were identified using electronic search programs with manual verification. patients in nursing homes, patients receiving diabetes care from specialists, and patients with less than 6 months of follow - up were excluded, leaving 1,470 subjects (49.5% of whom were male) in 1995 and 2,699 (51.2% male) in 2005 for analysis. variables included demographic data, processes of care, outcomes of care, and medications. differences between means in table 1 were tested using an anova analysis adjusted for age and sex. other results were adjusted for clustering by using practice - specific proportions or means as observations in student 's t test. the uk prospective diabetes study risk - engine model (3) was used to calculate the 10-year risk reduction for coronary disease. mean age decreased (69.1 to 66.3 years ; p < 0.001), mean diabetes duration increased (6.6 to 7.0 years ; p = 0.047), and mean weight increased (81.1 to 86.1 kg ; p < 0.001), whereas mean height was comparable between both surveys. the proportion of patients for whom important processes of care had been recorded improved as follows : cholesterol 46 to 88% (difference [] 42% [95% ci 3548 ] ; p < 0.001), hdl cholesterol 18 to 61% (43% [3649 ] ; p < 0.001), smoking habits 13 to 57% (44% [3950 ] ; p < 0.001), height 13 to 39% (26% [1636 ] ; p < 0.001), weight 38 to 56% (18% [828 ] ; p = 0.001), and referral to eye examination 30 to 74% (44% [3750 ] ; p < 0.001). table 1 shows mean values for risk factors related to treatment groups and the proportion of patients achieving national targets. treatment was more intensive in 2005 compared with 1995. among patients using antihyperglycemic therapy, 23 vs. 12% (11% [95% ci 716 ] ; p < 0.001) received two or more oral hypoglycemic agents and 13 vs. 1% (12% [914 ] ; few patients used glitazones (2%) or acarbose (1%) in 2005. of patients using antihyperglycemic therapy, the percent of patients with a1c < 6.5% increased from 10 to 22% (12% [717 ] ; p < 0.001). among patients treated for hypertension, 64 vs. 29% (35% [3041 ] ; p < 0.001) received two or more antihypertensive agents. more patients received aspirin (35 vs. 19% ; 16% [1220 ] ; p < 0.001) and statins (45 vs. 4% ; 41% [3845 ] ; p < 0.001). national targets for treatment of hyperglycemia, blood pressure, and cholesterol were achieved by significantly more patients in 2005 (table 1). there were no clinically important sex differences in the improvement of risk factor control or treatment. coronary risk reduction was calculated for an average patient (age 67 years, nonsmoking, with 7 years diabetes duration) using mean values for major risk factors in 1995 and 2005. the absolute 10-year risk reductions were from 42 to 28% for men and from 39 to 27% for women. reductions in mean values for a1c, blood pressure, and cholesterol were 0.6%, 10/4 mmhg, and 1.3 mmol / l, respectively, despite weight gain. this could be explained by more intensive treatment due to increased impact of guidelines after the publication of the uk prospective diabetes study results, changes in diagnostic criteria, increased efforts to diagnose diabetes earlier, or a trend toward healthier living because blood pressure and cholesterol have decreased in the general population of norway (4). some of these factors together with an increase in the prevalence of diabetes due to obesity may explain the marked increase in the number of patients with type 2 diabetes attending the practices. very similar reductions in a1c and blood pressure among patients with type 2 diabetes in general practice have been reported by the swedish national diabetes register between 1996 and 2005 (5). a decline in a1c from 7.8 to 7.2% between 1999 and 2004 among adult patients with diabetes in the u.s. has been reported in an analysis of national health and nutrition examination survey data (6). other studies (510) that have performed the latest survey since 2002 report improvements in risk factor control, whereas studies reporting in 2002 or earlier (1114) find little or no improvement. improvements in risk factor control, processes of care, and the more widespread use of metformin suggest that practitioners are responding to guidelines. apart from educational meetings, an independent study has confirmed that the guidelines are used by 52% of general practitioners (15). our findings are probably representative of norway, given that similar mean values were reported from a norwegian primary care survey in 2004 of 975 patients between 18 and 75 years of age with type 2 diabetes (8). furthermore, nearly identical mean values for a1c, cholesterol, and blood pressure were found in two other regions of norway in 2005 (n = 2,764) at primary care centers that did not participate in the 1995 survey. we conclude that there have been substantial improvements in type 2 diabetes primary care in norway that are potentially related to major improvements in health outcomes. | objective to assess changes in the quality of care in norway for patients with type 2 diabetes.research design and methods two cross - sectional surveys were examined that identified all patients (n = 1,470 in 1995 and n = 2,699 in 2005) with type 2 diabetes attending 33 general practices in 1995 and 2005.resultsbetween 1995 and 2005, there were significant improvements in the proportion of patients for whom important laboratory analyses, smoking habits, height, weight, and referral to eye examination were recorded. mean a1c declined from 7.74 to 7.15%, systolic blood pressure from 150.0 to 140.4 mmhg, and cholesterol from 6.28 to 5.0 mmol / l (p < 0.001, age and sex adjusted). the 10-year risk of coronary heart disease for an average male patient declined from 42 to 29%.conclusions there have been substantial improvements in type 2 diabetes primary care in norway that are potentially related to major improvements in health outcomes. |
rotavirus constitutes a major cause of severe gastroenteritis in young children worldwide [13 ]. studies have shown that, by the age of two years, almost all children stand the risk of being infected with rotavirus, with children living in the industrialized countries experiencing their first infection at comparatively older age compared to those in developing countries [4, 5 ]. in sub - saharan africa, gastroenteritis remains a major cause of childhood morbidity and mortality and a leading cause of childhood illness as a result of poor economy, infrastructure, and political instability. in a review of 43 studies from 15 countries, including nigeria, cunliffe. reported that, of the 25 million children born each year in sub - saharan africa, 4.3 million (about 1 in 6) dies by the age of 5 years and about 1/5 of these deaths (850,000) are associated with diarrhoea. among all the identified causes of infantile diarrhoea, rotavirus has been ranked as the single most important pathogen associated with diarrhoea in both hospitalized and outpatient cases. in nigeria, for example, a high incidence of childhood diarrhoea is estimated to account for over 160 000 of all deaths in children less than 5 years of age annually and approximately 20% are associated with rotavirus infection. although it was speculated that an effective and properly administered rotavirus vaccine in africa could potentially prevent 170,000210,000 deaths (about 1 in 20) annually based on the assumptions that 2025% of all childhood diarrhoea deaths are due to rotavirus, there is a lack of information on the epidemiology and genetic characteristics of the circulating human retrovirus in most of the developing nations. this will be crucial to guide control and prevention strategies so as to ensure that rotavirus infection is reduced. thus, the objectives of this study are to determine the occurrence and molecular characteristics of human rotavirus among children in sokoto state, nigeria. samples were collected from major hospitals in sokoto which lies between longitude 11 30 to 13 50 east and latitude 4 to 6 north. the formula of campbell was used to estimate the minimum of 189 samples ; however, in order to increase the precision and chances for the detection of infection, the samples were increased to 200. diarrhoea samples were collected from diarrheic children less than 5 years of age presented at the study hospitals. the samples were obtained following parental consent and ethical approval from the medical research ethics committee of the hospitals. diarrhoea in this study was defined as the passage of more than 3 looser than normal stools within 24 hours. the stool samples were collected aseptically in sterile commercial bijou bottles adequately labeled (patient i d and date of collection) and transported on ice to the veterinary microbiology laboratory of usmanu danfodiyo university, sokoto, where they were stored at 20c until being transported on ice to the noguchi memorial institute for medical research (nmimr) accra, ghana, where they were also stored at 20c until tested. the stored stool specimens were retrieved after freeze thawing and a 10% stool suspension was prepared by pipetting 1.5 ml of supply specimen preparation buffer (included in the kit). rotavirus antigens in stool samples were detected by a commercially available dako rotavirus elisa kit according to the manufacturer 's instructions. the result was read spectrophotometrically in 30 minutes after stopping the reaction on multiskan elisa reader (multiskan plus, labsystems helsinki, finland) at the reference wavelength of 450 nm. all elisa positive samples (n = 51) were subjected to sds - page according to the technique described by herring.. the g - typing was conducted according to the method of gouvea. and das. the protocols for viral rna extraction from the stools and the rt - pcr typing protocols for rotavirus gene 7 (g - serotyping) and gene 4 (p - genotyping) were kindly provided by dr. mab serotyping was carried out with monoclonal antibodies specific for group a human rotavirus strains using the mab serotyping kit according to manufacturer 's instructions. the mab serotyping kit was kindly provided by the national institute of health, atlanta, georgia, usa. the method employed the elisa protocol and the procedure was basically as described by taniguchi. with the monoclonal antibody as the capture reagent but skim milk was used to reduce the background as described by coulson.. phenol / chloroform method described by steele and alexander was used to extract the viral rna from all page positive specimens and then purified with the rnaid kit (bio 191, carlsbad, usa). the vp7 and vp4 genotyping was performed as described by gouvea. and gentsch. briefly, viral rna was subjected to reverse transcription (rt) step and polymerase chain reaction (pcr) step was performed to amplify the transcribed gene. finally a seminested pcr was performed using genotype specific primers as a mean to determine the viral genotype (table 1). the purified viral rna was reverse transcribed according to the method of gouvea.. briefly, 1 l each of the specific primer pair (sbeg9/end9) was added to 8 l of previously extracted dsrna and put in a 500 l pcr tube. the solution was heated for 5 minutes at 94c to denature the dsrna and chilled immediately in an ice bath for 2 minutes. the denatured dsrnas were then reverse transcribed by adding 3.2 l of master mix (0.2 l of 10 mm datp, 0.2 l of 10 mm dctp, 0.2 l of 10 mm dgtp, 0.2 l of 10 mm dttp, 0.4 l of avian myeloblastosis (amv), reverse transcriptase, and 20 l of 5x amv buffer) and incubated for 20 minutes at 42c in a water bath. the cdna was then amplified by pcr in a 40 l reaction mixture containing 1 l of 10 mm datp, 1 l of 10 mm dctp, 1 l of 10 mm dgtp, 1 l of 10 mm dttp, 4 l of 10x taq buffer, 2.4 l of 25 mm mgc12, 30 l of double distilled water, and 0.3 l taq polymerase prior to use. no template control (ntc) thirty cycles of pcr (1 minute denaturing at 94c, 2 minutes annealing at 42c, and 3 minutes extension at 72c) and a final extension cycle at 72c for 7 minutes was performed on a gene amp pcr primus 25 system machine the amplified samples were analysed onto 2% agarose (seakem, usa) and visualised on the geldoc system. a second round polymerase chain reaction was carried out to determine the genotype of the rotavirus strains. briefly, 1 l of the first round reaction products was added to 40 l pcr - mm containing 1 l of each of six serotype - specific primers (abt-1, act-2, aet-3, adt-4, aat-8, and aft-9) and the primer rvg9, 1 l of 10 mm datp, 1 l of 10 mm dctp, 1 l of 10 mm dgtp, 1 l of 10 mm dttp, 4 l of 10x taq buffer, 2.4 l of 25 mm mgcl2, 30 l of ddh2o, and 0.3 l of taq polymerase and pcr performed according to the condition stated above. the amplified products were analysed on 2% agarose gel and the genotype determined based on the size of the resultant amplicon. genotyping of vp4 gene of human rotaviruses used a cocktail of gentsch primers, con3 + [1t-1, 2t-1, 3t-1, 4t-1, and 5t-1 ], to determine genotypes p, p, p, p, and p, respectively (table 2). amplification for both sets of primers was carried out by initial denaturation at 94c for 2 mins, denaturation at 94c for 1 minute, annealing at 42c for 2 minutes, extension at 72c for 5 minutes, and final extension at 72c for 7 minutes. genotyping of vp4 gene of human rotaviruses used a cocktail of gentsch primers, con3 + [1t-1, 2t-1, 3t-1, 4t-1, and 5t-1 ], to determine genotypes p, p, p, p, and p, respectively (table 2). amplification for both sets of primers was carried out by initial denaturation at 94c for 2 mins, denaturation at 94c for 1 minute, annealing at 42c for 2 minutes, extension at 72c for 5 minutes, and final extension at 72c for 7 minutes. the rt - pcr typing method used for p - typing was similar to that used for g - typing. the consensus primers used were con2 and con3 as described by gentsch.. a second round polymerase chain reaction to determine the p - types of the rotavirus strains was similarly performed using 5 p genotype - specific primers (1-t1, 2t-1, 3t-1, 4t-1, 5t-1) and the primer con3. the pcr amplification was then performed according to the conditions earlier stated for g - typing. the amplified products were analysed on 2% agarose gel and the genotype determined from the size of the amplicons. table 3 provides the expected band sizes of different g- and p - types used as a criteria for interpretation of human rotavirus g- and p - types results based on the primer types used (table 3). out of the 200 human diarrhoea stools examined by elisa, rotavirus was detected in 51 of the samples, thus indicating a prevalence of 25.5%. of the 51 human elisa positive samples tested by page, 38 (74.5%) yielded electrophoretic patterns typical of rotavirus. 29 of the rotavirus strains (76.3%) had classical long rotavirus rna electrophoretic patterns and 9 (23.7%) exhibited classical short rna profile as shown in (table 4). the representative of human electrophoretic patterns of rotavirus strains from rotavirus strains from the sds - page results is shown in figure 1. of the 38 page positive samples further subjected to pcr genotyping method, first round rt - pcr revealed a total of twenty - nine (76.3%) positives for vp7 genotype. following second round amplification, 27 (71.1%) of the rotavirus isolates were successfully assigned a vp7 (g - type) specificity (figure 2(a)), while 11 (28.9%) of the isolates were nontypeable. similarly, vp4 genotyping of the 38 page positive samples after first round amplification showed 27 (71.1%) positives for vp4 genotype. however, only 23 (60.5%) samples produced second round pcr products that could be assigned a vp4 genotype (figure 2(b)). five different rotavirus vp7 genotypes including the rare human rotavirus g genotype (g12) were detected. the five predominant genotypes observed in circulation were sixteen g4 (59.3%), four g1 (14.8%), three g2 (11.1%), three g3 (11.1%), and one g12 (3.7%) (figure 3). g1, g2, and g3 are both common human g - types while the g12 is a rare human g - type. the rare g - serotype could be animal rotavirus strains that accidently infected human or reassortant viruses between animal and human rotavirus generated in nature. the distribution of vp7 serotypes according to electrophoretic profile of the isolates is summarized in table 5. twenty - nine samples exhibited classical long rna electrophoretic profile on page and 22 (75.9%) were typed and assigned to particular g - types while 7 (24.1%) could not be assigned any vp7 genotype. thirteen (59.1%) of these were typed as g4, three (13.6%) as g1, three (13.6%) as g2, two (9.1%) as g3, and one (4.5%) as g12. nine samples exhibited classical short rna electrophoretic profile on page and 5 (55.6%) were typed and assigned to particular g - types while 4 (44.4%) could not be assigned any vp7 genotype. of the 5 that were typed, three (60%) were typed as g4, one as g3 (20%), and one (20%) as g1. the monoclonal antibody serotyping detected g1 and g3 with consistency both on visual examination and spectrophotometrically. the od values of the assigned serotypes were twice greater than the values obtained from the samples with the same mab. the mab typing of the g1 and g3 serotypes was consistent with the result of the rt - pcr serotyping of these types. the vp4 genotypes detected during the study included two of the recognized human rotavirus vp4 gene alleles, p and p, and a rare human p. the most predominant p4 genotype was p 11 (47.8%), followed by p 9 (39.1%) and p 3 (13%), and none of the strains was carrying a p vp4 gene (figure 4). the data showed a high percentage of rare p genotype, p which is considered as an indication of either animal rotavirus strains that accidently infected human or reassortant viruses between animal and human rotavirus generated in nature. four distinct strains were identified with the common g and p combinations being strains bearing the genotype g4 p (56%), g4 p (11%), g1 p (22%), and g3 p (11%) (figure 5). similarly, three distinct strains with unusual combinations or rare g or p genotypes were observed ; these included g12 p (14%), g2 p (29%), and g4 p (57%) as summarized in figure 6. rotavirus has been identified to be the single most important pathogen associated with diarrhoea cases in both hospital patients and outpatients [4, 18 ]. in this study, 51 (25.5%) out of the 200 diarrhoeic children tested were found to be positive for rotavirus while 149/200 (74.5%) tested negative for rotavirus. thus, the prevalence of rotavirus diarrhea accounted for 25.5% of diarrhea cases among children younger than five years presented to hospitals in sokoto metropolis. the result of this study is consistent with the sentinel based rotavirus surveillance system and hospital based study results within the african region. interestingly, earlier studies carried out in different parts of the northern nigeria reported low prevalence. the report of pennap and umoh showed rotavirus infection prevalence of 15.6% among children 060 months old that were presented with diarrhea in northeastern nigeria. similarly reported rotavirus prevalence of 18% among diarrheic children and 7.2% among nondiarrheic children in a hospital setting in northern nigeria and prevalence of 9% in children under five years of age in a community based study in the same region. similarly, other investigators reported lower prevalence of the infection in the northern region. the low prevalence reported in the community based study is expected as higher prevalence of rotavirus infection more likely to be encountered in hospital based studies since rotavirus positive cases are often severe and likely represented in hospitals. however, generally, studies from southern nigeria had shown higher rotavirus prevalence values than those from northern nigeria [2427 ]. the differences in the prevalence recorded by different investigators had been attributed to differences in time of sample collection, method of screening samples, geographical location of the study, or changing trends of the burden of the rotavirus disease over the years. the possibility of direct transmission of animal rotavirus to human host and the uncommon serotypes detected in this study may explain the high prevalence of the disease among children with often contact with animals. the analysis of electropherotypes of rotavirus isolates provides information on genetic diversity of the virus and heterogeneity of circulating strains and can be useful in tracing spread through a population. indeed, viruses of the same serotype could exhibit different electropherotypes and those of the same electropherotypes different serotypes. in this study, analyses using page showed that, of the 51 human elisa positive samples tested by page, 38 (74.5%) yielded electrophoretic patterns typical of rotavirus while 13 showed no profile. of the 38 rotavirus strains that yielded rna profile, 29 (76.3%) had classical long rotavirus rna electrophoretic patterns and 9 (23.7%) exhibited classical short rna profile. the inconsistency between elisa results and page results might be as a result of rna degradation in which case strains will not yield rna profile on page. the absence of rna bands had also been attributed to too little rna or its degradation during the phenol / chloroform extraction stage. strains with long rna profiles were the most prevalent strains in circulation in the study area. interestingly, however, there were no unusual electropherotypes even in the samples containing the g12 strains. the rt - pcr vp7 serotyping results revealed that twenty - seven (71.1%) of the rotavirus isolates were successfully assigned a vp7 (g - type) specificity. while 11 (28.9%) of the isolates were nontypeable. it was likely that the nontypeable strains did not contain enough rna to permit typing similar to the observation of adah.. it may also be as a result of the existence of serotypes which the serotype - specific primers used in the study could not detect. the monoclonal antibody serotyping detected g1 and g3 with consistency both on visual examination and spectrophotometrically. but no other g - serotype was typed using mab apart from the two serotypes (g1 and g3). the od value recorded against each of these two serotypes with the specific mab was consistently more than twice than what was recorded in the reaction of other samples with the same mab. the mab typing of the g1 and g3 serotypes was consistent with the result of the rt - pcr serotyping of these types. previous studies had shown that the most common global human g - serotypes were g1, g2, g3, g4, and g9, with g1 being most prevalent and g9 the fastest emerging genotype worldwide [3133 ]. in this study, five different rotavirus vp7 serotypes including the rare human rotavirus g - serotype (g12) were detected. the predominant serotypes observed in circulation in this study were sixteen g4 (59.3%), four g1 (14.8%), three g2 (11.1%), three g3 (11.1%), and one g12 (3.7%). in earlier studies reported by adah, no g2 or g4 serotypes were detected ; however, aminu. the absence of g9 serotype in this study could be a result of genetic shift since g9 serotype was previously reported in northern region of the country [21, 22 ]. although g12 rotaviruses were first identified in the philippines in 1987, it emerged over the past few years in numerous countries with the first incidence in africa reported in johannesburg, south africa. indeed, the detection of g12 serotype may suggest either accidental human infection by animal rotavirus strains or reassortant viruses between animal and human rotavirus generated in nature. the results of vp4 genotyping revealed that the vp4 genotypes detected during the study included two of the recognized human rotavirus vp4 gene alleles, p and p, and a rare human p genotype (p). the most predominant p - type detected in this study was p 11 (47.8%) followed by p 9 (39.1%) and p 3 (13%). the predominance of p strains in this study agreed with the worldwide distribution of this genotype as epidemiological studies had shown p strains to be the most commonly identified worldwide [38, 39 ]. it is noted that similar to the result of the study conducted in nigeria by of adah. however, in disagreement with the work of adah., in which p was reported as the predominant type and p as the second most common type, p strains were the least detected in this study and p was the most predominant. in fact, p genotype was previously thought to be associated with neonatal rotavirus strains and hence thought to be associated with asymptomatic infection. but in this study, it was associated with symptomatic diarrhea similar to what was observed in earlier studies conducted in cameroon, nigeria [21, 22 ], and ghana. in fact, rotavirus with vp4 p genotype was the only genotype detected in the study conducted by aminu.. for example, in the surveys which detected p strains, only 0.2% of 1,316 rotaviruses between 1996 and 1999 in the united states and 3.8% of 282 rotaviruses between 1991 and 1994 in israel were of this p specificity. furthermore, all the strains with p specificity had been associated with g3 or g1 specificity [44, 45 ]. therefore, the high percentage of this genotype (p) detected in this study is considered as an indication of either animal rotavirus strains that accidently infected human or reassortant viruses between animal and human rotavirus generated in nature. studies in many countries have shown that g1 p, g2 p, g3 p, and g2 p are the g - p combinations most commonly found worldwide [38, 39, 46 ]. in this study, nine distinct strains were identified with the common g and p combinations bearing the combination g4 p (56%), g4 p (11%), g1 p (22%), and g3 p (11%). similarly, seven distinct strains with unusual combinations or rare g or p genotypes were observed. these were g12 p (14%), g2 p (29%), and g4 p (57%). numerous studies had reported an unexpectedly high diversity of rotavirus strains in most developing and developed countries including nigeria [34, 39, 42, 4754 ]. this had been attributed to natural reassortment which appears to be detected more frequently in developing countries than in developed world owing to low levels of hygiene and poor immunological defence in infants which facilitate mixed infections and hence more reassortment. in addition more close contact among humans, livestock, and other animals in developing countries makes the possibility of emergence of virulent rotavirus strains very high as a result of gene reassortment. apart from gene reassortment, interspecies transmissions of rotaviruses involving a whole genome constellation evidenced by molecular characterization of rotaviruses isolated from different species have been suggested. indeed, studies had indicated that uncommon human rotavirus strains are emerging as global strains, which has important implications for effective vaccine development [4, 56 ]. the detection of unusual g / p combinations in the present study adds to this pool of information and further confirms the emergence of these unusual strains. the study reported a high prevalence of rotavirus infection among diarrheic children attending health care in sokoto state, nigeria. both common, uncommon, and combinations of various genotypes were identified as revealed by their electrophoresis pattern based on the vp4 and vp7 gene. overall, this study contributed to the global understanding of the molecular epidemiology of human rotavirus which will be useful in guiding the choice and application of rotavirus vaccines for effective control and preventions. | this study was conducted to detect and characterize prevalent human group a rotavirus strains from 200 diarrheic children in sokoto, nigeria, by elisa, monoclonal antibody (mab) serotyping and reverse transcription - polymerase chain reaction (rt - pcr) techniques. rotavirus was detected in 25.5% of the children. the g - serotypes observed in circulation were g4 : 16 (59.3%), g1 : 4 (14.8%), g2 : 3 (11.1%), g3 : 3 (11.1%), and g12 : 1 (3.7%). the monoclonal antibody (mab) serotyping detected g1 and g3 but did not detect g4 and g2 serotypes. the mab typing of the g1 and g3 serotypes was consistent with the result of the rt - pcr. the vp4 genotypes detected were p[6 ] 3 (13%), p[8 ] 11 (47.8%), and the rare human p genotype (p[9 ]), found in 9 patients (39.1%). nine strains identified with the common g and p combinations were g4 p[8 ] 5 (56%), g4 p[6 ] 1 (11%), g1 p[8 ] 2 (22%), and g3 p[8 ] 1 (11%), while seven strains with unusual combinations or rare g or p genotypes identified were g12 p[8 ] 1 (14%), g2 p[8 ] 2 (29%), and g4 p[9 ] 4 (57%). to our knowledge this is the first molecular study of human rotavirus and report of rare human g and p serotypes in sokoto state. |
in the design of novel organic materials for nonlinear optical applications, it initially appears irrational to consider approaches using molecular building blocks in which second harmonic generation (shg) activity is strictly forbidden by symmetry. however, several recent studies have reported the observation of bright shg from appropriately arranged assemblies of centrosymmetric or nominally centrosymmetric molecules. the rational use of purely centrosymmetric molecules as building blocks for performing frequency doubling and mixing has the potential to open up entirely new synthetic strategies for the design of organic nonlinear optical (nlo) materials. rational design hinges first on elucidation of the dominant mechanisms driving the nonlinear optical response. however, the macromolecular mechanism underlying the emergence of shg activity still remains a somewhat open question. in one example using squaraines, shg activity was observed from langmuir blodgett films prepared using centrosymmetric chromophores. an intermolecular charge transfer mechanism was proposed in the case of the squaraines, in which two monomers form a t - shaped dimer. however, the actual structures of the squaraine multimers are not known, given the challenges of obtaining high - resolution structures of single - monolayer organic films. while charge transfer is a sufficient condition for the presence of shg, it is difficult to exclude alternative chromophore dimer architectures that may produce shg activity through coupled interactions without additional molecular - level information on the structures produced through intermolecular interactions. within crystals of related squaraines, the monomers adopt -stacked dimer structures, or -stacked herringbone structures within the extended lattice, rather than t - shaped intermolecular structures. in other work, vibrational sum - frequency generation (sfg) was observed from the liquid / air interface of benzene and other centrosymmetric liquids, studied both experimentally and theoretically. the observation of sfg from the benzene / air interface was first reported by hommel and allen, who attributed the signal to the symmetry breaking from intermolecular coupling and/or benzene dimer formation. in work by morita and co - workers, molecular dynamics calculations were coupled with interfacial hyperpolarizability and normal - mode analysis, concluding that symmetry breaking within the interfacial benzene molecules themselves was sufficient to explain the observed vibrational sfg without the need for dimerization, although bulk quadrupole contributions were also predicted to be of comparable magnitude. more recently, tahara and co - workers reported experimental results suggesting that the observed vibrational sfg from benzene may be dominated by bulk quadrupole effects. in related shg microscopy studies of centrosymmetric carotenoids, bright shg has recently been reported from h - aggregates of astaxanthin. the astaxanthin monomers are centrosymmetric with little conformational freedom, with the known thermodynamically stable crystal form also adopting a centrosymmetric shg - inactive lattice. as such, the nature of the intermolecular interactions driving shg are not trivially obvious. irrespective of the particular structure adopted by the dimer / multimer in the squaraines or the carotenoids, the shg activity can likely be attributed to electronic perturbations as a consequence of intermolecular interactions. given that the intermolecular interactions are relatively weak compared to the intramolecular interactions driving bond formation, a perturbation theoretical approach is likely to be appropriate for treating the emergence of shg activity. using the nonlinear optical properties of the unperturbed monomer as a starting point, the introduction of perturbations to the electronic structure can be described within the context of exciton coupling theory. in the present work, this simple exciton coupling approach is developed to provide a framework for describing the emergence of nonzero hyperpolarizability in noncentrosymmetric dimers of centrosymmetric molecules, serving as the foundation for predictions of larger extended clusters and aggregates. dimer interactions form the foundation for interpreting extended multimeric intermolecular interactions, in addition to being interesting in their own right. they also have the advantage of being the smallest computationally tractable unit for describing intermolecular interactions. quantum chemical calculations in a simple model system composed of two coupled butadiene monomers provide a framework for evaluating the strengths and limitations of the zero - order exciton coupling description. based on the predictions of the model, crystals were prepared from centrosymmetric 2,6-di - tert - butylanthraquinone (taq) and tested experimentally by shg microscopy. a framework for interpreting the predicted emergence of shg activity due to coupling is proposed based on molecular orbital descriptions of the exciton states in the dimer. in centrosymmetric molecules, all vibrational and electronic transitions are exclusively one - photon- or two - photon (including raman)-allowed, but not both. the absence of shg can be interpreted within the context of this one - photon versus two - photon exclusivity. the molecular hyperpolarizability tensor underlying shg can be described by a summation over products of one - photon transition moments and two - photon transition matrices, provided the contributing high - energy excited states correspond to frequencies near or above the second harmonic frequency.1 the preceding equation will break down in systems exclusively exhibiting one - photon resonance enhancement or in systems not initially in the ground state, but can be considered to be an excellent approximation under most practical experimental conditions. in eq 1, s(2) is a complex - valued line shape function. in the case of lorentzian line shapes, s(2) is given by the following equation.2 in eq 2, n is the transition energy between the ground state and the nth excited state, and n is the damping constant, related to the homogeneous line width. from inspection of eq 1, the requirement that transitions be either one - photon- or two - photon - allowed clearly results in zero values for each term in the summation. formally, each dimer state (indicated by the subscript d) is given by summations over all of the monomer excited states (indicated by m), but with the largest contributions arising from those closest in energy.3 so far, nothing yet has helped describe the emergence of shg activity. if mixing only arose between the states as indicated by the solid lines in figure 2, the and terms for each exciton state in the dimer could be recovered from the sums and differences of and from the corresponding transitions in the monomers. in this limit, the dimer would still exhibit no shg, since the exciton states arising from one - photon - allowed transitions in the monomer would exhibit negligible two - photon absorption, and vice versa. however, minor contributions from the other monomer excited states are also generally expected (eq 3), such that the shg activity of the a and b states can be turned on through mixing in of two - photon absorption character into one - photon - allowed monomer transitions and vice versa. electronic structure of 1,3-butadiene monomers and dimers were calculated the using gamess package separately. geometry optimization calculations were used to determine the energy minimized molecular geometry, and then avogadro software was used to orient the molecule(s) such that the z - axis was the primary axis of rotation. configuration interaction singles (cis) calculations were used to compute the electronic resonances of the monomer and dimer separately. time - dependent hartree fock (tdhf) calculations were used to compute first hyperpolarizability tensor elements on both the monomer and the dimer at 430, 450, and 1000 nm, with the highest energy incident frequency being within 4 nm of the first electronic resonance calculated using cis after frequency doubling. also, at 450 nm incident frequency on the dimer, tdhf calculations were used to compute first hyperpolarizability tensor elements at dimer distances of 3.8, 6.0, 8.0, and 60. all tdhf calculations obtained both iterative and noniterative tensor elements, which were in good agreement with each other. tdhf was selected as it has been shown to recover and describe resonant interactions, unlike conventional hf or density functional theory (dft). the tdhf calculations were all performed for optical frequencies approaching resonance at the second harmonic frequency consistent with the measurements but still far enough below to avoid complications from singularities that can arise near resonance. shg microscopy measurements of taq crystals were performed using an instrument described previously. in brief, all images were acquired with a built - in - house beam scanning shg microscope. beam scanning was performed with a resonant vibrating mirror (8 khz, eopc) along the fast - axis scan, and a galvanometer (cambridge) for slow - axis scanning. the 800 nm excitation wavelength by a 80 mhz ti : sapphire pulsed laser (spectra - physics mai tai) of 100 fs pulse width was directed through the scan mirrors and focused onto the sample using a 10 objective of working distance 1.6 cm (nikon, numerical aperture (n.a.) = 0.30). shg signals were collected, with dichroic mirrors and narrow band - pass filters (chroma hq400/20 m-2p) centered around 400 nm placed prior to the photomultiplier tube detectors (burke, xp 2920pc). an in - house - written matlab code was used to digitize each synchronous laser pulse with strict timing, to control the scanning mirrors and to communicate with the data acquisition electronics. laser transmittance images were made by recording the intensity of the incident fundamental beam using a photodiode. before considering the butadiene dimer, it is useful to start with a review of the electronic structure of the monomer. butadiene conforms to the c2h point group, which is centrosymmetric and shg - inactive by symmetry. based on quantum chemical calculations, the two lowest energy transitions correspond to a highest occupied molecular orbital lowest unoccupied molecular orbital (homo lumo) transition of bu symmetry, with the next highest energy transition corresponding to bg symmetry. as required by symmetry in centrosymmetric molecules, each transition must be allowed for either one - photon or two - photon excitation, but not both. in this case, the bu transition is one - photon - allowed and two - photon - forbidden, while the bg state is one - photon - forbidden and two - photon - allowed. quantum chemical calculations of the butadiene monomer confirm these expectations, even when symmetry is not rigorously imposed. when positioned in a -stacking configuration such as that shown in figure 1, the symmetry of the dimer becomes c2, with the a and b states generated from linear combinations of the monomer states. because of the odd symmetry of the -orbitals, the difference states are lower in energy than the sum states in -stacked dimers, consistent with the exciton coupling diagram depicted in figure 2. 1,3-butadiene dimer used in quantum simulations, arranged so that the z - axis is the primary axis of rotation. the z - axis is blue, the x - axis is red, and the y - axis is green. the monomers are stacked on top of each other to form a y shape as can be seen from the top - down view of the second image. the exciton coupling model of a dimer is fully rigorous in the limit of inclusion of all excited states in the summation. in brief, the set of excited states serves as a basis set for recovering the new states in the coupled system. since the excited states themselves are constructed from a linear combination of fundamental basis set functions, so too are the states produced from exciton coupling. in the limit of weak coupling consistent with intermolecular interactions (as opposed to covalent bond formation), each exciton state of a dimer can be reasonably described by the interactions between just one or two excited states of the monomer. however, the practical need to consider a finite number of excited state couplings can potentially introduce uncertainties in the approach. consequently, the approach is likely to be most accurate when the coupling between monomers is relatively weak (such that only a few excited states are required to recover the exciton states) and for molecular systems with a relatively sparse population of spectrally overlapping excited states capable of participation in coupling. these are both reasonable assumptions in the present case. unlike the c2h point group, the a and b states of the dimer can in principle however, in practice the core nature of the monomer transitions is carried over when describing the excited state transitions in the dimer arising from exciton coupling. within the validity of this simple exciton coupling description, the most significant contributions to the dimer states will be produced from the sums and differences of the corresponding orbitals of the monomers. for example, considering just the two excited state transitions shown in figure 2, the one - photon transition moment to the first excited b state should be recovered from the vector difference between the two monomer transition moments, resulting in a predominantly y - polarized transition with an oscillator strength equal to the y - component of the monomer multiplied by 2. the total wave function describing the lowest excited state transition in the dimer can be written as a linear combination of both the major one - photon - allowed bu contributions and the minor two - photon - allowed bg contributions.4the corresponding transition moments as well as the matrices describing two - photon absorption can be similarly produced from appropriately weighted sums and differences.5 although |cbu| > |cbg|, the presence of a nonzero contribution from the bg transition provides some two - photon transition character that can drive nonzero values of the hyperpolarizability tensor. in this simplified three - state model for the monomer, the hyperpolarizability tensor for the lowest - lying b state is approximated by the following expression.6the corresponding tensor contributions for the a states are given by the summation (rather than the difference) between the monomer and terms. this model suggests several specific predictions that can be compared directly with computational and experimental results. (1) the dominant tensor elements driving the hyperpolarizability in the dimer can be predicted based on the symmetries of the corresponding monomer states contributing to exciton coupling. (2) in the limit of weak interchromophore coupling, the shg activity should approach zero. (3) the shg activity of the dimer should be substantially enhanced close to resonance but approach zero far from resonance. (4) significant charge transfer is not expected for the observation of shg activity in the dimer. the second is clear conceptually, but potentially less so mathematically. in the limit of weak coupling, the excited state energies of an exciton pair converge to nearly degenerate values. in this limit, it becomes nearly mathematically equivalent to describe the dimer in a basis set consisting of two uncoupled monomers rather than as a coupled dimer. the key criterion has already been established for assessing whether the hyperpolarizability can be considered through the coherent summation of two uncoupled monomers, or if coupling and exciton state descriptions are required. specifically, coupling should be considered if the energy splitting is comparable or greater than the experimental line width of the transition and can safely be neglected under conditions in which it is not. the third prediction is closely related to the second. from inspection of eq 2, the weighting of each exciton state in the net hyperpolarizability is related to the energy difference between the exciton state and 2, where is the fundamental frequency. as the second harmonic frequency moves away from resonance, the contribution from each of the exciton states for example, the two exciton transition moments from the pair of bu monomer states each contribute with approximately equal weight, such that the net result is closely approximated by the direct coherent sum of the uncoupled monomers. correspondingly, in this limit far from resonance the perturbation from exciton coupling becomes negligible. since the unperturbed system of two centrosymmetric monomers is shg - inactive, the nonresonant result should also converge to that same outcome far from resonance. since neither of the monomers possesses a net dipole nor charge transfer character in any of the transitions, little or no charge transfer is expected in the exciton states produced from sums and differences of those same monomer states. the predictions of the exciton coupling model were compared with the results of quantum chemical calculations of the linear and nonlinear optical properties of the butadiene monomer as a point of reference for interpreting the nlo properties of the dimer structures. cis calculations for the monomer were performed and are summarized briefly in the supporting information. in brief, the lowest lying excited state corresponds to a transition of bu symmetry, consistent with the presence of a transition moment polarized within the xz - plane using the coordinate system indicated in figure 2. the next highest excited state is one - photon - forbidden, suggesting either ag or bg symmetry. the symmetry is tentatively assigned as bg based on trends in the dimer detailed in following text. the butadiene structure considered computationally was one in which just one pair of carbon atoms were coparallel and -stacked, as shown in figure 2. in this configuration, the butadiene dimer has c2 symmetry. a summary of the linear optical properties of the dimer is provided in the supporting information. as a simple confirmatory test, the hyperpolarizability as a function of intermolecular separation is shown in figure 3. as one might expect, the magnitude of each hyperpolarizability tensor element uniformly decreases as the intermolecular distance is increased, asymptotically approaching a value of zero in the limit of negligible interchromophore coupling consistent with the second prediction of the exciton coupling model. the hyperpolarizability tensor elements as a function of fundamental wavelength are summarized in figure 4. results for the frequency - dependent dimer calculations clearly demonstrate a trend in which the tensor elements are rapidly reduced in magnitude as the incident wavelength is shifted further from resonance. again, this observation is in good agreement with the predictions of the exciton coupling model. interestingly, the largest magnitude for the shg activity is given in the chiral zxy tensor element with the largest relative enhancement close to resonance. the dominance of this contribution can be understood within the context of the exciton coupling model by considering just the two lowest excited states in the butadiene monomer. the monomer bu (homo lumo) transition is polarized within the yz - plane of the chromophore and oriented largely along the long z - axis of the molecule. the lowest energy b exciton state in the dimer should be formed from the difference of the two monomer wave functions (given the sign difference between the p - orbitals), with symmetry dictating that it be y - polarized, and with a transition moment roughly 2 larger in magnitude than the monomer, in excellent agreement with the quantum chemical calculations. similarly, the next highest excited state in the dimer should consist of the sum of the monomer wave functions, corresponding to an a state with a z - polarized transition moment. the major contributions to this pair of a and b states will arise from coupling primarily from just the two one - photon - allowed monomer bu states. however, the dimer a and b states can also borrow minor contributions from the next highest two - photon - allowed excited state of bg symmetry. for a transition of bg symmetry, the nonzero tpa tensor elements in the monomer will be xy and xz, the first of which can contribute exclusively to a states in the dimer, and the second exclusively to b states. combining the nonzero elements of and according to eq 1, the lowest energy dimer transition should be dominated by the yxz tensor element (nonzero y and borrowed xz) and the next highest transition dominated by the zxy tensor element (large z and borrowed xy). given the larger one - photon transition moment along the long monomer z - axis, it is not surprising that the second excited state in the dimer corresponding to the zxy tensor element drives much of the nlo activity near resonance. these combined conditions predict relatively large contributions from the chiral tensor elements, in reasonably good agreement with the computational results. the tensor elements zyx and yxz are larger in magnitude than all other tensor elements (at all three wavelengths considered). for example, the next most significant tensor element was zzz, presumably arising from the large z from the bu monomer transition coupled with zz contributions from the next higher excited states of bg symmetry. the steep sensitivity of the calculated hyperpolarizability with fundamental wavelength indicated in figure 3 is noteworthy. this trend is consistent with the molecular orbital diagram depicted in figure 2, assuming the borrowing of the one - photon and two - photon contributions goes both ways in this two excited state limit. while the lowest two excited states of the dimer yield nonzero values for yxz (nonzero y and borrowed xz) and zxy (large z and borrowed xy), the next highest exciton pair will similarly be driven by a large, but equal and opposite, contribution to those same tensor elements yxz (borrowed y and nonzero xz) and zxy (borrowed z and nonzero xy). the requirement that they sum to approximately zero in the two excited state model arises simply by the nature of the centrosymmetry of the monomers from which the dimer states were generated. of course, additional excited states are also present and contributing, but the general sensitivity to resonance enhancement in the dimer can still be qualitatively understood within the context of this argument. crystals of taq form a particularly useful benchmark to test the exciton coupling model. the particular set of nonzero tensor elements generated from exciton coupling depend solely on the relative orientation, and not their relative position. the magnitudes of the tensor elements are affected by the degree of coupling, but not which tensor elements are nonzero. consequently, the allowed tensor elements are arguably most easily identified by considering first structures for the taq dimer with different relative positions between the monomers. based on a previously published crystal structure, taq forms a centrosymmetric, shg - inactive crystal structure of symmetry, in which every monomer is in exactly the same orientation within the lattice and each monomer is centrosymmetric. considering a dimer formed from two monomers of identical orientation, the wave functions for the sum states will simply be identical but rescaled, and all of the difference states will be zero - valued. as such, the shg activity of the taq dimer and crystal is interesting to interpret within the context of the exciton coupling model. considering a dimer composed of two monomers offset in space by not being rotated, the symmetry of the dimer is formally ci and should result in no shg activity. in shg measurements of taq powders as received (figure 5), the large majority (92.6% of the total area in the field of view) was shg - inactive as expected based on the known crystal form. consequently, the absence of significant shg from the large majority of the taq powder is in excellent agreement with both the established bulk crystal symmetry and the exciton coupling arguments. laser transmitted images of taq from different crystallization conditions are presented (top row) along with the corresponding shg images (bottom row). panels a and b correspond to the powder as received, c and d correspond to the crystals grown by the solvent evaporation over the time course of a few minutes, and e and f are the images of the same sample following enclosure in a chamber containing high solvent vapor pressure for 3 days. the shg images are all presented using a common intensity scale relative to a batio3 nanoparticle reference. since the established crystal structure for taq material is symmetry - forbidden for shg, it is particularly noteworthy that strong shg is nevertheless observed from localized domains within the powdered sample. while the large majority of the taq powder is shg - inactive consistent with expectations, approximately 7.4% of the total area in figure 5a is occupied by shg - active domains, representing a small but significant total volume fraction of the material. the shg activities of the taq crystals rival those of batio3, used as a reference material. recrystallization by rapid desolvation resulted in a 10-fold increase in the integrated shg activity of the taq powder per unit area, shown in figure 5d. following recrystallization, the shg - active taq crystals were placed in a sealed container with a saturated vapor pressure of 1,4-dioxane (the solvent used in the initial crystallization) and then reimaged after 3 days at room temperature (figure 5e, 5f). over this time frame, the shg activity of the sample within the same field of view was reduced 27-fold to levels similar to those observed initially within the crystalline powder. the observation of such a reduction in shg from an identical region of the powder strongly suggests the absence of significant bulk - allowed quadrupolar or magnetic dipole origins for the observed shg signals. both higher order effects arise with comparable efficiency for both centrosymmetric and noncentrosymmetric media. as such, their contributions would be unlikely to be perturbed by the solvent - mediated recrystallization. this observation is in noteworthy contrast to vibrational sfg measurements of the benzene / air interface, in which calculations and measurements suggest quadrupole effects may be significant. furthermore, shg arising from trace impurities can similarly be excluded, as they would be present in equal quantities before and after exposure to solvent vapor. in addition, the shg intensity produced by taq rivals that of the noncentrosymmetric bulk dipole - allowed batio3 reference, which strongly suggests a bulk - allowed electric dipole origin of the observed signal. given the steep dependence on the preparation method, the shg arising from the taq following recrystallization is attributed to the production of at least one alternative new noncentrosymmetric crystal form. in previous studies, it has been shown that rapid solvent evaporation can promote the formation of metastable polymorphs by placing crystallization under kinetic control rather than thermodynamic control. the observed loss in shg activity shown in figure 5 following exposure of the crystals to solvent vapor is in good agreement with this explanation, as adsorbed solvent films can facilitate the interconversion between different crystalline solvates and/or polymorphs. two possible mechanisms for the observed bright shg activity within the taq crystals are considered.. this mechanism can be excluded by inspection of the structure of taq, which consists of a rigid ring with significant flexibility only in the tert - butyl rotation angles. it is unlikely that the relatively weak intermolecular interactions driving crystal packing will substantially distort the centrosymmetric ring structure driving the nonlinear polarizability of taq. it is equally unlikely that a noncentrosymmetric eclipsed configuration for the tert - butyl groups as opposed to the centrosymmetric staggered configuration would exhibit substantially enhanced nonlinear optical activity of the monomer. consequently, the observation of shg activity is attributed to intermolecular exciton coupling interactions within a noncentrosymmetric lattice. the observation of bright shg from taq crystals confirms the presence of significant intermolecular interactions within the lattice, but is not alone sufficient to exclusively confirm the exciton coupling model and exclude alternative mechanisms such as charge transfer. of course, a charge transfer complex is really just a specific example of exciton coupling. without more detailed knowledge, we can only state that the observation of shg is consistent with the predictions of the model and that the exciton model imposes the least requirements in terms of specific structures produced than alternative hypotheses, such as charge transfer. it is interesting that the regions of high shg in taq were brighter than the batio3 reference materials. given that the molecular building block is forbidden by symmetry to produce shg, such bright signals are clear indicators of intermolecular interactions within the lattice as a key driving influence. the low - lying transitions in taq approach energies corresponding to the twice the incident photon energy, while batio3 is transparent throughout the visible spectrum. the presence of an shg - active form for the taq crystals is in excellent qualitative agreement with the exciton coupling model described herein. while the packing arrangement within this new polymorph is not yet established, for the present purposes it is sufficient to note that it is clearly and strongly shg - active, despite being produced from a centrosymmetric molecular building block. a model based on exciton coupling theory was developed for interpreting the emergence of shg in assemblies of centrosymmetric monomers. from the one - photon transition moments and two - photon absorption tensors within the monomer, the relative magnitudes and polarization dependences of the hyperpolarizability tensor elements describing the exciton states the degree of energy splitting between the resulting exciton states is dependent on the coupling strength between the monomers. this approach was tested computationally using tdhf and cis calculations on both the monomer and dimer of 1,3-butadiene, with good agreement between the predictions of the model based on the monomer optical properties and the quantum chemical calculations of the dimers. specifically, the signs and relative magnitudes of the different tensor elements predicted from the monomer and calculated for the dimer were in good agreement, indicating zxy as the dominant tensor contribution in the dimer at optical wavelengths. additional experimental support for the exciton model was found in studies of taq crystals, in which both the shg - active and shg - inactive forms were found experimentally. since taq is itself centrosymmetric with little conformational flexibility within the chromophore, the observation of relatively strong shg from the metastable crystals is consistent with an exciton coupling mechanism to produce shg - active crystalline forms. | a simple model is presented for interpreting the presence of substantial second harmonic generation (shg) activity from assemblies of centrosymmetric molecular building blocks. using butadiene as a computationally tractable centrosymmetric model system, time - dependent hartree fock calculations of the nonlinear polarizability of butadiene dimer were well - described through exciton coupling arguments based on the electronic structure of the monomer and the relative orientation between the monomers within the dimer. experimental studies of the centrosymmetric molecule 2,6-di - tert - butylanthraquinone suggest the formation of a combination of shg - active and shg - inactive crystal forms. the structure for the centrosymmetric form is known, serving as a negative control for the model, while the presence of an additional shg - active metastable form is consistent with predictions of the model for alternative molecular packing configurations. |
the 3t3-l1 cells procured from the national centre for cell science (nccs), pune, india, and cells (45 10) were cultured in dulbecco 's modified eagles medium (dmem) supplemented with 10% fetal bovine serum (fbs) in 25 cm tissue culture flasks. the cells were treated with various concentrations of oil (25, 50, 75, 100, and 125 g / ml) for 60 min in a carbon dioxide (co2) incubator. after pretreatment, the cells were exposed to 100 m of h2o2 for 30 min on ice. the cells were harvested, centrifuged for 5 min at 1500 rpm, and resuspended in phosphate - buffered saline (pbs). twenty - five microliter of cell suspension was mixed with 75 l of 0.6% low melting agarose. the suspension was spread on a frosted microscopic slide precoated with 0.8% of normal melting agarose. the cell suspension was covered with a cover slip and kept on ice for 10 min. the coverslips were removed and the slides were incubated overnight in a lysis solution containing 1% sodium dodecyl sulfate, 2.5 m sodium chloride, 100 mm ethylene diamine tetraacetic acid disodium (na2edta), 1% triton x-100, and 10% dimethyl sulfoxide (dmso) at 4c. the slides were arranged in an electrophoresis tank filled with prechilled electrophoretic buffer (1 mm na2edta and 300 mm naoh) and incubated for 20 min. electrophoresis was carried out at 25 v (300 ma) for 20 min using a power supply (cbs scientific company, usa). after electrophoresis, the slides were washed with 0.4 m tris (ph 7.5) and stained with ethidium bromide (etbr) (20 g / ml). the comet tail length was measured using an eyepiece micrometer, and dna damage was calculated as follows : comet tail length (m) = (maximum total length) (head diameter). hela cell line procured from nccs, pune, india, was maintained in a humidified incubator at 37c and in a 5% co2 atmosphere. dmem containing 10% fbs was supplemented with antibiotics penicillin (100 units / ml) and streptomycin (100 g / ml). the assay detects the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (mtt) by mitochondrial dehydrogenase to blue formazan product which reflects the normal functioning of mitochondria and cell viability. briefly, after being harvested from culture flasks, the cells were counted using a hemocytometer and cell viability was determined by trypan blue exclusion. 1 10 cells were incubated in a 96-well plate containing 100 l of the medium per well. cells permitted to adhere for 24 h and then treated with different concentration of essential oil dissolved in the medium for 48 h ; 20 l of 5 mg / ml mtt in pbs was added to each well and the plate was incubated at 37c for 4 h. the medium was removed, and 100 l of dmso was added to each well. after incubation at 37c for 10 min, absorbance at 570 nm of the dissolved solution was measured by a microplate reader (cyberlab). apoptotic cells were identified with an annexin v - fitc apoptosis detection kit (sigma - aldrich, usa). briefly, 1 10 cells / ml was treated with different concentrations of essential oil for 12 h at 37c. cells were stained with 10 l of annexin v - fluorescein isothiocyanate (fitc) and 5 l of propidium iodide (pi) for 15 min at room temperature in the dark and viewed under olympus ckx42 fluorescence microscope and photographed. hoechst 33258 stain (sigma - aldrich, usa) was used to observe the apoptotic morphology of cells. briefly, 1 10 cells / ml were seeded in six - well plates and incubated for 24 h. afterward, the cells were treated with different concentrations of essential oil for 48 h. then, the cells were collected and fixed with 4% formaldehyde in pbs for 15 min and stained with hoechst 33258 (10 mg / ml) at room temperature for 10 min. after washed with pbs, morphological changes including a reduction in volume and nuclear chromatin condensation were observed under olympus ckx42 fluorescence microscope and photographed. morphological analysis of apoptosis by acridine orange / etbr (ao / etbr ; hi - media, india) dual staining was performed. briefly, 2 10 cells per well were seeded in 96-well plate, and treated with different concentrations of essential oil for 48 h. after incubation, the plates were centrifuged. ten microliter of 1 mg / ml ao and etbr mixture was added to each well. nuclei were visualized and photographed under olympus ckx42 fluorescence microscope and photographed. the date obtained from the cytotoxicity study the 3t3-l1 cells procured from the national centre for cell science (nccs), pune, india, and cells (45 10) were cultured in dulbecco 's modified eagles medium (dmem) supplemented with 10% fetal bovine serum (fbs) in 25 cm tissue culture flasks. the cells were treated with various concentrations of oil (25, 50, 75, 100, and 125 g / ml) for 60 min in a carbon dioxide (co2) incubator. after pretreatment, the cells were exposed to 100 m of h2o2 for 30 min on ice. the cells were harvested, centrifuged for 5 min at 1500 rpm, and resuspended in phosphate - buffered saline (pbs). twenty - five microliter of cell suspension was mixed with 75 l of 0.6% low melting agarose. the suspension was spread on a frosted microscopic slide precoated with 0.8% of normal melting agarose. the cell suspension was covered with a cover slip and kept on ice for 10 min. the coverslips were removed and the slides were incubated overnight in a lysis solution containing 1% sodium dodecyl sulfate, 2.5 m sodium chloride, 100 mm ethylene diamine tetraacetic acid disodium (na2edta), 1% triton x-100, and 10% dimethyl sulfoxide (dmso) at 4c. the slides were arranged in an electrophoresis tank filled with prechilled electrophoretic buffer (1 mm na2edta and 300 mm naoh) and incubated for 20 min. electrophoresis was carried out at 25 v (300 ma) for 20 min using a power supply (cbs scientific company, usa). after electrophoresis, the slides were washed with 0.4 m tris (ph 7.5) and stained with ethidium bromide (etbr) (20 g / ml). the comet tail length was measured using an eyepiece micrometer, and dna damage was calculated as follows : comet tail length (m) = (maximum total length) (head diameter). hela cell line procured from nccs, pune, india, was maintained in a humidified incubator at 37c and in a 5% co2 atmosphere. dmem containing 10% fbs was supplemented with antibiotics penicillin (100 units / ml) and streptomycin (100 g / ml). the assay detects the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (mtt) by mitochondrial dehydrogenase to blue formazan product which reflects the normal functioning of mitochondria and cell viability. briefly, after being harvested from culture flasks, the cells were counted using a hemocytometer and cell viability was determined by trypan blue exclusion. 1 10 cells were incubated in a 96-well plate containing 100 l of the medium per well. cells permitted to adhere for 24 h and then treated with different concentration of essential oil dissolved in the medium for 48 h ; 20 l of 5 mg / ml mtt in pbs was added to each well and the plate was incubated at 37c for 4 h. the medium was removed, and 100 l of dmso was added to each well. after incubation at 37c for 10 min, absorbance at 570 nm of the dissolved solution was measured by a microplate reader (cyberlab). apoptotic cells were identified with an annexin v - fitc apoptosis detection kit (sigma - aldrich, usa). briefly, 1 10 cells / ml was treated with different concentrations of essential oil for 12 h at 37c. cells were stained with 10 l of annexin v - fluorescein isothiocyanate (fitc) and 5 l of propidium iodide (pi) for 15 min at room temperature in the dark and viewed under olympus ckx42 fluorescence microscope and photographed. hoechst 33258 stain (sigma - aldrich, usa) was used to observe the apoptotic morphology of cells. briefly, 1 10 cells / ml were seeded in six - well plates and incubated for 24 h. afterward, the cells were treated with different concentrations of essential oil for 48 h. then, the cells were collected and fixed with 4% formaldehyde in pbs for 15 min and stained with hoechst 33258 (10 mg / ml) at room temperature for 10 min. after washed with pbs, morphological changes including a reduction in volume and nuclear chromatin condensation were observed under olympus ckx42 fluorescence microscope and photographed. morphological analysis of apoptosis by acridine orange / etbr (ao / etbr ; hi - media, india) dual staining was performed. briefly, 2 10 cells per well were seeded in 96-well plate, and treated with different concentrations of essential oil for 48 h. after incubation, the plates were centrifuged. ten microliter of 1 mg / ml ao and etbr mixture was added to each well. the date obtained from the cytotoxicity study was analyzed using the spss (16.00, spss inc. h2o2-induced dna damage in 3t3-l1 cells, and different classes of comets were formed, the longer tail length indicates more dna damage [figure 1 ]. of the five different concentrations tested, 125 g / ml oil showed considerable dna protection with reduced comet tail formation [figure 2 ]. h2o2-induced genotoxicity in 3t3-l1 cells shows different classes of comets (a) class 0 and 1, (b) class 2, (c) class 3, (d) class 4 comet tail length expressed in m, (e and f) normal cells dna - protecting activity of atalantia monophylla essential oil against h2o2-induced genotoxicity in 3t3-l1 cells we investigated the effect of a. monophylla essential oil on hela cell proliferation. hela cells were exposed to different doses of oil for 24 h, and cell viability was checked by mtt assay. the concentration required to inhibit 50% of cell growth was calculated as 43.08 0.02 g / ml. to confirm the apoptosis induction of oil in hela cells, annexin v - fitc / pi double staining method was used. after treatment with oil at different concentrations for 12 h, apoptosis was induced in a concentration - dependent manner. the double positive staining of the majority of cells revealed that those cells were at the late apoptotic stage. at the same time, cells were negative to pi indicate that they were at an early stage of apoptosis [figure 3 ]. the ability of oil to induce apoptosis in hela cells, hoechst 33258 staining was used. after 24 h of treatment, bright fluorescence was detected at 2575 g / ml concentration which indicates apoptotic morphology followed by nuclear condensation and apoptotic body formation was observed at 100150 g / ml concentration at 48 h [figure 4 ]. further apoptosis - inducing activity of oil was tested using ao / etbr - staining technique. fluorescence microscopic image clearly showed majority of the oil - treated cells were at early apoptosis with morphological changes such as cell breakage, shrinkage, and membrane blebbing [figure 5 ]. annexin v - fluorescein isothiocyanate / propidium iodide double staining of hela cells after 12 h treatment with different concentrations of atalantia monophylla essential oil. (a - c) early apoptosis (25, 50, and 75 g / ml, respectively), (d - f) more number propidium iodide - stained nuclei indicates cell necrosis (100, 125, and 150 g / ml, respectively) hoechst 33258 staining of different concentrations (25, 50, 75, 100, 125, and 150 g / ml) of atalantia monophylla essential oil - treated hela cells. (a - c) morphological changes (25, 50, 75 g / ml, respectively), (d - f) apoptotic body formation (100, 125, and 150g / ml, respectively) acridine orange / ethidium bromide staining of different concentrations (25, 50, 75, 100, 125, and 150 g / ml) atalantia monophylla essential oil - treated hela cells. (a - f) cells were at early apoptosis stage (25, 50, 75, 100, 125, and 150g / ml, respectively) h2o2-induced dna damage in 3t3-l1 cells, and different classes of comets were formed, the longer tail length indicates more dna damage [figure 1 ]. of the five different concentrations tested, 125 g / ml oil showed considerable dna protection with reduced comet tail formation [figure 2 ]. h2o2-induced genotoxicity in 3t3-l1 cells shows different classes of comets (a) class 0 and 1, (b) class 2, (c) class 3, (d) class 4 comet tail length expressed in m, (e and f) normal cells dna - protecting activity of atalantia monophylla essential oil against h2o2-induced genotoxicity in 3t3-l1 cells hela cells were exposed to different doses of oil for 24 h, and cell viability was checked by mtt assay. the concentration required to inhibit 50% of cell growth was calculated as 43.08 0.02 g / ml. to confirm the apoptosis induction of oil in hela cells, annexin v - fitc / pi double staining method was used. after treatment with oil at different concentrations for 12 h, apoptosis was induced in a concentration - dependent manner. the double positive staining of the majority of cells revealed that those cells were at the late apoptotic stage. at the same time, cells were negative to pi indicate that they were at an early stage of apoptosis [figure 3 ]. the ability of oil to induce apoptosis in hela cells, hoechst 33258 staining was used. after 24 h of treatment, bright fluorescence was detected at 2575 g / ml concentration which indicates apoptotic morphology followed by nuclear condensation and apoptotic body formation was observed at 100150 g / ml concentration at 48 h [figure 4 ]. further apoptosis - inducing activity of oil was tested using ao / etbr - staining technique. fluorescence microscopic image clearly showed majority of the oil - treated cells were at early apoptosis with morphological changes such as cell breakage, shrinkage, and membrane blebbing [figure 5 ]. annexin v - fluorescein isothiocyanate / propidium iodide double staining of hela cells after 12 h treatment with different concentrations of atalantia monophylla essential oil. (a - c) early apoptosis (25, 50, and 75 g / ml, respectively), (d - f) more number propidium iodide - stained nuclei indicates cell necrosis (100, 125, and 150 g / ml, respectively) hoechst 33258 staining of different concentrations (25, 50, 75, 100, 125, and 150 g / ml) of atalantia monophylla essential oil - treated hela cells. (a - c) morphological changes (25, 50, 75 g / ml, respectively), (d - f) apoptotic body formation (100, 125, and 150g / ml, respectively) acridine orange / ethidium bromide staining of different concentrations (25, 50, 75, 100, 125, and 150 g / ml) atalantia monophylla essential oil - treated hela cells. (a - f) cells were at early apoptosis stage (25, 50, 75, 100, 125, and 150g / ml, respectively) plant products are well known for the treatment of cancer and other disease since long ago. many plant species have been reported to have significant antimicrobial, antioxidant, and anticancer properties. phytochemicals have emerged as drugs upon modification, and many are available in the form of crude, and more are yet to reach the market. essential oil is one among the known secondary metabolites with immense commercial and medicinal values. essential oils are complex mixtures of volatile substances mainly contains mono-, sesqui-, diterpenes, and phenylpropanoids. numerous reports are available on chemical composition, biological activities, and possible applications of essential oils in food, pharmaceutical, and cosmetic industries. excess ros formation results in oxidative stress and tissue injury. using water channels, h2o2 cross cell membrane, reach nucleus, attack dna, and single strand breaks occur. in the present study, we treated 3t3-l1 cells with 100 m of h2o2 for 30 dna protecting the activity of oil may be associated with scavenging of h2o2-induced hydroxyl radical formation through fenton reaction. we assumed that observed antigenotoxic activity of a. monophylla essential oil might be due to the presence of active constituents such as sabinene, trans - asarone, and myrcene. apoptosis regulators have been suitable targets for cancer therapy over several decades. however, chemotherapeutic agents have many undesirable effects on patients. last one - decade research in apoptosis induction has been performed with plants, and their products are gaining much importance than chemopreventive agents with little side effects. a significant cytotoxic effect and apoptosis were noted in hela cells following treatment with different concentrations of a. monophylla essential oil. anticancer activity of each compound present in the oil was not evaluated against hela cell line ; therefore, at this stage, it is not possible to say which compound is responsible for the observed activity. it is possible to hypothesize that observed cytotoxic and apoptotic activity of oil could be related to combined action of compounds present. although synergism is a reason, it is unavoidable to discuss about the cytotoxic and apoptotic activities of major components of the oil. this work was financially supported by the department of science and technology - science and engineering research board, new delhi, india, under grant no. this work was financially supported by the department of science and technology - science and engineering research board, new delhi, india, under grant no. | objective : to study antigenotoxic and apoptotic activities of hydrodistilled essential oil from the leaves of atalantia monophylla correa.materials and methods : antigenotoxic activity of essential oil was tested against hydrogen peroxide (100 m)-induced deoxyribonucleic acid (dna) damage in 3t3-l1 cells. cervical cancer cell (hela) growth inhibitory effect of essential oil was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. annexin v - fluorescein isothiocyanate / propidium iodide (fitc / pi), hoechst 33258, and acridine orange / ethidium bromide (ao / etbr) staining techniques were used to identify apoptosis.results:dna protecting the activity of a. monophylla essential oil was high at 125 g / ml. hela cell growth was inhibited dose - dependently and inhibitory concentration 50% was calculated as 43.08 0.02 g / ml. annexin v - fitc / pi double staining showed membrane breakage and nuclei staining. further, hoechst 33258 and ao / etbr stain also confirmed the apoptosis in essential oil - treated hela cells.conclusion:the results obtained suggest that a. monophylla essential oil is a promising natural agent which may be used in preparation of herbal medicine to treat cancer and other diseases. |
the definitive hosts are canines ; intermediate hosts are sheep, goats, cattle and humans. 70% of the larvae are trapped in the liver and those which pass through the hepatic filter disseminate to the lungs, heart and other organs. if the larvae pass through the first filter, they reach the lungs which are the second most frequently involved site (10 - 25%). there are preliminary data on the possibility of dissemination through lymphatic channels. in this paper, we present a case in which a patient had a hydatid cyst at an uncommon site. a 54-year - old female farm worker came with chief complaints of a painless mass [figure 1 ] in left iliac fossa gradually increasing in size over 6 months. a mass of 16 8 cm was noted in suprapubic region and left iliac fossa, it was diagonally placed in lower abdomen. the swelling was smooth, bi - lobed with well defined margins, no change in dimensions on lying down position. it was cystic in consistency, not reducible, nor compressible, not freely mobile, fixed to pubic crest and was situated in parietal plane. chest x - ray was also normal. figure 1abdominal wall hydatid abdominal wall hydatid surgery was planned. on exploration, a smooth cystic swelling was present in between left internal oblique and transversusabdominis muscle, adhered to pubic bone inferiorly and peritoneum posteriorly by separating transversusabdominis muscle [figure 2 ]. the swelling was dissected carefully, adhered portion of transversusabdominis muscle and peritoneum was sacrificed. defect in abdominal wall was repaired by hernioplasty using polypropylene mesh [figure 3 ] ; suction drain was kept in situ and the specimen [figure 4 ] was sent for histopathology for the diagnosis of hydatid cyst and it was confirmed. parenteral antibiotics were given for 5 days ; the drain was removed on 3 day. the patient was adviced albendazole 400 mg bd for a period of one month and discharged after 8 days. hydatid disease is endemic in cattle rearing regions, such as central europe, mediterranean, middle east, south america, australia, new zealand and south africa. lactic acid produced by the underlying muscles may assist in hatching of the ova, allowing the parasite embryos to form hydatid cysts. clinically, a hydatid cyst in the soft tissues might mimic teratomas, abscesses, or fibromatosis. ultrasound is an important imaging modality for hydatid disease and may clearly demonstrate the floating membranes and daughter cysts. preoperative diagnosis of subcutaneous hydatid disease is also possible by fnac.[1246 ] serology and radiological imaging establish the diagnosis in most of the cases. serological test consists of immuno - electrophoresis, immuno - hemagglutination test and complement fixation test. arc 5 immunoelectrophoresis is confirmatory test, it detects antibodies against immune - dominant and specific antigen, antigen 5 of the cestode. for detecting the particular species of echinococcus a polymerase chain reaction (pcr) ct scan is better than mri. on changing thepatient 's posture under real time scanner, falling snowflake pattern of hydatid sand the ova are ingested by the intermediate hosts, herbivores and humans. the ova hatch in the small intestine, and they are trapped in the sinusoids ; therefore, the liver is the most frequently involved organ (70%). the larvae which pass through this first filter, reach the lung via the right heart. the interesting aspect of cases with solitary cysts in uncommon sites is the absence of disease in the liver and lungs. it is very possible that systemic dissemination via the lymphatic route accounts for cases with solitary cysts in uncommon sites. however, a review of the english medical literature also revealed cases involving the muscles of the chest wall, sartorius, biceps brachii, supraspinatus and gluteus. the clinical manifestation of the disease is formed by localization and pressure effect of the slowly growing cyst in the infected organ. hydatid disease should be considered in the differential diagnosis of all cystic masses in all anatomic locations especially in endemic areas. en - bloc resection alone is curative for isolated muscular hydatid cysts but postoperative adjuvant therapy with albendazole is given. we did not used scolicidal agent for disinfecting the wound as there was no evidence or previous history of incisional biopsy of the lesion. the patient has to be kept on regular follow up paying attention to the possibility of complications. in conclusion, subcutaneous hydatid cyst is rare and should be kept in differential diagnosis of a cystic lesion especially in regions where hydatid disease is endemic. it is better not to biopsy the lesion if one suspects that it could be a hydatid cyst and should be excised totally to avoid recurrence. care should be taken at the time of operation of cystic lesion situated anywhere in the body, to avoid accidental incision over the cyst, as there are chances of leak, anaphylactic shock, re - infection and dissemination of hydatid cyst. if it is proven radiologically it should be resected enbloc without traumatizing the cyst wall and postoperative albendazole should be given to prevent recurrence and further complications. | hydatid cyst is the disease of liver and lungs and is common in some regions especially sheep rearing countries of the world, but this disease may occur in any part of world and anywhere in the body. this report presents primary hydatid cysts located in intramuscular region of left side of the abdomen. a 54-year - old female patient from central india, farmer by occupation, non vegetarian by diet came with chief complaints of a painless mass in the left iliac fossa, gradually increasing in size over a period of 6 months. superficial ultrasound revealed a lesion resembling a hydatid cyst. surgical excision was done without injuring the cyst. diagnosis was confirmed on histo - pathological examination and was compatible with a hydatidcyst. ahydatid cyst should be considered in the differential diagnosis of subcutaneouscystic lesions, it should be excised totally, with an intact wall and postoperative albendazole should be given to avoid recurrence. |
blood samples were obtained from volunteers of horizonte project (pbh) in belo horizonte, between 1996-june 2012. this project was an open cohort of hiv - negative homosexual and bisexual men supported by the brazilian ministry of health and it is one of the components of the minas gerais hiv - aids vaccine centre of the federal university of minas gerais (ufmg) in brazil (carneiro. 2000). the project objectives included the evaluation of the role of counselling and condom distribution in the incidence of hiv infection in this population. every six months, the volunteers were examined, received counselling and preservatives and underwent serologic tests for hiv (elisa) and other sexually transmitted diseases, such as syphilis and viral hepatitis. the eligibility criteria for enrolment included being homosexual or bisexual, 18 years or older, consenting to hiv testing and testing negative for hiv-1. all the available hiv+ samples collected after enrolment in pbh were genotyped. from the beginning of the project in 1994 until june 2012, 1,328 subjects were followed and 119 were infected with hiv-1 during this period. genotyping was carried out on 64 samples of seroconverters between 1996-june 2012. in the remaining 55 individuals, genotyping was not possible either because the samples were not available or the pol gene was not amplified with the available kits. the elapsed time was the time between the last negative hiv-1 test and the first positive hiv-1 test. we considered an infection to be a recent hiv-1 infection when the elapsed time between the last negative hiv test and the first positive test was less than 12 months. the viroseq genotyping system version 2.0 (celera diagnostics, alameda, california, usa) was used for genotypic analysis until 2007 and the trugene hiv-1 genotyping assay (siemens diagnostics, usa) was subsequently used. all sequences obtained were subjected to quality control assessments using the blast - renageno resistance analysis program (aids.gov.br/renageno) to exclude sample mix - up or contamination. drug resistance was analysed using 297 nucleotides of protease (pr) (positions 1 - 99) and the first 1,003 nucleotides of reverse - transcriptase (rt) (positions 1 - 335). the amino acid sequences of the rt and pr genes were deduced from the nucleic acid sequences and compared to a subtype b consensus sequence from the stanford hivdb program genotypic resistance interpretation algorithm version 6.2.0 (hivdb.stanford.edu/) rt and the pr sequence database. in this study, the world health organization sdrm criteria (bennett. 2009) were used to determine the significant mutations for tdr calculation. tests for mutations associated with at least two classes of drugs were considered in multi - drug - resistant (mdr) samples. for all the individuals included, the initial viral load (vl) and cd4 t lymphocyte (cd4) counts were obtained from control system laboratory tests of the national lymphocyte count cd4 /cd8 and viral load (siscel), a brazilian aids program database, or from the patient files. all the seroconverters are being followed at the ufmg hiv / aids outpatient clinic training and reference center in infectious and parasitic diseases. the data were analysed using stata for windows (version 11.2, statacorp, college station, tx, usa). the chi - square and mann - whitney u tests were used as appropriate. this study was conducted in accordance with the brazilian research ethics guidelines and approved by the ethical review committee (ufmg). thirty - five patients (54.7%) had documented recent infections (< 12 months). the overall tdr prevalence was 14.1% (9 patients) according to the sdrm criteria (bennett. three (4.7%), five (7.8%) and four (6.2%) patients presented pi, nrti and nnrti resistance mutations, respectively, as shown in table ii. among the patients with tdr, four (44.4%) had single resistance mutations and two (22.2%) had mdr hiv-1. patients with and without tdr had similar cd4 cell counts (median, 531 and 476 cell / mm, respectively, p = 0.355, mann - whitney u test) and vl levels (17,746 and 14,250 copies / ml, respectively, p = 0.952, mann - whitney u test). 2009total [n (%) ] 64 (100)9 (14.1)male [n (%) ] 64 (100)9 (14.1)age (years)mean30.630.1range19 - 5422 - 42hiv subtype [n (%) ] b44 (68.8)9f11 (17.2)-c4 (6.3)-bc, bf, fb5 (7.8)-elapsed time (months) mean14.529.4range3 - 746 - 36laboratory variables - vl (copies / ml) median17.74614.250range590 - 610.000700 - 610.000cd4 count (cells / ml) median531476range110 - 1.440394 - 1.364 a : protease and reverse transcriptase subtypes / number of samples (bc/1, bf/3 and fb/1) ; b : from last negative human immunodeficiency virus (hiv) elisa test and the first positive test. first positive hiv elisa test sample was used for drug - resistance testing ; c : for six patients there was no information on viral load (vl) and cd4 t lymphocytes count ; srdm : surveillance drug resistance mutations. a : protease and reverse transcriptase subtypes / number of samples (bc/1, bf/3 and fb/1) ; b : from last negative human immunodeficiency virus (hiv) elisa test and the first positive test. first positive hiv elisa test sample was used for drug - resistance testing ; c : for six patients there was no information on viral load (vl) and cd4 t lymphocytes count ; srdm : surveillance drug resistance mutations. table iihorizonte project, open cohort of human immunodeficiency virus negative males having sex with males in belo horizonte, brazil between 1996-june 2012reverse transcriptaseproteasepatientelapsed time (months)age (years)cd4/vlseroconversion datenrtinnrtipisubtype stanford arv susceptibility renageno arv susceptibility 1632665/610,000november 9670r, 77l -- bazt, d4t, tdf = iazt = i22223688/700august 98 - -90mbs to all drugss to all drugs37341364/ -september 200241l, 184v,210w, 215y188l - br to all nrtiefv, nvp = retv = sr to all nrtiefv = retv = s4628- / -august 200241l, 184v,210w, 215y188l90mbr to all nrtiefv, nvp = retv = rr to all nrtiefv = retv = s5822462/201,309october 200370e -- babc, ddi = i, tdf = iddi = i, tdf = r6623458/78,460october 2005 - -73abs to al drugss to all drugs7628476/4,744september 201041l, 67n,69d, 210w, 215d -- b3tc = sabc, tdf = iazt, d4 t, ddi = r3tc = iazt, d4 t, ddi, tdf = r83633394/14,250september 2010 - 103n - betv = sefv, nvp = refv, nvp = r9642732/8,238august 2011 - 103n - betv = sefv, nvp = refv, nvp = r a : susceptibility was predicted using the stanford hivdb program genotypic resistance interpretation algorithm version 6.2.0 (last updated 29 sep 2012) (hivdb.stanford.edu/) ; b : susceptibility was predicted using the renageno resistance interpretation algorithm version 12 (05 - 2012) (algoritmo.aids.gov.br/) ; arv : antiretroviral therapy ; azt : zidovudine ; d4 t : stavudine ; ddi : didanosine ; efv : efavirenz ; etv : etravirine ; i : intermediate ; nnrti : resistant against non - nucleoside reverse - transcriptase inhibitors ; nrti : resistant against nucleos(t)ide transcriptase inhibitors ; nvp : nevirapine ; pi : protease inhibitor ; r : resistant ; s : susceptible ; tdf : tenofovir ; vl : viral load ; 3tc : lamivudine. a : susceptibility was predicted using the stanford hivdb program genotypic resistance interpretation algorithm version 6.2.0 (last updated 29 sep 2012) (hivdb.stanford.edu/) ; b : susceptibility was predicted using the renageno resistance interpretation algorithm version 12 (05 - 2012) (algoritmo.aids.gov.br/) ; arv : antiretroviral therapy ; azt : zidovudine ; d4 t : stavudine ; ddi : didanosine ; efv : efavirenz ; etv : etravirine ; i : intermediate ; nnrti : resistant against non - nucleoside reverse - transcriptase inhibitors ; nrti : resistant against nucleos(t)ide transcriptase inhibitors ; nvp : nevirapine ; pi : protease inhibitor ; r : resistant ; s : susceptible ; tdf : tenofovir ; vl : viral load ; 3tc : lamivudine. subtype b was the most prevalent (44, 68.8%), followed by f (11, 17.2%) and c (4, 6.3%). tdr was only found in subtype b, with statistically significant differences between the b and non - b subtypes and the presence of sdrm (p = 0.029, chi - square test). this study presents data collected from msm in belo horizonte, over a broad timespan (1996 - 2012) that encompasses different eras of hiv treatment. the tdr rate of 14.1% was moderate, but higher than in other studies in the general global population and in brazil. (2003) found tdr in 7% of chronically infected patients (2.2%, 2.4% and 2.1% prevalence of resistance against pis, nrtis and nnrtis, respectively), while inocncio. (2009) reported tdr in 8.1% of recently diagnosed individuals from six brazilian capital cities among 210 samples. there were intermediate levels of tdr (5 - 15%) in the cities of belm, braslia, so paulo and rio de janeiro and the last two cities are in the southeast region of brazil, which is relatively close to belo horizonte (inocncio. patients have been more exposed to art, which is a plausible explanation for the higher rates of tdr. our results do not differ from the overall national data (inocncio. 2009). studies with msm have shown moderate rates of tdr with an apparently stable prevalence (truong. (2012) showed a tdr prevalence of 14.3% in a predominantly msm cohort with recent / acute hiv infection from 1995 - 2010 in new york city. a swedish study performed in nine cities between 2003 - 2010 showed a 9.5% tdr prevalence (karlsson. a recent study revealed a high tdr level (21.4%) in the city of so paulo in community - recruited samples from msm (bermdez - aza. according to the brazilian genotyping interpretation system renageno version 05 - 2012 (algoritmo.aids.gov.br) and the stanford hivdb program, seven of the patients in our study would be predicted to have some degree of resistance to the initial art regimen commonly used in brazil (table ii), which consists of two nrtis (azt + 3tc or tdf + 3tc) and one nnrti (efv or nvp). all three patients with single pi - related mutations were predicted to be fully susceptible to all the pis. interestingly, there were no pi - related mutations after 2005, most likely because ritonavir boosted the potency of this class. this is in agreement with tdr incidence studies that show a tendency of stabilisation or even reduction of resistance in this class (vercauteren. our sample has limitations that may have affected the rate of tdr prevalence in this population. the major problem was the loss of 55 samples during the study ; in some cases, the pol gene was not amplified with the available kits and in other cases, samples were not available after seroconversion. another problem was that some of the patients had up to 74 months between the last hiv - negative elisa and the first positive test and we could not determine how long they had been infected. subsequently, more than half of the patients were found to be recently infected, that is, less than 12 months). studies have shown that resistance mutations persist in the absence of art, which may have been the case for those with a long elapsed time (brenner. another limitation is the relatively small sample size, which may not accurately reflect the newly hiv - infected patients in the region. however, in belo horizonte, the hiv epidemic is still more prevalent among msm. we chose the sdrms as our standard, which may have limited direct comparisons to other studies that utilised the ias list of drug resistance mutations (ias - drms). some potentially relevant mutations are absent in the sdrm, but green. (2008) showed that compared to sdrms as the reference standard, the overall sensitivity of the ias - drms was inferior for detecting tdr. in fact, when we applied the main ias - drms in our study, there was no significant difference in the tdr rate (data not shown). viral subtype b was the most prevalent in this study, as has been observed in most regions of brazil except the southern region, where subtype c is more common (soares. tdr was present only among subtype b patients, with a statistically significant difference between b and non - b subtypes and the presence of sdrm (p = 0.029, chi - square test). it is worth noting that subtype c was present in 6.3% (4/64) of patients, which indicated that this subtype is spreading throughout the southeast (soares. there were 11 patients with subtype f and an increase in its prevalence has already been reported in the northeast region of brazil ; cavalcanti. (2012) showed that 37.7% of patients had subtype f in metropolitan city of recife. (2005) showed that genotypic testing is cost - effective for art - nave patients when the tdr prevalence is 1%. in our study, there were no statistically significant differences related to the cd4 count and vl with or without resistance mutations at the time of infection. however, we did not evaluate the clinical data from patients with sdrms to verify how tdr may have affected the clinical outcome. the impact of tdr on surrogate markers of clinical progression (cd4 count and vl) remains uncertain. the eurocoord - chain project evaluated the effect of tdr on first - line art. they estimated a virological failure of 4.7% at 12 months for those with tdr and fully active art and 15.1% for those with tdr and resistance to at least one prescribed drug (wittkop.. a slower rate of cd4 decline has been reported in patients who present tdr mutations (bhaskaran. 2004), while others have not found a favourable effect on vl and cd4 counts in the presence of tdr mutations (chan., this study shows that the tdr prevalence among recently infected msm in belo horizonte from 1996 - 2012 remained moderate and comparable with other similar studies. while the prevalence remained moderate in belo horizonte in this specific population, the profile of the transmitted mutations in the overall population may be different, underscoring the need for continued and improved measures for targeted interventions. thus, tdr surveillance studies must be maintained over time, as recommended by who (who 2012), with additional national surveillance studies to identify the true rate of tdr and evaluate if genotyping tests should be performed prior to the beginning of art in this specific population. | the presence of transmitted human immunodeficiency virus (hiv)-1 drug - resistance (tdr) at the time of antiretroviral therapy initiation is associated with failure to achieve viral load (vl) suppression. here, we report tdr surveillance in a specific population of men who have sex with men (msm) in belo horizonte, brazil. in this study, the rate of tdr was evaluated in 64 hiv - infected individuals from a cohort of msm between 1996-june 2012. fifty - four percent had a documented recent hiv infection, with a seroconversion time of less than 12 months. the median cd4+t lymphocyte count and vl were 531 cells / mm3and 17,746 copies / ml, respectively. considering the surveillance drug resistance mutation criteria, nine (14.1%) patients presented tdr, of which three (4.7%), five (7.8%) and four (6.2%) had protease inhibitors, resistant against nucleos(t)ide transcriptase inhibitors and against non - nucleoside reverse - transcriptase inhibitors mutations, respectively. two of the patients had multi - drug - resistant hiv-1. the most prevalent viral subtype was b (44, 68.8%), followed by subtype f (11, 17.2%). this study shows that tdr may vary according to the population studied and it may be higher in clusters of msm. |
the role of epigenetic dysregulation in cancer has motivated interest in dna methylation and methods for its modulation. in mammals, dna methylation occurs in the major groove of dna at the 5 position of both cytosine residues in the palindromic cg dyad (cpg). cpgs are rare in the genome and 70% methylated, with nearly all unmethylated cpgs clustered in g, c - rich regions called cpg islands. approximately 60% of rna polymerase ii transcribed human genes contain cpg islands, and their methylation causes transcriptional repression. in cancer, for example, otherwise functional tumor suppressor genes can be silenced by hypermethylation in their associated cpg island. importantly, inhibition of dna methylation at tumor suppressor genes has been shown to reactivate apoptotic pathways and sensitize cancer cells to previously ineffective chemotherapy. the most effective demethylation agents are cytidine analogues such as 5-aza - deoxycytidine which find limited use due to significant side effects. these cytidine analogues are suicide inhibitors incorporated into dna to form covalent methyltransferase - dna adducts. the methyltransferase is sequestered and unavailable to methylate cpgs resulting in genome - wide demethylation. dna binding molecules, such as the bis - intercalating natural product echinomycin, can disrupt cpg methylation in vitro but have dose - limiting toxicities that have abrogated further clinical advancement. while other cpg methylation inhibitors are under investigation, none of these agents have demonstrated the ability to inhibit dna methylation in a sequence - specific fashion. hairpin pyrrole - imidazole (py - im) polyamides are a class of sequence - specific oligomers that bind in the minor groove of dna. programmable sequence preference is accomplished by side - by - side pairings of aromatic amino acids that distinguish the edges of the four watson crick base pairs. referred to as the pairing rules, im / py codes for gc base pair, hp / py codes for ta base pairs, and py / py binds both ta / at in preference to gc / cg. eight - ring hairpin oligomers linked by a central aliphatic -aminobutyric acid unit have affinities for match sites with ka 10 to 10 m. these binding energetics are comparable to natural transcription factors and, like natural dna binding proteins, are sensitive to differences in the sequence - dependent microstructure of dna. to relax the curvature of all ring hairpins, alanine () can be substituted for py - rings in some cases such that / pairs replace py / py for ta / at specificity, and im/ replaces im / py pairs in strategic locations while retaining specificity for gc base pair. hairpin py - im polyamides usually bind with the n - to - c terminus aligned in the 5-to-3 direction of dna, referred to as forward orientation. this modest forward binding preference can be enforced by substitution of the prochiral position in the -turn, i.e., replacement of -aminobutyric acid by (r)-2,4-diaminobutyric acid. hairpin architectures containing / pairs and /ring pairs have been found in some cases to prefer the n to c terminus aligned in a 3-to-5 direction of dna. while adhering to the pairing rules, this reverse hairpin orientation would bind a different dna sequence. recently, we used massively parallel sequencing methods in conjunction with biotin - tagged hairpins, termed bind - n - seq, to scan genome - size dna sequence space for hairpin high affinity sites. although the canonical pairing rules are remarkably predictive of polyamide dna binding specificity, we identified high affinity dna binding sites in the reverse orientation for several polyamides containing /im pairs. structure of py - im polyamide s2 previously reported to bind methylated 5-cgcg-3 oligonucleotide duplex. eight - ring hairpin py - im polyamides have been shown to discriminate 5-gggg-3, 5-gcgc-3, and 5-ggcc-3 with appropriate arrangement of four im / py pairs. from experience, sequences with cpg steps such as 5-cgcg-3 are not as readily accessed for reasons not well understood. in an effort to improve the affinity of an eight - ring hairpin polyamide for the sequence 5-cgcg-3, sugiyama and co - workers replaced two im / py pairs with im/ pairs. a change from pyimpyim--pyimpyim (s1) to pyimim--pyimim (s2) afforded a 65-fold increase in affinity for 5-cgcg-3. both hairpins conform to the pairing rules and would bind 5-cgcg-3 in the forward orientation. in this study, we employ a high - throughput sequencing assay of polyamide - dna association to revisit targeting the 5-cgcg-3 sequence. our findings indicate that hairpin polyamides of sequence pyimim--pyimim s2 favor 5-gcgc-3, a reverse binding mode. the issue of designing a hairpin polyamide sequence that prefers 5-cgcg-3 to 5-gcgc-3 remains to be solved. using bind - n - seq methods as our screen for a library of polyamide biotin conjugates, we find that replacement of one alanine with py to afford pyimpyim--pyimim restores the preference for forward binding 5-cgcg-3. recent structural work has shown that a cyclic py - im polyamide binding in the minor groove causes significant widening of the minor groove width of dna, and provides a mechanistic rationale for disruption of dna - binding proteins in the major groove. we demonstrate the ability of our 5-cgcg-3 specific minor groove binding hairpin polyamides to inhibit enzymatic cpg methylation in the major groove of a 5-cgcg-3 sequence. biotin conjugate is incubated in a genome - sized library of all possible 21mers, enriched, sequenced, and the resulting data set analyzed with motif - finding software. (b) polyamide 1 could potentially bind in the forward orientation or the reverse orientation. the 5-cgcg-3 sequence is a compelling dna target for an 8-ring hairpin py - im polyamide because it is one of the least represented 6-bp sequence patterns in the human genome, potentially promoting greater genomic specificity. minoshima and co - workers have previously targeted this sequence and shown that polyamide s2 (figure 1) can bind the fully methylated sequence. in their study, the substitution of two s for py moieties resulted in improved affinity for 5-cgcg-3 over the eight - aromatic ring architecture s1 (si figure s1). in light of recent bind - n - seq studies, however, we wondered whether these changes may have also had the unintended effect of reducing the preference of the polyamide for binding in the forward orientation. bind - n - seq is a high - throughput sequencing method that allows facile identification of high affinity binding sites of biotin - labeled py - im polyamides by affinity purification followed by sequencing (figure 2a). as a first step, biotin conjugate 1 of sequence pyimim--pyimim (figure 2b), which has a biotin affinity tag appended at the c - terminus of the heterocyclic oligomer. polyamide biotin conjugate 1 was incubated at 50 nm in a library of all possible 21 base pair dna sequences, enriched, and sequenced to identify polyamide - bound sequences. this data set was then analyzed by the dreme algorithm to construct a motif logo summarizing the highest affinity sequences. a binding preference for 5-gcgc-3 was revealed, suggestive of a reverse binding mode (table 1). (a) scheme of py - im polyamide binding in the minor groove of dna. (b) single position changes made to hairpin polyamide 1 to afford 2 and 3. positions are highlighted in yellow. in order to restore the preference for binding 5-cgcg-3 in the forward orientation, we considered two possible points of modification (figure 3a). first, we made a single modification to 1 at the turn unit, replacing the gaba turn to a chiral -amino gaba, affording 2 (figure 3b). the -amino gaba turn has previously been shown to restore forward orientation and increase affinity, including in -containing polyamides. this effect is thought to arise from a steric interaction with the floor of the minor groove when the chiral -amino gaba turn unit is bound in the reverse orientation. assessment of polyamide 2 by bind - n - seq found that this modification improved the reverse / forward ratio but was insufficient to restore a preferred forward orientation binding preference (table 1). to confirm the high - throughput sequencing findings, we performed a thermal dna denaturation study, as previous studies have shown that thermal stabilization (tm) of duplex dna by py - im polyamides correlates well with binding affinity. assays were performed with dna oligonucleotides differing only in the central binding sequence (5-cgcg-3 versus 5-gcgc-3) to directly test the binding orientations identified by the bind - n - seq logos (table 1). this analysis substantiated a reverse orientation binding preference for polyamide 1, with a tm of 10.9 c in the reverse direction as compared to 3.4 c in the forward direction. modification at the turn to the -amino gaba in polyamide 2 resulted in increased stabilization of the forward 5-cgcg-3 oligomer by 4.5 c ; stabilization by polyamide 2 in the reverse 5-gcgc-3 orientation was diminished by 2.0 c. this indicated an improved forward preference for 5-cgcg-3. nonetheless, the relative magnitudes of the tm support an overall modest energetic preference for reverse orientation binding. panel of polyamides synthesized for assessment by bind - n - seq and dna thermal stabilization for binding the 5-cgcg-3 sequence. according to the pairing rules, polyamides 14 target 5-cgcg-3 in the forward orientation and polyamides 58 target 5-cgcg-3 in the reverse orientation. the inability of the -amino gaba turn to enforce forward orientation binding led us to investigate alternative solutions for the molecular recognition of 5-cgcg-3 (figure 3). we posited that reverse binding is abetted by the flexibility afforded by the two units in the core binding region, as had been similarly noted in polyamides containing a / pair. we thus considered whether removing one residue might reinstitute sufficient rigidity in one of the polyamide strands to limit reverse binding while retaining the specificity and affinity provided by the other. of the two moieties in the core of polyamide 1, the c - terminal in the core binding region was retained based on previous studies that have shown it is necessary for high affinity recognition of the 5 cg base pair. to isolate the effect of each modification, we returned to parent polyamide 1 and replaced the n - terminal with a py while retaining the achiral gaba turn, to provide polyamide 3 (figure 3b). the assessment of 3 by bind - n - seq followed by dreme analysis generated a high affinity motif consistent with forward binding 5-cgcg-3 (table 1). this was corroborated by tm measurements showing considerable preference for the forward 5-cgcg-3 direction. polyamide - mediated thermal stabilization (tm) of 12 base pair oligonucleotides of the forward (5-cgcg-3) and reverse (5-gcgc-3) sequences were used to validate the revealed motifs. we further examined whether a hairpin polyamide designed to target a reverse orientation sequence may productively bind cpgs with high specificity. to test this, we expanded the library of compounds to include polyamides 48, single modifications targeting the 5-gcgc / cgcg-3 core (figure 4). in contrast to our findings with 5-cgcg-3 targeting polyamide 2, we confirmed that the incorporation of an -amino gaba turn in polyamide 6 restores forward orientation binding for the 5-gcgc-3 sequence. this difference is striking given that the two polyamides are composed of nearly identical amino acid sequences. bind - n - seq data and tm assays of polyamides 4, 5, 7, and 8 together suggest that all other modifications preferentially bind the reverse orientation, and 5, 7, and 8 do so with poor specificity (table 1). indeed, among all variations tested of both 5-cgcg-3 forward binding and 5-gcgc-3 reverse binding cores, polyamide 3 displayed the highest specificity for the 5-cgcg-3 sequence (table 1). next, we considered the potential for minor groove binding hairpin py - im polyamides to prevent dna methylation undergoing dna replication. to do so, they must be able to bind the hemi - methylated dna of daughter strands that have not yet undergone maintenance methylation. dna thermal stabilization analysis was used to pursue evidence of the above trends of binding orientation with hemi - methylated dna sequences. the sense strands of each of the 12 base pair oligomers containing 5-cgcg-3 or 5-gcgc-3 cores were methylated on both cytosines, whereas the antisense strands were left unmethylated. flanking sequences were modified to lack self - complementarity and enforce hemi - methylated duplex formation. analysis of tm of the hemi - methylated dna oligomers confirmed the above magnitudes of stabilization and trends of reverse and forward binding modes for 1, 2, and 3 (table 2a). with a specific polyamide capable of binding hemi - methylated dna in hand, we evaluated its application as a sequence - specific inhibitor of dna methyltransferases. the biotin enrichment tag was deleted from the c - terminus by resynthesis to afford parent hairpins 1b and 3b. melting temperature analyses confirmed that these molecules show comparable binding preference to biotin conjugates 1 and 3, respectively (table 2b). we developed an in vitro assay to probe the methylation state of specific sites employing the methylation - sensitive restriction enzyme mlui to compare sequence specific effects of 1b, 3b, and at - binding distamycin d as a control (si figure s2a). in this assay, we measured the ability of these compounds to inhibit the methylation activity of m.sssi, a robust prokaryotic methyltransferase that operates in a processive manner like human methyltransferases and shares structural similarities with the catalytic core of human dnmt1. we employed the methylation - sensitive enzyme mlui, which cleaves at seven 5-acgcgt-3 sites, to interrogate methylation of the -phage dna (48.5 kb), of which 5 bands were visualized by agarose gel electrophoresis. both 1b and 3b were titrated from increasing concentrations 1 nm to 1 m, while d was dosed 10-fold higher from 10 nm to 10 m. full digestion of the dna by mlui indicates a lack of cpg methylation at 5-acgcgt-3 restriction sites, and is demonstrated by positive control lane 2 (si figure s2b). in contrast, full methylation would protect dna from mlui digestion, as in lane 1 where no compound was added to dna prior to exposure to m.sssi for methylation. (a) scheme of in vitro dna methyltransferase (dnmt) inhibition assay. generic polyamide shown in ball - and - stick notation and cpg sites represented by red squares. dna (7.5 kb) with a single mlui restriction site was incubated at 50 pm with inhibitor and subjected to methylation by m.sssi. dna was isolated for restriction digest by mlui to reveal methylation at the target site. (b) single changes in polyamides 2 and 3 that promote forward orientation binding were combined in designing 9b as a third generation candidate for improved methylation inhibition. (c) representative gel image of the polyamides 1b, 3b, and 9b in the assay described in (a), suggestive of differential inhibitory activity. values are determined from band intensities in the in vitro as assay shown in (c) and normalized against maximal methylation with no inhibitor. ic50 values were calculated from at least three replicates and fit to a four - variable, dose response model. consistent with our biophysical characterization of the compounds, polyamide 3b showed the most robust inhibition of cpg methylation (si figure s2b, lanes 710) at 5-acgcgt-3 sites. in lane 10, full mlui digestion comparable to positive control lane 2 was observed at 1 m of 3b, indicating this concentration was sufficient to block all methylation at the cognate binding sites. further, incomplete protection was evidenced at 100 nm of 3b by additional, partially digested bands in lane 9. in contrast, polyamide 1b showed weak inhibition of m.sssi and was active only at the highest concentration (si figure s2b, lanes 36). this reflects its weaker affinity for the 5-cgcg-3 forward binding orientation, also observed by thermal duplex denaturation analysis. inhibition by 1b at 1 m, however, is reduced relative to that observed at 100 nm of 3b, consistent with the binding preferences of the two molecules. there was no inhibition by distamycin d at all concentrations tested, even at the highest concentration of 10 m, underscoring the importance of cpg specificity of py - im polyamides in preventing cpg methylation. to enable quantitation of enzyme activity inhibition, the substrate dna was changed to a 7.5 kb fragment containing a single 5-acgcgt-3 site (figure 5a). we were encouraged by these results to consider the design of an improved methylation antagonist at 5-cgcg-3. we revisited the single modifications to 1 in polyamide 2 and 3 that had promoted forward orientation binding. we combined the -amino modification at the gaba turn that had encouraged 2 to bind in the forward orientation, albeit insufficiently, with the py substitution in the top strand, as in 3, to afford 9b (figure 5b). analysis by thermal denaturation assays revealed that the effects of the modifications were additive, and 9b displayed increased affinity and preference for forward orientation binding (table 2b). we then sought to determine ic50 values for the three generations of 5-cgcg-3 methylation inhibitors : 1b, 3b, and 9b. with consideration for their dna binding affinities, compounds 1b, 3b, and 9b were titrated from 10 nm to 33 m, 330 pm to 10 m, and 33 pm to 1 m, respectively (figure 5c). it is worth noting that an additional sds wash step was necessary in this assay to remove the higher affinity 9b from the dna before resolution by the mlui restriction enzyme. prior to the addition of this sds incubation, inhibition was maximally revealed to approximately 40%, due to polyamide inhibition of the mlui restriction enzyme. overnight incubation of dna in 2% sds removed additional polyamide and improved the revealed inhibition, suggesting the compressed inhibitory range is an artifact of this method and the high affinity of 9b. the ic50 values of 1b, 3b, and 9b were determined to be 2.2 m (95% confidence : 1.23.9 m), 117 nm (95% confidence : 65210 nm), and 2.6 nm (95% confidence : 1.06.7 nm), respectively (figure 5d). this is in good correlation with the iterative improvement shown in the biophysical analyses of these compounds, as well as the previous qualitative in vitro assay. polyamide 9b shows nearly 1000-fold improvement over 1b as a 5-cgcg-3 methylation antagonist. this study provides a basis for design of sequence - specific dna - binding molecules for targeted inhibition of cpg methylation. the disparity in methyltransferase inhibition between at - binding distamycin d and hairpin polyamide 3b suggests that the specific cpg - binding capability and widening of the minor groove by bound py - im polyamides are critical for disrupting dna methylation in the major groove. at the same time, applying the pairing rules demands caution in the design of imidazole and -rich polyamides as the inherent conformational flexibility of the subunit can support unintended reverse dna - binding modes. while previous studies have shown an -amino gaba turn unit can be used to restore the forward orientation binding preference of -containing polyamides, we found that 5-cgcg-3 binding py - im polyamides required an alternative solution. specifically, restoring the rigidity of the n - terminal strand via substitution of its -subunit with a py appears necessary to resolve the undesired reverse - binding of these architectures. the lack of inhibition of cpg methylation by the reverse binding 1b as compared to 3b at the interrogated 5-cgcg-3 sites suggests that inhibition of the preceding m.sssi enzyme is a sequence - specific, localized event. that swings the target cytosine out of the double helix and into its catalytic core. structural studies of mouse dnmt1, the relevant mammalian methyltransferase for maintenance methylation, show that enzyme residues enter the double helix from both the major and minor grooves in an intercalative - manner around the target cpg. these residues disrupt local base pairing and rotate the substrate cytosine around the sugar phosphate backbone and into the catalytic core of the enzyme. a py - im polyamide bound to the target dna site likely acts as a stabilizing clamp in the minor groove and prevents the intrusion of these residues. the increased dna stability disallows the conformational reorganization of the cpg substrate necessary for catalysis and results in the inhibition of methyltransferase activity. in this study, we examined programmable py - im polyamides targeting the 5-cgcg-3 sequence as a model for sequence - specific inhibition of cpg methylation. the unbiased bind - n - seq method was critical for revealing unanticipated binding modes of the polyamides. through deliberate, incremental synthetic modifications, we were able to discern structure activity relationships that guided improved design of cpg methylation antagonists. further work will be necessary to understand whether this represents a more general solution for controlling py - im polyamide orientation or is specific to the 5-cgcg-3 sequence. this study demonstrates that high affinity minor groove binding py - im polyamides can inhibit major groove cpg methylation by methyltransferase in a sequence - specific manner. it will be the focus of future research to assess these molecules as antagonists of cpg methylation in cells and its utility in the desilencing of specific genes. it will be of interest whether the intrinsic rarity of the cpg dinucleotide sequence and noncovalent binding of polyamides will reduce off - target effects. polyamides were synthesized by microwave - assisted, solid - phase synthesis on pam resin (peptides international) according to previously described protocols. the polyamides were cleaved from resin with 3,3-diamino - n - methyldipropylamine and purified by reverse phase hplc. for biotin - conjugated polyamides, the free amine at the c - terminus was allowed to react with 2 equiv of preactivated peg4-biotin nhs ester (thermo scientific) and 4 equiv of diea for 1 h at 55 c in dmf. purity and identity of compounds were verified by analytical hplc and matrix - assisted laser desorption / ionization time - of - flight (maldi - tof) mass spectrometry. biotin conjugate was equilibrated at 50 nm concentration for 15 h with a uniquely barcoded library of all possible 21mers. dna associated with polyamide biotin conjugates were affinity purified with streptavidin magnetic beads (m-280 dynabeads) and eluted. isolated dna was amplified by touchdown pcr and sequenced at the california institute of technology millard and muriel jacobs genetics and genomics laboratory on an illumina hiseq 2000 genome analyzer. the generated data set was then distributed by barcode using scripts in the mermade pipeline and a fasta file of a random 25% of sequences for each compound submitted for dreme motif analysis. unmethylated dna duplexes and hairpin polyamides were mixed to a final concentration of 2 and 3 m, respectively, for polyamides 18, 1b, and 3b in 1 ml total volume. for experiments with hemi - methylated oligonucleotides, dna duplexes and hairpin polyamides were mixed to a final concentration of 1 and 1.5 m, respectively. an aqueous solution of 10 mm sodium cacodylate, 10 mm kcl, 10 mm mgcl2, and 5 mm cacl2 at ph 7.0 was used as analysis buffer. all oligonucleotides (100 m solutions dissolved in 10 mm tris - cl, 0.1 mm edta, ph 8.0) were purchased from integrated dna technologies. the assay was conducted on a varian cary 100 spectrophotometer equipped with a thermocontrolled cell holder with a cell path length of 1 cm. samples were heated to 90 c and cooled to a starting temperature of 25 c prior to heating at a rate of 0.5 c / min to 90 c. denaturation profiles were recorded at = 260 nm and melting temperatures were defined as the maximum of the first derivative of the denaturation profile. reported data represents the average of four measurements. in pcr tubes, serially diluted concentrations of polyamides 1b, 3b, and distamycin d control were incubated in 96 l of 10 pm unmethylated -phage dna (promega) and 1 neb2 buffer (new england biolabs) in depc - treated water (usb) for 12 h at 25 c. after incubation, s - adenosyl methionine (new england biolabs) and m.sssi (new england biolabs) or water was added to all samples to a final concentration of 320 m and 0.25 units, respectively, to afford 100 l of total solution. samples were then incubated for 3 h at 37 c on a biorad mycycler thermal cycler and heat inactivated for 15 min at 65 c. dna was ethanol precipitated in a centrifuge at 4 c for 15 min with the addition of 10 l of 3 m naoac, 1 l of glycogen, and 2.5 volumes of ethanol at 20 c. dna was washed once with 75% aqueous ethanol at 20 c and allowed to air - dry for 30 min. samples were dissolved in 35 l of water and 15 l taken for mlui restriction enzyme digestion. samples were prepared in pcr tubes per manufacturer s protocol with 1 unit of mlui per sample and incubated at 37 c for 1 h. blue loading buffer 6 (new england biolabs) was added to samples and 20 l added to a 0.7% agarose gel in 0.5 tbe buffer. dna was visualized with sybr gold (invitrogen) and a typhoon fla9000 scanner (ge healthcare). the in vitro assay was run as described above with 1b, 3b, and 9b at concentrations titrated at 10-fold and 3-fold intervals ranging from 10 nm to 33 m, 330 pm to 10 m, and 33 pm to 1 m, respectively, and dna at 50 pm. the substrate dna fragment (7.5 kb) was pcr amplified from ptyb21 (new england biolabs) after linearization with bamhi (new england biolabs). primers 5-acttttcggggaaatgtgcg-3 and 5-ttagaggccccaaggggtta-3 (idt dna) were used for amplification with the expand long template pcr system (roche). the amplicon was isolated with qiaquick pcr purification kit (qiagen), and the amplicon size was verified by agarose gel electrophoresis. after the ethanol precipitation step which follows methylation, the dna pellet was dissolved in 100 l of 2% sds and incubated overnight at 55 c to wash off residual polyamide. the high affinity of 9b made this additional wash step necessary prior to mlui digestion. to the solution, 10 l of 2 m nacl followed by 2.5 volumes of ethanol were added to reprecipitate the dna. the pellet was washed twice with cold 75% ethanol before submission to mlui digest, as described above. gels were scanned on a typhoon fla scanner (ge healthcare) and the bands quantitated using imagequant software (ge healthcare). ic50 curves and 95% confidence intervals were determined using graphpad prism by variable - slope, nonlinear regression fit to a dose response model with a bottom constraint of 0. | the cpg dyad, an important genomic feature in dna methylation and transcriptional regulation, is an attractive target for small molecules. to assess the utility of minor groove binding oligomers for cpg recognition, we screened a small library of hairpin pyrrole - imidazole polyamides targeting the sequence 5-cgcg-3 and assessed their sequence specificity using an unbiased next - generation sequencing assay. our findings indicate that hairpin polyamide of sequence pyimim--pyimim (1), previously identified as a high affinity 5-cgcg-3 binder, favors 5-gcgc-3 in an unanticipated reverse binding orientation. replacement of one alanine with py to afford pyimpyim--pyimim (3) restores the preference for 5-cgcg-3 binding in a forward orientation. the minor groove binding hairpin 3 inhibits dna methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence - specific antagonists of cpg methylation. |
parkinson s disease (pd) is a progressive neurodegenerative disorder caused due to dopamine depletion. it is estimated that the prevalence of pd in thailand is 424.57 per 100,000 population.1 pd is mainly a movement disorder with four typical cardinal symptoms, including bradykinesia, rigidity, tremor, and postural instability.24 the gold standard treatment of pd is dopamine replacement.58 levodopa is the first - line treatment and is highly effective for improving the symptoms of pd. long - term levodopa treatment may cause several motor complications such as dyskinesia or dystonia.9 thailand and other developing countries have an issue with the price of the original medications including levodopa. the original levodopa or madopar has a higher price as compared to the generic levodopas, vopar or levomet. as a result, original levodopa is limited for clinical use and only available in university or tertiary care hospitals. generic levodopa is widely used in community or general hospitals, but its efficacy compared with that of original is still of concern. studies comparing original and generic compounds are very difficult to perform, as most patients at the university hospital are on the original compound while the community and general hospitals are mandated to use only the generic compound with a few exceptions. this study was aimed to investigate whether the generic levodopa was as effective as the original levodopa in improving the symptoms of pd. also, the prevalence of motor complications between the original and generic levodopa was compared. this study was a multicenter consecutive case cohort study of patients with pd enrolled from three northeast hospitals in thailand, including udonthani hospital, khon kaen hospital, and srinagarind hospital of khon kaen university. the first two hospitals are general hospitals. patients who had been diagnosed with pd according to the uk pd brain bank clinical diagnostic criteria and the study protocol was approved by the ethics committees for human research of all three hospitals. all eligible patients were treated by the attending physicians and were categorized by the type of levodopa given, original or generic. yahr (h - y) stage, duration of disease, treatment, dosage of levodopa, and motor complications were recorded. the dose of levodopa could be adjusted, but the type of levodopa for each patient remained the same throughout the study period. the modified h - y stages were used to classify the involvement of pd as follows : 1) unilateral involvement only ; 1.5) unilateral and axial involvement ; 2) bilateral involvement without impairment of balance ; 2.5) mild bilateral disease without recovery on pull test ; 3) mild to moderate bilateral disease, some postural instability but physically independent ; 4) severe disability, still able to walk or stand unassisted ; and 5) wheelchair bound or bedridden unless aided. the responses to treatments were classified for each patient using the ratings for subjective improvement as good or poor. motor fluctuations are the variable and unpredictable benefits of treatment of parkinsonian features in response to a dose of levodopa. dyskinesia is the levodopa - induced involuntary movement that commonly occurs at the time of peak concentration of the dose of levodopa. the time of peak concentration was from the start of levodopa use to the first report of a motor complication. levodopa therapy, however, is associated with motor complications, particularly at higher doses. therefore, an alterative strategy is to use lower doses of levodopa to reduce the motor complications of the monotherapy and using a dopamine agonist or monoamine oxidase type b inhibitor or adjunct therapy combining a dopamine agonist, a monoamine oxidase type b inhibitor, or a catechol - o - methyltransferase inhibitor with a lower dose of levodopa. to facilitate the comparisons between each strategy of treatment, a levodopa equivalent dose (led) the total levodopa dose equivalents were calculated as follows : (regular levodopa dose 1) + (levodopa continuous - release dose 0.75) + (pramipexole dose 67) + (ropinirole dose 16.67) + (pergolide dose 100) + (bromocriptine dose 10) + [regular levodopa dose + (continuous - release levodopa dose 0.75) ] 0.25, if the patient is taking entacapone.10 assuming that a 20% difference between original and generic levodopa treatment response is significant, the minimal number of subjects needed was 89. this calculation was performed by using the compared hypothesis for calculating the least number needed for each group. after providing written informed consent, all patients were categorized into two groups by the type of levodopa : original and generic. numerical factors were compared by either student s t - test or wilcoxon rank - sum test where appropriate, whereas categorical factors were compared by using chi - square or the fisher s exact test where appropriate. all statistical analyses were performed by the statistical package for the social sciences statistical package version 22.0 (ibm corporation, armonk, ny, usa) and stata version 10.0 (statacorp lp, college station, tx, usa). this study was a multicenter consecutive case cohort study of patients with pd enrolled from three northeast hospitals in thailand, including udonthani hospital, khon kaen hospital, and srinagarind hospital of khon kaen university. the first two hospitals are general hospitals. patients who had been diagnosed with pd according to the uk pd brain bank clinical diagnostic criteria and the study protocol was approved by the ethics committees for human research of all three hospitals. all eligible patients were treated by the attending physicians and were categorized by the type of levodopa given, original or generic. yahr (h - y) stage, duration of disease, treatment, dosage of levodopa, and motor complications were recorded. the dose of levodopa could be adjusted, but the type of levodopa for each patient remained the same throughout the study period. the modified h - y stages were used to classify the involvement of pd as follows : 1) unilateral involvement only ; 1.5) unilateral and axial involvement ; 2) bilateral involvement without impairment of balance ; 2.5) mild bilateral disease without recovery on pull test ; 3) mild to moderate bilateral disease, some postural instability but physically independent ; 4) severe disability, still able to walk or stand unassisted ; and 5) wheelchair bound or bedridden unless aided. the responses to treatments were classified for each patient using the ratings for subjective improvement as good or poor. motor fluctuations are the variable and unpredictable benefits of treatment of parkinsonian features in response to a dose of levodopa. dyskinesia is the levodopa - induced involuntary movement that commonly occurs at the time of peak concentration of the dose of levodopa. the time of peak concentration was from the start of levodopa use to the first report of a motor complication. levodopa therapy, however, is associated with motor complications, particularly at higher doses. therefore, an alterative strategy is to use lower doses of levodopa to reduce the motor complications of the monotherapy and using a dopamine agonist or monoamine oxidase type b inhibitor or adjunct therapy combining a dopamine agonist, a monoamine oxidase type b inhibitor, or a catechol - o - methyltransferase inhibitor with a lower dose of levodopa. to facilitate the comparisons between each strategy of treatment, a levodopa equivalent dose (led) the total levodopa dose equivalents were calculated as follows : (regular levodopa dose 1) + (levodopa continuous - release dose 0.75) + (pramipexole dose 67) + (ropinirole dose 16.67) + (pergolide dose 100) + (bromocriptine dose 10) + [regular levodopa dose + (continuous - release levodopa dose 0.75) ] 0.25, if the patient is taking entacapone.10 assuming that a 20% difference between original and generic levodopa treatment response is significant, the minimal number of subjects needed was 89. this calculation was performed by using the compared hypothesis for calculating the least number needed for each group. after providing written informed consent, all patients were categorized into two groups by the type of levodopa : original and generic. numerical factors were compared by either student s t - test or wilcoxon rank - sum test where appropriate, whereas categorical factors were compared by using chi - square or the fisher s exact test where appropriate. all statistical analyses were performed by the statistical package for the social sciences statistical package version 22.0 (ibm corporation, armonk, ny, usa) and stata version 10.0 (statacorp lp, college station, tx, usa). statistical significance was defined as p - value 0.999), but the development of motor complications was significantly greater in the original than in the generic group as shown in table 3. the times to develop motor fluctuations at 48.1233.20 versus 62.1039.55 months (p - value 0.07) and dyskinesia at 62.4637.72 versus 83.5252.74 months (p - value 0.13) were not significantly different between the original and generic groups (table 2). clinical features of pd in the present study were quite similar to those in a previous study on thai population by kulkantrakorn.11 sex and average ages were nearly the same in the original formulation and generic groups. the present study compared original and generic levodopa groups for symptomatic responses in order to determine if the generic form was as good as or inferior to the original levodopa (original group 82.0%, generic group 81.9% ; p - value > 0.999). the total dosage of levodopa used in the original group was lower than in the generic group (original group 199.97 mg, generic group 305.58 mg ; p - value < 0.001). the led used in the original group was 198.10 mg versus 308.85 mg in the generic group (p - value < 0.001). the present study showed that the dosage of levodopa used in treatment of patients with pd who had developed motor complications in the generic group was higher than the dosage used in the original group by approximately 1.5 times. gasser reported on the pharmaceutical quality of generic levodopa / benserazide products compared with original madopar / prolopa (roche, switzerland) and found deviations in the active ingredients, a marked excess in degradation, and some problems with the dissolution times of generic compounds. therefore, the higher dose of generics may be due to lower efficacy of the generic levodopa than the original. the us food and drug administration published draft guidelines on levodopa / carbidopa showing that the bioequivalent base is at 90% confidence interval;13 therefore, the dosage used in the generic group was higher than that in the original group of the cited study. to date, there has been no long - term study on the bioequivalence of levodopa / benserazide in patients with pd. pahwa who compared single doses of sinemet and atamet (generic carbidopa / levodopa) in patients with pd found that there was no statistically significant difference between sinemet and atamet in the time taken to reach the maximum concentration (tmax), the maximum concentration (cmax), area under the curve, and the motor performance tests, but there has been no study comparing the original and generic forms in the long - term use of levodopa. patients with pd who often use multiple drugs and have low gastric motility may experience drug drug interactions and slow absorption of orally administered compounds in the morning.15 madopar, the original levodopa, was slightly more expensive than vopar and levopar, the generic levodopa (us$ 0.32 and 0.23 per tablet, respectively). the generic drugs, however, are used at higher doses than the original drug (305.58138.27 versus 199.97127.08 mg) ; therefore, it may not be cost - effective to substitute generic drugs for the brand name product. the present study showed that a number of patients developed complications similar to the previous study.11 the incidence of complications in the original group was 41.50% and in the generic group was 29.92%. the original group reported more motor complications (p - value 0.035), but used lower doses of total levodopa and leds than the generic group. further study on the pharmacokinetics and pharmacodynamics of original and generic levodopa in patients with pd will need to be carried out before considering the use of generic levodopa as a cost - effective treatment. even though this study was of cohorts and similar to real - life clinical practice, a further large randomized controlled trial should be performed to confirm the results of this study. the lower rate of motor complications in the generic group may be due to the lower efficacy on a dosage basis of the generics versus the original levodopa. the evaluation of motor complications by asking patients rather than critical clinical evaluation using the unified pd rating scale as used in real clinical practice could result in underdiagnosis. the prevalence of motor complications in the original compound group, at a lower dose of levodopa equivalent, was higher than in the generic group. | backgroundin general, a generic drug is considered interchangeable with the original formulated drug. in parkinson s disease (pd), generic drug use remains debated. this study was aimed to investigate whether the generic drug was as effective as the original in improving the symptoms of pd and the prevalence of motor complications.methodsthis study was a multicenter cohort study of patients with pd enrolled from three northeast hospitals in thailand between february 2013 and february 2014. the patients were categorized into original and generic levodopa groups. the clinical characteristics, efficacy, and motor complications were compared between the groups.resultsthere were 400 eligible patients. of these, 327 patients (81.75%) met the study criteria and were classified as the original levodopa group (200 patients, 61.16%) and the generic levodopa group (127 patients, 38.84%). the average age of all patients with pd was 65 years. the duration of pd and the modified hoehn yahr stages were not different between the groups. the total doses of original and generic levodopa - equivalent doses were significantly different (199.97127.08 versus 305.58138.27 mg ; p - value 0.999), but the development of motor complications was significantly greater in the original than in the generic group.conclusiongeneric levodopa was effective in improving the symptoms of pd. the prevalence of motor complications in the original compound group, at a lower dose of levodopa equivalent, was higher than in the generic group. |
chronic obstructive pulmonary disease (copd) is the fourth leading cause of death and is estimated to rise to be the third most common cause of death worldwide by 2020 (rielly 2005). many patients with copd have concomitant conditions, mostly coronary artery disease (cad), that require the use of beta - blockers (bbs). however, despite the clear evidence of bbs effectiveness, there is a general reluctance to use them in patients with copd due to a perceived contraindication and fear of inducing adverse reactions and bronchspasm (kennedy and rosenson 1995 ; viskin and barron 1996 ; gottlieb 1998 ; chafin 1999). it is a common practice of physicians to consider copd as contraindication to the use of bbs, based mainly on anecdotal evidence and case reports citing acute bronchospasm following the administration of bbs (tattersfield 1986, 1990 ; belli and topol 1995 ; craig 1996 ; kendall 1997). the most common comorbid conditions associated with withholding bbs in elderly patients after myocardial infarction (mi) are copd and asthma (heller 2000), while peripheral arterial and bronchial problems are reported to be the leading side effects (frishman 1979 ; frishman 1998). on the other hand, many patients are diagnosed and treated for copd with no objective evidence, such as pulmonary function tests or specialist assessment, to confirm the diagnosis, as recommended by most thoracic societies. this may indicate that a significant number of patient are deprived the prognostic benefits of using bbs (chen 2001). bbs are well tolerated in patients with cardiac disease and concomitant copd with no evidence of worsening of respiratory symptoms or fev1 (formgren 1976 ; george 1983 ; quan 1983 ; krauss 1984 ; falliers 1985 ; dorrow, bethge 1986 ; mooss 1994) and the safety of bbs in patients with copd has been demonstrated, but their use in this group of patients remains low (salpeter 2001, 2002a, 2002b, 2003). the cumulative evidence from trials and meta - analysis indicates that cardioselective bbs should not be withheld in patients with reactive airway disease or copd (salpeter 2001, 2002a, 2002b, 2003). patients with copd have a high incidence of cardiac events necessitating careful consideration of prophylactic treatment. the benefits of beta blockade in this group appear to outweigh any potential risk of side effects according to the available evidence. for instance, bbs are well tolerated by the large majority of patients with heart failure, even in those with comorbid conditions such as diabetes mellitus, copd, and peripheral vascular disease (hfsa 2006). in this article, we will discuss the use of bbs in patients with copd and review the evidence for their use and safety in this group of patients. beta - blockers were originally designed by the nobel prize winner sir james black to counteract the adverse effects of adrenergic stimulation. he demonstrated that, by blocking the cardiac - receptors, these agents could cause inhibitory effects on the sinus node (chronotropic effect), atrioventricular node (dromotropic effect), and on myocardial contractility (inotropic effect). if activated, they lead to an increase in the rate and force of myocardial contraction (positive inotropic effect) by opening the calcium channels. on the other hand, 2-adrenoceptors are found mainly in bronchial and vascular smooth muscles. if activated, they cause broncho- and vaso - dilatation. there are, however, sizable populations of 2-adrenoceptors in the myocardium, of about 20%25%, which leads to the cardiac effects of any 2-adrenoceptors stimulation. there is a relative up - regulation of these receptors to about 50% in heart failure. the role of 3-adrenoceptors in the heart is not yet fully identified and accepted (kalinowski 2003). the first generation agents (such as propranolol, sotalol, timolol, and nadolol), are nonselective and block 1 and 2 receptors. blocking 1-receptors affects the heart rate, conduction and contractility, while blocking 2-receptors, tends to cause smooth muscle contraction, therefore, bronchospasm in predisposed individuals. the second - generation agents or the cardioselective agents (such as atenolol, bisoprolol, celiprolol, and metoprolol) block 1-receptors in low doses but are capable of blocking 2-receptors in higher doses. this selective mode of action makes the use of these agents more suitable in patients with chronic lung disease (wellstien 1987) or those with insulin - requiring diabetes mellitus. there action is either selective (nebivolol) or nonselective (carvidolol and labetolol). the vasodilatory properties are mediated either by nitric oxide release as for nebivolol or carvidolol (kalinowski 2003) or by added alpha - adrenergic blockade as in labetolol and carvidolol. a third vasodilatory mechanism, as in pindolol and acebutolol, acts via 2-intrinsic sympathomimetic activity (isa). these beta - blockers therefore have the capacity to stimulate as well as to block adrenergic receptors and tend to cause less bradycardia than the other beta - blockers and may cause less coldness of the extremities. beta - blockers are used extensively and have a proven morbidity and mortality benefits in the management of patients with cardiac disease (frishman 1984 ; the ipppsh collaborative group 1985 ; wadworth 1991 ; stienbeck 1992 ; mangano 1996 ; doughty 1997 ; jnc vi 1997 ; lechat 1998 ; freemantle 1999 ; heidenreich 1999 ; poldermans 1999). they are a standard therapy for hypertension, angina, unstable angina, post myocardial infarction, tachyarrhythmias, and congestive heart failure (the merit - hf study group 2000 ; packer 2001). many patients with copd have concomitant conditions such as cad (coexists in up to 27% of copd patients (karoli and rebrov 2005)) that require the use of bbs. bbs are often avoided in these patients (kennedy and rosenson 1995 ; viskin and barron 1996 ; gottlieb 1998 ; chafin 1999) because of fear of bronchospasm and possible adverse reactions, despite the known cardiovascular mortality and morbidity benefit (frishman 1984 ; the ipppsh collaborative group 1985 ; wadworth 1991 ; stienbeck 1992 ; mangono 1996 ; doughty 1997, jnc vi 1997 ; lechat 1998 ; freemantle 1999 ; heidenreich 1999 ; polderman 1999). this is mainly based on anecdotal evidence and case reports citing acute bronchospasm following the administration of beta - blocker (tattersfield 1986, 1990 ; omalley 1991 ; belli and topol 1995 ; craig 1996 ; kendall 1997). copd patients are at greater risk of ischaemic heart disease than asthmatics, so would benefit from the use of bbs. on the other hand, they also have more severe airway obstruction, so may be more sensitive to small changes in fev1 due to beta - blockade. the use and effectiveness of bbs therapy after mi for elderly patients with copd or asthma was evaluated (chen 2001). the study was rationalised by the fact that patients with copd and asthma have largely been excluded from clinical trials of bbs therapy for ami. the authors used the data from the cooperative cardiovascular project (marciniak 1998) to examine the relationship between discharge use of bbs and one - year mortality in patients with copd or asthma who were divided into three groups : those who are not using beta - agonists, those who are using beta - agonists, and those with severe disease (on prednisone or previous hospitalization) in comparison with patients free from copd or asthma. of 54,962 patients without contraindications to beta - blockers, patients with copd or asthma (20%) were significantly less likely to be prescribed beta - blockers at discharge after ami. patients with copd or asthma who were not on beta - agonist had lower one - year mortality if they were on bbs. this mortality benefit was not found among patients using beta - agonists or those with severe copd or asthma. large meta - analyses were published by salpeter (2001, 2002a, 2002b) where randomized, blinded, placebo - controlled trials that studied the effects of cardioselective bbs on fev1, symptoms, and the use of inhaled 2-agonists in patients with reactive airway disease were selected, of which, there were 19 single dose treatment studies and 10 continued treatment studies. the outcomes measures were the change in fev1 from baseline, the number of patients with respiratory symptoms, and the use of inhaled 2-agonists with active treatment compared with placebo. the results were that no significant treatment effect in terms of fev1 was found in patients with concomitant copd, whether single doses (change in fev1, 5.28% [ci, 10.03% to 0.54% ]) or continued treatment (change in fev1, 1.07% [ci, 3.3% to 5.44% ]) was used. the conclusion was that cardioselective bbs do not produce clinically significant adverse respiratory effects in patients with mild to moderate reactive airway disease, and that they should not be withheld from these patients. the studies were not designed to make recommendations about people with significant chronic airway obstruction. a similar review on a similar cohort of patients 11 single dose treatment studies (schanning and vilsvik 1976 ; beil and ulmer 1977 ; mcgavin and williams 1978 ; perks 1978 ; sinclair 1979 ; anderson 1980 ; sorbini 1982 ; von wichert 1982 ; adam 1984 ; dorow, bethge 1986 ; macquin - mavier 1988) and 8 continued treatment studies (tivenius 1976 ; wunderlich 1980 ; ranchod 1982 ; buttand 1983 ; fenster 1983 ; lammers 1985 ; dorow, clauzel 1986 ; fogari 1990) was also published by the previous group (salpeter 2003). this demonstrated that cardioselective bbs produced no significant change in fev1 or respiratory symptoms compared to placebo and did not significantly affect the fev1 treatment response to 2-agonists. subgroup analysis revealed no significant change in results for those participants with severe copd or for those with a reversible obstructive component. the conclusion was again that cardioselective bbs given to copd patients do not produce a significant reduction in airway function or an increase in the incidence of copd exacerbations. a retrospective study (egred 2005) assessed the use of bbs in patients with copd admitted with acute coronary syndrome (acs). a diagnosis of copd was the most common cause for withholding bbs and only 62% of patients with copd have been reviewed by a chest physician or had a previous pulmonary function test. of these copd patients, only 16% were prescribed bbs and many patients with a diagnosis of copd have no objective evidence to support this diagnosis. the conclusion was that, these patients are being denied the prognostic benefits of bbs when presenting with acs, and the recommendation was that before withholding bbs, copd and reversibility should be ascertained by pulmonary function testing and that the overall use of bbs was sub - optimal in this setting. the use of bbs in congestive heart failure patients with copd and/or asthma was also assessed in a retrospective analysis (peters 2004). one thousand sixty seven patients with chf were included and reviewed over 18 months period. medications, non - routine office visits, emergency room visits, and hospitalizations for respiratory events were assessed. of the 1067 patients, 19.6% had obstructive pulmonary disease (old) : 5.9% asthma, 11.2% copd and 2.5% asthma / copd. only 35.9% of the patients with old were on bbs half of which were cardioselective. the use of bbs did not result in any increased respiratory events, respiratory encounters, emergency room visits, or hospitalizations. the result showed that the long - term use of bbs did not increase the risk of respiratory complications and there was no difference in outcomes with the use of cardioselective or noncardioselective bbs and the conclusion was that cardio - selective bbs without intrinsic sympathomimetic activity are preferred until future studies resolve this issue. most of the evidence presented is meta - analysis ; therefore, it has many limitations like any other meta - analysis (ionnidis and lau 1999). a few studies did not have placebo controls, and many did not provide standard deviations for fev1 treatment effects. it is possible that a longer study period may be required in order to detect clinically important side effects of bbs. in the recent publication of the heart failure society of america (hfsa) on heart failure practice guidelines (hfsa 2006), bbs therapy is recommended in the great majority of patients with lv systolic dysfunction, even in the presence of concomitant copd. they recommend that bbs are initiated at a low dose and uptitrated gradually in two weeks intervals. the guidelines also recommend that patients with difficulties in initiating, uptitrating, or maintaining bbs therapy to be referred to a physician with expertise in the management of these patients. this is to ensure that no patient is denied the benefits of bbs (hfsa 2006). in this article, we presented a review of the data supporting the use of cardioselective bbs in patients with mild to moderate copd. the evidence suggests that cardio - selective bbs are not only safe but also beneficial in patients with co - existing airways and coronary disease and can significantly improve prognosis. due to the proven mortality benefit of bbs in numerous cardiac conditions, many of the other relative or absolute contraindications traditionally listed for bbs have been questioned, including impaired left ventricular function, peripheral vascular disease, and diabetes mellitus (kjekshus 1990 ; wichkmayr 1990 ; radack and deck 1991 ; rosenson 1993 ; jonas 1996 ; gottlieb 1998 ; lechat 1998). finally, although we believe physicians should feel more comfortable prescribing cardioselective bbs to copd patients, we can not make a generalised recommendation that bbs can be used in all copd patients. this is due to the fact that almost all of the evidence is from retrospective analysis of data available and more prospective studies are needed. physicians should use their clinical judgment and use bbs where they think the benefits outweigh the risks. we suggest that patients who are admitted with any cardiac condition requiring bbs and who have concomitant copd should be tried on bbs. a safe approach is to initiate cardio - selective bbs at a low dose and metoprolol is cardioselective bbs with short half - life and has been shown to be safe and effective in patients with copd (camsari 2003) and may be the bbs of choice to initiating therapy. this will allow close observation and assessment of tolerance of these medications and will ensure that these patients are not denied the prognostic benefits of a well - tolerated and effective treatment. it may be necessary to discontinue the drug in few patients due to bronchconstriction, but the potential benefit appears large enough to warrant this small risk. further randomised studies may be required to assess the use of the new bbs (nebivolol) with its nitric oxide (no) releasing property in patient with copd. this will allow the assessment of weather it is safe and similarly will help define weather no has any role to play in reducing the effect of bbs on bronchospasm. a drug called pimobendan which is a phosphodiesterase (pde) iii - inhibitor, with vasodilatory and bronchodilatory effects, has been used in two patients with heart failure who were unable to tolerate bbs. the use of pimobendan allowed the start and maintenance of bbs without worsening copd or heart failure with evidence of progressive decline in brain natriuretic peptide (bnp) (shiga 2002). this may present an interesting way of dealing with bbs intolerant patients, where the bronchdilatory effects of pimobendan would allow the initiation of bbs. this is only two case reports and the safety and efficacy of this drug has not been tested in a controlled manner. bbs reduce mortality in patients with copd and coexisting cad and should be used whenever possible. cardioselective bbs are safe in patients with copd who have an indication for their use. nonselective bbs are better avoided in general, except in patients with heart failure who may benefit from the use of carvedilol. a short acting cardioselective bbs (such as metoprolol) should be started at a low dose and uptitrated slowly. once metoprolol is established, it can be changed to a once daily longer acting product such as bisoprolol or atenolol. in case of uncertainty or difficulty, specialist opinion should be sought to insure that these patients are not denied the prognostic benefit of bbs therapy. | the mortality and incidence of chronic obstructive pulmonary disease (copd) and coronary heart disease increase with age. despite the clear evidence of beta blockers (bbs) effectiveness, there is a general reluctance to use them in patients with copd due to a perceived contraindication and fear of inducing adverse reactions and bronchspasm. bbs are well tolerated in patients with cardiac disease and concomitant copd with no evidence of worsening of respiratory symptoms or fev1, and the safety of bbs in patients with copd has been demonstrated, but their use in this group of patients remains low. the cumulative evidence from trials and meta - analysis indicates that cardioselective bbs should not be withheld in patients with reactive airway disease or copd.patients with copd have a high incidence of cardiac events necessitating careful consideration of prophylactic treatment. the benefits of beta blockade in this group appear to outweigh any potential risk of side effects according to the available evidence.in this article, we will discuss the use of bbs in patients with copd and review the evidence for their use and safety in this group of patients. |
raw and processed data for the studies described here may be found at the following links : somatic tissue : http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse49222 es and neuro2a cells : http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60007 whole brain, liver and kidney from a 1-week - old male c57bl6/n mouse were lysed overnight in 10x w / v tail lysis buffer (100 mm tris cl [ph 8.5 ], 5 mm edta [ph 8.0 ], 200 mm nacl, 0.2% w / v sodium dodecylsulfate [sds ]) and 0.01x v / v 20 mg / ml proteinase k (sigma - aldrich). the tissue - lysis products were divided into 300 l aliquots and stored at 20 c. isoamyl alcohol followed by ethanol precipitation. a similar dna isolation protocol, involving cell lysis following by phenol chloroform extraction and ethanol precipitation, the isolated dna was resuspended in 50100 l 1x te and its concentration quantified using the nanodrop (thermo - scientific). approximately 1 g of the isolated dna was sonicated using the bioruptor ngs (diagenode) to obtain fragments which were approximately 700 bp in length (4 cycles, on / off cycle time : 15/90) (fig. 1). next, these fragments were end - repaired using nebnext end repair module (new england biolabs), and cleaned using the dna clean and concentration kit (zymo research) with 5x v / v binding buffer and eluted in 30 l hplc water. the end - repaired dna was then incubated at 20c for 2 h with 50 pmols of custom - made methylated - c ion torrent a adaptors (integrated dna technologies) and 800 units of t4 dna ligase (new england biolabs). unligated adaptors were removed using the dna clean and concentration kit (zymo research) with 5x v / v binding buffer and the resulting a adaptor - ligated fragments were eluted in 50 l hplc water. the adaptor - ligated dna fragments were further size - selected to remove any excess adaptors and dna fragments smaller than 300 bp in length using the agencourt ampure xp beads (beckman coulter) using a dna : bead ratio of 1:0.7 (100 l dna + 70 l beads) (fig. 1). this library was then quantified using bioanalyzer (agilent) and subsequently subjected to one round of bisulfite conversion using the ez dna methylation kit (zymo research). this bisulfite - converted library (1 l) was then used to conduct quantitative real - time pcr using sybr - green (life technologies) to determine the appropriate cycle number to amplify the bisulfite - converted library for subsequent size selection and sequencing. the cycle number for pcr amplification was individually determined for each library to be the cycle number corresponding to the midpoint of the exponential portion of the amplification curve (fig. 1). the forward primer used for pcr was complementary to the 5 end of the a adaptor region (5-ccatctc atccctgcgtgtctccgactcag-3). the reverse primer (5-ccactacgcc tccgctttcctctctatgggcagtcggtgat^ctccctaattaactacaacccatc-3) was designed to bind the 24-bp region within the u3 region of the ltr which is conserved between five subtypes of iap ltr (iapltr1, iapltr1a, iapltr2, iapltr2a, and iapltr2b) and is devoid of any cpg sites. the sequence at the 5 end of the carat (^) corresponds to the p1 adaptor which is a part of the amplification scheme used by ion torrent (life technologies) (fig. 1). the amplified libraries were then size - selected using agarose gel extraction (megaquick - spin total fragment dna purification kit, intron biotechnology) to have approximately 250300 bp insert length flanked by the this was achieved by excising the gel fragment corresponding to 330380 bp in order to account for the ~ 80 bp combined length of the two adaptors (fig approximately 25 l of the size - selected library at 10 pm was used for the emulsion pcr and subsequent next - generation sequencing using the ion torrent personal genome machine (pgm) sequencer and ion 318 chips (ion torrent, life technologies). we have implemented the following bioinformatics pipeline to process the raw sequence reads from the ngs platform (fig. 2). we have used bowtie2 for mapping and biq analyzer ht for dna methylation analyses. we have used several unix - based command lines along with custom - made perl scripts for the pipeline. first, we used the following steps for the construction of a custom database (below and supplemental material 1).1)download the sequence of 9282 iap ltrs containing 330-bp ltr along with two 350-bp flanking regions in a fasta format from the table database at ucsc genome browser.2)reverse complement half of the iapltrs in an opposite direction so that the entire set will have an identical forward direction with a custom perl script.3)convert all the iap ltrs into bisulfite - converted sequences with a custom perl script.4)compile all the bisulfite - converted sequences into one large sequence with a unix command line.5)construct a searchable index file with a large compiled sequence with bowtie2-build. download the sequence of 9282 iap ltrs containing 330-bp ltr along with two 350-bp flanking regions in a fasta format from the table database at ucsc genome browser. reverse complement half of the iapltrs in an opposite direction so that the entire set will have an identical forward direction with a custom perl script. convert all the iap ltrs into bisulfite - converted sequences with a custom perl script. compile all the bisulfite - converted sequences into one large sequence with a unix command line. the execution of these steps will provide the following files : (i) a directory containing the 9282 files with the original sequences in a fasta format (step 2) and (ii) a directory containing 6 indexable file (step 5). second, we used the following steps for the mapping of sequence reads and subsequent dna methylation level analyses (below and supplemental material 1).1)remove raw sequence reads smaller than 40 bp in length.2)map the raw reads against the custom - made database using bowtie2.3)remove headerlines from the sam file derived from mapping with a unix command line.4)sort each line of the sam file based on each locus of iap ltr and generate a directory containing each iap ltr file containing mapped raw reads with a custom perl script.5)filter out raw reads from each iap ltr file that are not qualified based on the insufficient overlap with the flanking unique region (greater than 10 bp in length) with a custom perl script.6)execute biq analyzer ht with two directories, the directory containing all the sequences of iap ltrs (step 2 of database construction) and the directory containing all the mapped bisulfite sequence reads, with a custom perl script.7)extract the necessary information (number of reads used, % methylation, standard deviation) from the output of biq analyzer ht with a custom perl script. remove raw sequence reads smaller than 40 bp in length. sort each line of the sam file based on each locus of iap ltr and generate a directory containing each iap ltr file containing mapped raw reads with a custom perl script. filter out raw reads from each iap ltr file that are not qualified based on the insufficient overlap with the flanking unique region (greater than 10 bp in length) with a custom perl script. execute biq analyzer ht with two directories, the directory containing all the sequences of iap ltrs (step 2 of database construction) and the directory containing all the mapped bisulfite sequence reads, with a custom perl script. extract the necessary information (number of reads used, % methylation, standard deviation) from the output of biq analyzer ht with a custom perl script. the execution of these steps will finally derive one text file containing the dna methylation level for the entire set of iap ltrs, which can be imported into an excel file for further calculation and inspection (fig. 2). the following are the supplementary data related to this article.supplemental material 1perl scripts and unix command lines used for ht - trebs data analysis. whole brain, liver and kidney from a 1-week - old male c57bl6/n mouse were lysed overnight in 10x w / v tail lysis buffer (100 mm tris cl [ph 8.5 ], 5 mm edta [ph 8.0 ], 200 mm nacl, 0.2% w / v sodium dodecylsulfate [sds ]) and 0.01x v / v 20 mg / ml proteinase k (sigma - aldrich). the tissue - lysis products were divided into 300 l aliquots and stored at 20 c. isoamyl alcohol followed by ethanol precipitation. a similar dna isolation protocol, involving cell lysis following by phenol chloroform extraction and ethanol precipitation, the isolated dna was resuspended in 50100 l 1x te and its concentration quantified using the nanodrop (thermo - scientific). approximately 1 g of the isolated dna was sonicated using the bioruptor ngs (diagenode) to obtain fragments which were approximately 700 bp in length (4 cycles, on / off cycle time : 15/90) (fig. 1). next, these fragments were end - repaired using nebnext end repair module (new england biolabs), and cleaned using the dna clean and concentration kit (zymo research) with 5x v / v binding buffer and eluted in 30 l hplc water. the end - repaired dna was then incubated at 20c for 2 h with 50 pmols of custom - made methylated - c ion torrent a adaptors (integrated dna technologies) and 800 units of t4 dna ligase (new england biolabs). unligated adaptors were removed using the dna clean and concentration kit (zymo research) with 5x v / v binding buffer and the resulting a adaptor - ligated fragments were eluted in 50 l hplc water. the adaptor - ligated dna fragments were further size - selected to remove any excess adaptors and dna fragments smaller than 300 bp in length using the agencourt ampure xp beads (beckman coulter) using a dna : bead ratio of 1:0.7 (100 l dna + 70 l beads) (fig. 1). this library was then quantified using bioanalyzer (agilent) and subsequently subjected to one round of bisulfite conversion using the ez dna methylation kit (zymo research). this bisulfite - converted library (1 l) was then used to conduct quantitative real - time pcr using sybr - green (life technologies) to determine the appropriate cycle number to amplify the bisulfite - converted library for subsequent size selection and sequencing. the cycle number for pcr amplification was individually determined for each library to be the cycle number corresponding to the midpoint of the exponential portion of the amplification curve (fig. 1). the forward primer used for pcr was complementary to the 5 end of the a adaptor region (5-ccatctc atccctgcgtgtctccgactcag-3). the reverse primer (5-ccactacgcc tccgctttcctctctatgggcagtcggtgat^ctccctaattaactacaacccatc-3) was designed to bind the 24-bp region within the u3 region of the ltr which is conserved between five subtypes of iap ltr (iapltr1, iapltr1a, iapltr2, iapltr2a, and iapltr2b) and is devoid of any cpg sites. the sequence at the 5 end of the carat (^) corresponds to the p1 adaptor which is a part of the amplification scheme used by ion torrent (life technologies) (fig. 1). the amplified libraries were then size - selected using agarose gel extraction (megaquick - spin total fragment dna purification kit, intron biotechnology) to have approximately 250300 bp insert length flanked by the this was achieved by excising the gel fragment corresponding to 330380 bp in order to account for the ~ 80 bp combined length of the two adaptors (fig approximately 25 l of the size - selected library at 10 pm was used for the emulsion pcr and subsequent next - generation sequencing using the ion torrent personal genome machine (pgm) sequencer and ion 318 chips (ion torrent, life technologies). we have implemented the following bioinformatics pipeline to process the raw sequence reads from the ngs platform (fig. 2). we have used bowtie2 for mapping and biq analyzer ht for dna methylation analyses. we have used several unix - based command lines along with custom - made perl scripts for the pipeline. first, we used the following steps for the construction of a custom database (below and supplemental material 1).1)download the sequence of 9282 iap ltrs containing 330-bp ltr along with two 350-bp flanking regions in a fasta format from the table database at ucsc genome browser.2)reverse complement half of the iapltrs in an opposite direction so that the entire set will have an identical forward direction with a custom perl script.3)convert all the iap ltrs into bisulfite - converted sequences with a custom perl script.4)compile all the bisulfite - converted sequences into one large sequence with a unix command line.5)construct a searchable index file with a large compiled sequence with bowtie2-build. download the sequence of 9282 iap ltrs containing 330-bp ltr along with two 350-bp flanking regions in a fasta format from the table database at ucsc genome browser. reverse complement half of the iapltrs in an opposite direction so that the entire set will have an identical forward direction with a custom perl script. convert all the iap ltrs into bisulfite - converted sequences with a custom perl script. compile all the bisulfite - converted sequences into one large sequence with a unix command line. construct a searchable index file with a large compiled sequence with bowtie2-build. the execution of these steps will provide the following files : (i) a directory containing the 9282 files with the original sequences in a fasta format (step 2) and (ii) a directory containing 6 indexable file (step 5). second, we used the following steps for the mapping of sequence reads and subsequent dna methylation level analyses (below and supplemental material 1).1)remove raw sequence reads smaller than 40 bp in length.2)map the raw reads against the custom - made database using bowtie2.3)remove headerlines from the sam file derived from mapping with a unix command line.4)sort each line of the sam file based on each locus of iap ltr and generate a directory containing each iap ltr file containing mapped raw reads with a custom perl script.5)filter out raw reads from each iap ltr file that are not qualified based on the insufficient overlap with the flanking unique region (greater than 10 bp in length) with a custom perl script.6)execute biq analyzer ht with two directories, the directory containing all the sequences of iap ltrs (step 2 of database construction) and the directory containing all the mapped bisulfite sequence reads, with a custom perl script.7)extract the necessary information (number of reads used, % methylation, standard deviation) from the output of biq analyzer ht with a custom perl script. sort each line of the sam file based on each locus of iap ltr and generate a directory containing each iap ltr file containing mapped raw reads with a custom perl script. filter out raw reads from each iap ltr file that are not qualified based on the insufficient overlap with the flanking unique region (greater than 10 bp in length) with a custom perl script. execute biq analyzer ht with two directories, the directory containing all the sequences of iap ltrs (step 2 of database construction) and the directory containing all the mapped bisulfite sequence reads, with a custom perl script. extract the necessary information (number of reads used, % methylation, standard deviation) from the output of biq analyzer ht with a custom perl script. the execution of these steps will finally derive one text file containing the dna methylation level for the entire set of iap ltrs, which can be imported into an excel file for further calculation and inspection (fig. 2). the following are the supplementary data related to this article.supplemental material 1perl scripts and unix command lines used for ht - trebs data analysis. | dna methylation is a major epigenetic mark associated with multiple aspects of retrotransposons within the mammalian genome. in order to study dna methylation of a large number of retrotransposons on an individual - locus basis, we have developed a new protocol termed high - throughput targeted repeat element bisulfite sequencing (ht - trebs) (ekram and kim, 2014 [1 ]). we have used this technique to characterize the locus - specific patterns of dna methylation of 4799 members of the mouse iap ltr (intracisternal a particle long terminal repeat) retrotransposon family in embryonic stem, somatic and neuro2a cells (bakshi and kim, 2014 [2 ]). here we describe in detail the sample preparation and bioinformatics analyses used for these studies. the somatic cell data may be accessed under geo accession number gse49222. the es and neuro2a data are deposited under geo accession number gse60007. |
autism spectrum disorders (asds) are considered to be the result of a complex interaction between a genetic background and environmental factors [1, 2 ]. autism is a heterogeneous, behaviorally defined neurodevelopmental disorder affecting four times more males than females, with a clinical onset usually within the 2nd year of life and mainly consisting of social impairment ; communication difficulties ; and restricted, repetitive, and stereotyped patterns of behavior. classical autism is the overwhelmingly predominant asds phenotype, which also include other four disorders, that is, asperger syndrome and pervasive developmental disorder not otherwise specified (pdd - nos), and childhood disintegrative disorder. rett syndrome (rtt) is a genetically determined neurodevelopmental disorder with autistic features, and has been recently separated by the asds as a distinct nosological entity (diagnostic and statistical manual of mental disorders v - dsmv). the search for specific and reliable biomarkers is of paramount importance in autism, given its dramatically rising prevalence in the general population over the last two decades from 1 in 5000 in the mid-1970s to 1 in 88 in 2008 [4, 5 ]. a redox imbalance has been repeatedly reported in autism [68 ] although its role in the pathogenesis is still under question. in particular, it is unclear whether oxidative stress (os) is a cause or consequence of autism. recent studies indicate that autism is associated with deficits in glutathione antioxidant defense in selective regions of the brain, thus potentially contributing to os, immune dysfunction, and apoptosis, particularly in the cerebellum and temporal lobe. more recently, os and erythrocyte membrane alterations have been described in autistic children, including elevated erythrocyte thiobarbituric acid reactive substances, urinary isoprostane, and hexanoyl - lysine levels, associated with a significant reduction of na / k - atpase activity, a reduction of the erythrocyte membrane fluidity and an alteration in the erythrocyte fatty acid membrane profile linked to an increase in monounsaturated fatty acids, a decrease in eicosapentaenoic acid and docosahexaenoic acid, and a consequently increased -6/-3 ratio. the peculiar triad of os imbalance, mild chronic hypoxia, and an abnormally high frequency of leptocytes in the peripheral blood has been reported by our group in girls rtt, a relatively rare neurodevelopmental disorder almost affecting females and mainly due to de novo mutations of the x - linked methyl - cpg - binding protein 2 (mecp2) gene. these data indicate that redox imbalance and oxygen exchange could be key players in the pathogenesis of this particular human model of autism. the shape is critical to red blood cells (rbcs) function and blood rheological properties, and emerging evidence indicates that os is a key factors in modulating erythrocyte shape [1416 ]. although rbcs membrane lipidomics and rheological variables have been reported to be altered, and some suggestions of an increased lipid peroxidation in the erythrocyte membrane exist, to date no information is available on how the rbcs oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, erythrocyte shape in autism. in the present study, we investigated rbc morphology, in situ membrane oxidative damage, and cytoskeletal proteins in patients with classic autistic disorder a total of n = 15 patients (male : 9 ; female : 6), with classic autistic disorder (mean age at examination : 15.9 5.9 years, range 626), as well as n = 15 healthy controls of comparable age and a typical neurodevelopment (mean age : 16.3 6.2 years, range 530 ; male : 8 ; female : 7) participated in the study. furthermore, a third group of 15 patients (male : 7 ; female : 8) with nonautistic neurodevelopmental disorders (na - ndds) (mean age at examination : 15.8 6.0 years, range 528) was enrolled as a positive control population, including idiopathic mental retardation (n = 6), cerebral palsy (n = 3), attention - deficit / hyperactivity disorder (adhd) (n = 4), and language disorders (n = 2). childhood autism rating scale scores (cars) for the examined autistic patients and the na - ndds groups were estimated. the populations of patients and healthy subjects were recruited by the medical staff of the child neuropsychiatry unit of the azienda ospedaliera senese (siena, italy). the autistic patients were diagnosed by dsmv and evaluated using autism diagnostic observation schedule (ados), and autism behaviour checklist (abc). patients with rtt, x - fragile syndrome, or tuberous sclerosis, as well as subjects with clinical evident sideropenic anemia, perinatal adverse events, and/or brain abnormalities on magnetic resonance imaging, were excluded for the present study. the study was conducted after the approval by the institutional review board and all written informed consents were obtained from either the parents or the legal tutors of the enrolled patients. for these particular laboratory determinations, samples collected in tubes with k2edta were analyzed by an automated hematology system sysmex xe-2100 (sysmex corporation, japan) in the automated aspiration (i.e., closed) sampling mode, using 200 l sample volume. the instruments are in routine use for count blood cells and automated differential counts analyses and underwent periodic quality assessment in internal and external control programs. a 5-part differential count was performed by lysing erythrocytes and analyzing the light scatter / fluorescence. blood smears were stained with standard may - grnwald giemsa within 6 hours after blood sampling (sp1000 instrument) and visualized by an automated image recognition system cellavision dm96, an automated microscope with software showing digitalized images of the blood smears. for these aims, an aliquot of blood was collected in heparinized tubes, and manipulations were carried out within 2 hrs after sample collection. blood samples were centrifuged at 2400 g for 15 min at 4c, whereas the platelet poor plasma and the buffy coat were removed by aspiration. an aliquot of packed erythrocytes was resuspended in ringer solution (125 mm nacl, 5 mm kcl, 1 mm mgso4, 32 mm n-2-hydroxyethylpiperazine - n-2-ethanesulfonic acid (hepes), 5 mm glucose, 1 mm cacl2), ph 7.4 as a 50% (vol / vol) suspension for the determination of intraerythrocyte nonprotein - bound - iron (npbi). the remaining volume of packed rbcs was used for erythrocyte membranes preparations (i.e., hemoglobin - free ghosts) for 4-hydroxynonenal protein adducts (4-hne pas) determinations. an aliquot of each blood sample (1 ml) was centrifuged at 800 g for 10 minutes at 4c for scanning electron microscopy (sem) analysis of erythrocytes. as previously described, erythrocytes were plated on poly - l - lysine coated slides and fixed in karnowsky (2.5% glutaraldehyde4% paraformaldehyde in 0.1 m sodium - cacodylate buffer, ph 7.2) for 2 h at 4c, rinsed twice for 10 min with 0.1 m sodium cacodylate buffer and postfixed in 1% osmium tetroxide in 0.1 m sodium - cacodylate buffer for 1 h at 4c. specimens were then dehydrated through a graded ethanol series, dried in a co2 critical point dryer (cpd010, balzers union, liechtenstein), mounted on specimen stub, sputter coated with gold (sputter coater s150b, edwards, england), and examined in a xl 20 sem (philips, eindhoven, netherlands). altered rbcs shapes were evaluated by counting 800 cells (50 erythrocytes for each different sem field at a magnification of 3000) from all groups of subjects. intraerythrocyte npbi (nmol / ml) was determined as a desferrioxamine (dfo)-iron complex (ferrioxamine), as previously reported. briefly, 25 m dfo was added to the samples (1 ml erythrocyte suspension), the erythrocytes were then lysed by adding water (1 vol) and freezing (70c) and thawing. the hemolysate was ultrafiltered at 3373 g for 30 min in centrifugal filters with a 30 kda molecular weight cutoff (vivaspin 4, sartorius stedim biotech gmbh, goettingen germany) and the ultrafiltrate was stored at 20c until analysis. the dfo excess was removed by silica (silicagel ; 2540 m) column chromatography. the dfo - iron complex was determined by hplc and the detection wavelength was 229 nm. the calibration curve correlation for intraerythrocyte npbi was adequate (r = 0.994009), the minimum detection limit was 0.1 nmol / ml, and mean intra- and inter - observer coefficients of variation were 2.5% and 5%, respectively. an aliquot (600 l) of packed rbcs was lysed in dodge buffer, and erythrocyte membranes were prepared, according to dodge., by repeated washing until the ghosts were pearly white. samples were kept frozen at 70c until used for sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page), immunoprecipitation (ip), and esterified f2-isoprostanes (f2-isops). erythrocyte membrane (ghosts) proteins (200 g) were incubated with 5 g -actin antibody (millipore corporation, billerica, ma, usa) overnight at 4c on a rotator. then, immune complex was incubated with 50 l of protein a - sepharose (sigma - aldrich, milan, italy) and rotated at 4c for 2 h. samples were centrifuged at 10,000 g for 5 min and washed three times with 1 ml ice - cold pbs. the pellet was mixed with 2x reducing sample buffer, boiled, and loaded on sds - page gels for silver staining or western blotting analysis. erythrocyte membrane (i.e., ghosts) proteins (20 g protein, determined using biorad protein assay ; biorad, hercules, ca) or immunoprecipitated -actin were separated by the polyacrylamide gel electrophoresis (one - dimensional) method for discontinuous sds - page on 10% polyacrylamide gels in denaturing conditions, according to laemmli. at the end of the electrophoretic run, gels were stained with silver nitrate for protein visualization (sigma - aldrich, milan, italy). gel image was acquired using image scanner and the bands were automatically detected and analyzed using totallab software (nonlinear dynamics, version 1.0). band volume was expressed as a ratio of the total protein volume detected from the entire gel to minimize differences between band (band normalization) and to compare band measurements in different lanes. erythrocyte membrane proteins (40 g protein, determined using biorad protein assay ; biorad, hercules, ca) or the immunoprecipitated -actin were resolved on 10% sds - page gels and transferred onto a hybond ecl nitrocellulose membrane (ge healthcare europe gmbh, milan, italy). after blocking in 3% nonfat milk (biorad, hercules, ca, usa), the membranes were incubated overnight at 4c with goat polyclonal anti-4-hne adduct antibody (cod. ab5605 ; millipore corporation, billerica, ma, usa). following washes in tbs tween and incubation with specific secondary antibody (mouse anti - goat horseradish peroxidase - conjugated, santa cruz biotechnology, inc., ca, usa) for 1 h at rt, the membranes were incubated with ecl reagents (biorad, hercules, ca, usa) for 1 min. images were digitized (chemidoc xrs, biorad, hercules, ca) and band optical densities were quantified using a computerized imaging system (quantity one imaging system). a 100 l aliquot of the erythrocyte membrane samples was resuspended with h2o (0.9 ml) and 1 n koh (500 l) was added for the basic hydrolysis. after incubation at 45c for 45 min, the sample was acidified to ph 3 with hcl 1 n (500 l), spiked with tetradeuterated prostaglandin f2 (pgf2-d4) (500 pg in 50 l of ethanol ; cayman, ann arbor, mi, usa), as internal standard, and extracted with 10 ml of ethyl acetate. the upper organic layer, obtained after centrifugation at 1000 g for 5 min, was applied onto an aminopropyl (nh2) cartridge (waters, milford, ma, u.s.a.) preconditioned with 10 ml of hexane. after derivatization the determination of f2-isops was accomplished by gas chromatography / negative ion chemical ionization tandem mass spectrometry (gc / nici - ms / ms) analysis. esterified f2-isops were normalized for membrane proteins and quantified by biorad protein assay (biorad, hercules, ca) using 0.2% triton x-100 to dissolve the membranes. the measured ions were the product ions at m / z 299 and m / z 303 derived from the [m-181 ] precursor ions (m / z 569 and m / z 573) produced from 15-f2t - isops (the most represented f2-isop isomer) and pgf2-d4, respectively. all variables were tested for normal distribution (d'agostino - pearson test) and data were presented as means sd for normally distributed variables. differences between groups were evaluated using independent - sample t - test (continuous normally distributed data), mann - whitney rank sum test (continuous nonnormally distributed data), chi - square statistics (categorical variables with minimum number of cases per cell 5) or fisher 's exact test (categorical variables with minimum number of cases per cell na - ndds > healthy controls), thus indicating that classical autism and na - ndds are sharing erythrocyte oxidative membrane damage, although membrane proteins likely undergo different degrees of oxidation. in particular, a quantitative decrease in -actin (16 to 18.8% ; f - ratio = 314.5 ; p < 0.001) was evidenced and further confirmed in the western blot analysis carried out after actin - ip by using a specific antibody (figure 4(b)). in autistic patients the blotting study indicated detectable protein bands showing increased binding with 4-hne, that is, the major aldehyde product of lipid peroxidation, as compared to both negative and positive control subjects (figure 4(c)). -actin was found to be a major target for 4-hne binding, as indicated by densitometric analysis (+ 61.9 to + 78.8% ; f - ratio = 2622.8 ; p < 0.001) following -actin ip (figure 4(d)). the percentage of elliptocytes was positively correlated with ie - npbi, erythrocyte membrane 4-hne pas, and esterified f2-isops, as well as membrane 4-hne -actin adducts (4-hne -aas) levels. the examined os markers showed positive associations between them, while elliptocytes were inversely related to -actin membrane content, and a remarkably high inverse relationship between amount of -actin in the membrane and 4-hne -aas was observed (r = 0.960 ; p < 0.001) (table 3). our findings show the coexistence of a peculiar combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and -actin alterations, which represents a previously unrecognized triad in classical autism. several conditions, either congenital or acquired, are known to lead to abnormal erythrocyte shape. the maintenance of the discoid shape is of paramount importance for the rbc main physiological role (i.e., transport of respiratory gases to and from tissues), while the deformability of the circulating cells has critical effects on the rheological properties of blood. our observations indicate that elliptocytes are the predominant abnormal erythrocyte shape occurring in the peripheral blood from patients with classical autism, and, to the best of our knowledge, this is the first time that an abnormal erythrocyte shape is reported in asds other than rtt. the presence of abnormal rbc shapes appears to be associated with an altered redox status in erythrocytes and a decrease and/or oxidization for some of the main cytoskeletal proteins known to be critical for the maintenance of the horizontal interactions in the erythrocyte membrane architecture. although, abnormal rbc shape are reported in a series of associated conditions, this is likely the first time that an elliptocytosis is described outside of the known conditions of hereditary elliptocytosis and thalassemias. hereditary elliptocytosis is caused by weakened horizontal linkages in membrane skeleton due either to a defective spectrin dimer - dimer interaction or a defective spectrin - actin - protein 4.1r junctional complex. the severity of the disease is related to extent of decrease in membrane mechanical stability and resultant loss of membrane surface area. in contrast to the healthy population, in which elliptical rbcs shapes are up to 15% of erythrocytes, a diagnosis of elliptocytosis requires that at least 25% of erythrocytes in the specimen are abnormally elliptical in shape. therefore, it can be hypothesized that autism and hereditary elliptocytosis, an inherited blood disorder with an estimated incidence of 3 and 5 per 10,000 in the us population, may share common physiopathological mechanisms, although neither clinical signs of hemolytic anemia nor evidence of a shortened rbcs lifespan are known in autistic patients. the na - ndds group, on the other hand, shows a heterogenous variety of abnormal rbc shape changes, with only ~40% of discocytes and a slightly dominance of leptocytes which appears to be just below 25% of all erythrocyte shapes in the blood samples. in this patient group, the sem data were associated with a lower hemoglobin content and a higher degree of anisocytosis. interestingly, we have previously described a marked (~55%) leptocytosis in -3 pufas unsupplemented girls with rtt, a neurodevelopmental disorder known to be the result of a single gene mutation. in the near future, in our opinion, possible membrane cytoskeletal changes are to be carefully explored in this particular cause of cognitive impairment with autistic features. although iron deficiency in about a quarter of autistic children has been recently reported, resulting in iron deficiency anemia in about 15% of the cases and related to detective iron intake, none of our examined patients had clinically evident anemia. neuroacanthocytoses, a distinctly abnormal erythrocyte shape (i.e., acanthocytosis) is reported to be associated with neurodegeneration and specific rbcs protein defects. genetic analyses over the past two decades have linked a full host of rare mutations to increased risk for asds, with a list of hundreds of autism risk loci in the human genome, whereas also common genetic variants are reported to affect the asds risk, but their individual effects appear to be modest. nevertheless, to the best of our knowledge, estimated iq was significantly different between the two neurodevelopmental groups (i.e., autistic versus na - ndds), thus suggesting that the na - ndds group was actually composed by nonautistic patients with a demonstrated cognitive impairment, although of a less severe entity, as compared with that observed in the autistic population. therefore, it is possible that stepwise changes in red blood cell phenotype between the autistic, positive, and negative control groups may be more closely linked to iq than autism - specific differences. as a consequence, caution is needed before stating that an elliptic rbc shape is synonymous of autism, as we indicate that the triad rbc shape + membrane os damage + -actin alteration is associated with autism rather than elliptocytosis per se. furthermore, during our preliminary studies, we found that patients not currently included in the na - ndds nor in the autistic group showing mild autistic like features (i.e., a few patients with anorexia nervosa (nervous anorexia), currently classified as a psychiatric illness in the group of eating disorders and two patients with juvenile schizophrenia) showed an intermediate percentage of elliptocytes between the values here reported for the na - ndds and the control population (our unpublished data). therefore, a proportionality between percentage of elliptocytes in the peripheral blood and autistic features in the behavior might be present, although, at the present time, we have no definite proof for it. erythrocyte membrane integrity is critical for maintaining the erythrocyte characteristic shape and is based on both vertical and horizontal interactions among the cytoskeletal proteins, the integral membrane proteins, and the phospholipid bilayer. vertical interactions are based either on spectrin, ankyrin, and band 3 protein or spectrin, 4.1 protein, and glycophorin, while horizontal interactions are mainly based on spectrin, 4.1 protein and actin [3032 ]. in particular, -actin is a globular protein composed of filaments, weakly binding to the tail end of and spectrins. our present findings evidence a deficiency in -actin in the rbcs membranes from patients affected by classical autism, thus suggesting the coexistence of defective horizontal interaction forces in the cytoskeletal proteins of the erythrocyte membranes of these patients. besides the potential effects of -actin deficiency on the membrane cytoskeletal structure of classical autistic patients, the detected -actin alterations might be even more far - ranging given that emerging evidence indicated a role for -actin in motor neuron function and axonal regeneration. in particular, it has been suggested that distinct dynamics of ca--actin could be a critical player in mediating the localized actin polymerization required for cellular constriction events mediating tissue bending, synaptic plasticity, and behavior [34, 35 ]. emerging evidence indicates that the role of environmental factors acting on genome by epigenetic regulation is relevant in autism and other neurodevelopmental diseases. moreover, a large number of toxic substances are known to cause erythrocyte damage in both experimental (i.e., phenylhydrazine and dapsone) [14, 37 ] and human settings (i.e., lead, penicillins, methyl - dopa, and antiarrhythmics), often leading to haemolytic anemias through different mechanisms. in our patients no clues exist for a specific exogenous molecule potentially causing an increased prevalence of elliptocytosis, and no evidence of haemolytic anemias was detectable. to date, no specific gene mutations involving the cytoskeletal components of the erythrocyte membrane have been demonstrate. therefore, the demonstrated alteration of -actin in our autistic patients appears to be the effect of a posttranslational modification, rather than the result of a specific gene mutation in the progenitor erythrocyte cells. a link between oxidative stress and asd has been previously reported by several authors [3941 ]. over the last few years, our team has demonstrated that os is a key player in modulating the genotype - phenotype expression in rtt [20, 4246 ]. moreover, in previous reports, a correlation between os and asd has been widely explored by measuring different molecules, possibly coming from oxidative pathway as metabolic biomarkers of os, in biological fluids [10, 39 ]. in autistic patients, the immunochemical detection of 4-hne pas indicate that oxidative events are ongoing in the rbcs of autistic patients and individuals with several neurodevelopmental disorders without autistic features. the reduction in membrane -actin appears to be inversely related to the levels of 4-hne -aas, thus indicating that the apparently reduced protein expression in the erythrocyte membranes from autistic patients is rather the consequence of a peculiar posttranscriptional modification linked to lipid peroxidation than the results of a reduced protein synthesis. this event, triggered by npbi as a prooxidant factor, produces several compounds of degradation, 4-hne among them. this highly reactive aldehyde can covalently bind proteins, phospholipids, and dna ; in particular, 4-hne reacts readily with nucleophilic groups of amino acidic side chains, and its covalent attachment to proteins lead to alteration in their structure and biological activity. depending on its concentration and location, 4-hne may be therefore considered as a second toxic messenger, which disseminates and augments initial free radical events. although a protective physiological role for os for instance oxidative shielding has been recently underlined, there is little doubt that an oxidized protein is a damaged molecule with a likely reduced function. thus, within the red cell membrane environment, oxidized proteins contribute to alter the phospholipid bilayer integrity and weaken the membrane mechanical properties, including a loss of the membrane fluidity as already reported in autism. the report of significant alterations in the fatty acid profiles in individuals with asds in erythrocyte membrane appears to be in line with our morphological and biochemical observations. although an increased lipid peroxidation in the erythrocyte membrane from autistic patients has been suggested by using a thiobarbituric acid reactive substances (tbars) assay kit, to date no further information on the in situ lipid and protein membrane damage is available. our findings of increased erythrocyte membrane f2-isops, as measured by a specific spectrometric method, and increased membrane 4-hne - pas reveal an increased arachidonic acid peroxidation, with a subsequent protein posttranslational modification in the erythrocyte membrane of patients with classical autism and na - ndds. our findings indicate the presence of an unrecognized triad combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and -actin alterations in classical autism and provides new biological markers in the diagnostic workup of asds. firstly, the specificity of our findings to autistic disorders is unknown to date and needs to be further explored. secondly, the relationship between the abnormal erythrocyte shape and the neurological development in autistic children needs to be further investigated. the reported alteration in erythrocyte shape for classical autism could be theoretically translatable into a routine technology, such as fluorescence - activated cell sorting (facs) either testing the volume or the morphological complexity of cells, in addition to the membrane fluidity. in conclusion, our data shed new light on the concept of os as a key factor in the pathogenesis of neurodevelopmental disorders. interestingly, our data indicate that erythrocyte shape, either due to a defective rbc cytoskeletal scaffold or being the consequence of an oxidative cell damage, could be considered as a new potential physical biomarker for neurodevelopmental disorders. | autism spectrum disorders (asds) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. although red blood cells (rbcs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, rbcs shape in autism. here, we investigated rbc morphology by scanning electron microscopy in patients with classical autism, that is, the predominant asds phenotype (age range : 626 years), nonautistic neurodevelopmental disorders (i.e., positive controls), and healthy controls (i.e., negative controls). a high percentage of altered rbcs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. the rbcs altered morphology in autistic subjects was related to increased erythrocyte membrane f2-isoprostanes and 4-hydroxynonenal protein adducts. in addition, an oxidative damage of the erythrocyte membrane -actin protein was evidenced. therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and -actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of asds. |
there is little relationship between the incidence of a disease and the mechanistic insights that can be derived from elucidating its molecular mechanisms. most rare diseases are inherited, and advances in genomic technologies are rapidly increasing our knowledge of their genetic basis and the pathways that regulate key cellular processes. the exquisite phenotyping of human diseases is enabling intriguing genotype - phenotype relationships to be uncovered, as exemplified by a recent report by arboleda. in nature genetics on the role of cyclin - dependent kinase inhibitor 1c (cdkn1c, p57kip2) mutations in the growth retardation syndrome image and the overgrowth syndrome beckwith - wiedemann syndrome (bws). image syndrome (mendelian inheritance in man (mim) i d 300290) is a very rare inherited disorder characterized by intra - uterine growth restriction, abnormal bones (metaphyseal dysplasia), congenital adrenal gland insufficiency (adrenal hypoplasia congenita) and genital anomalies (table 1). arboleda. studied a very large family who have image syndrome and using array - based snp genotyping and targeted second generation resequencing methods, they identified a region on chromosome 11p that was shared by all affected individuals and detected a heterozygous cdkn1c missense mutation that all affected individuals had inherited from their mother. further analysis of unrelated patients with image syndrome revealed additional cdkn1c missense mutations and, remarkably, all five distinct missense mutations that were characterized occurred in the carboxy - terminal region of cdkn1c, within a cluster of six amino acids in the proliferating cell nuclear antigen (pcna)-binding domain. cdkn1c is a maternally expressed imprinted gene that maps within the 11p15.5 imprinted gene cluster and all individuals affected by image syndrome had inherited the mutation on the maternal allele. human inherited disorders associated with abnormal cdkn1c function bws is characterized by pre- and postnatal overgrowth, macroglossia (large tongue), anterior abdominal wall defects and susceptibility to embryonal tumors (most commonly wilms tumor). previously, maternally inherited cdkn1c mutations have been demonstrated to cause up to 50% of familial and 5% of sporadic cases of bws. furthermore, about 50% of sporadic bws cases have epigenetic silencing of maternal cdkn1c allele expression, secondary to epimutations at an 11p15.5 imprinting control center (ic2/kvdmr1). in contrast to the restricted type and location of cdkn1c mutations in image syndrome, those in bws - associated cdkn1c are most often truncating mutations distributed throughout the gene, or missense mutations in the amino - terminal cdk inhibitor domain, suggesting that the mutations causing image syndrome and bws have different functional effects. arboleda. found that whereas bws - associated cdkn1c mutations caused loss of cell cycle inhibition, image - associated mutations had no effect on the ability of cdkn1c to inhibit the g0-to - g1 cell cycle transition. evidence for a gain - of - function effect of image - associated cdkn1c mutations was detected in an in vivo drosophila melanogaster assay, in which expression of the mutant but not the wild - type human protein reduced eye and wing size. pcna has crucial roles in dna replication and cell cycle control (by facilitating ubiquitination and degradation of cell - cycle proteins). arboleda. found that image - associated cdkn1c mutations inhibited binding to pcna and monoubiquitination of cdkn1c. impaired ubiquitination of cdkn1c might have a number of functional consequences, and further studies are required to elucidate precisely how image - associated cdkn1c mutations produce a gain - of - function effect (including their effect on protein stability). although ubiquitin status can be an important determinant of protein stability, arboleda. point out that proteasomal degradation is typically induced by polyubiquitination, so alterations in monoubiquitination status probably influence function by an alternative mechanism. discovering of the exact pathogenic mechanisms of image cdkn1c mutations could provide a basis for developing new therapeutic strategies and targets, both for image and for other diseases associated with cdkn1c dysregulation. genomic imprinting refers to the expression of genes from only one of the two chromosome homologs depending on whether the copy is maternally or paternally inherited. imprinted genes account for less than 1% of all human genes, but those identified so far seem to be preferentially implicated in the regulation of growth and development. furthermore, in general, paternally expressed genes (such as that encoding insulin - like growth factor 2 (igf2)) promote growth, whereas maternally expressed imprinted genes (such as cdkn1c) suppress growth. these observations have been used to support the kinship (or parental conflict) theory for the evolution of parental imprinting in which it is suggested that paternally expressed imprinted genes will favor fetal growth over maternal health, whereas maternally expressed genes act to limit fetal growth so as to preserve maternal fitness and allow further progeny. image syndrome and bws are both associated with abnormal prenatal growth (under- and overgrowth, respectively). in addition, silver - russell syndrome (mim 180860 ; table 1), which is characterized by pre- and postnatal growth failure, is most commonly associated with epimutations leading to loss of igf2 expression, but has occasionally been associated with a maternal chromosome duplication that includes cdkn1c. interestingly, cdkn1c is highly expressed in the developing adrenal gland and different mutation types present different adrenal phenotypes : image syndrome is associated with congenital adrenal hypoplasia, whereas bws may be associated with adrenocortical tumors, and sporadic adrenocortical tumors can show cdkn1c inactivation. the application of high - throughput genomic analysis techniques enabled arboleda. to define the molecular basis of image syndrome. this breakthrough will facilitate diagnosis of the disorder and better assessment of genetic risk in affected families. in addition to illuminating cdkn1c growth regulatory mechanisms, arboleda. provide a further example of how the mutational diversity of inherited diseases in humans can provide a rich source of insights into basic biology and pathobiology - as extolled by william harvey more than 300 years ago : ' nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path ; nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature by careful investigation of cases of rare forms of disease '. bws : beckwith - wiedemann syndrome ; cdkn1c : cyclin - dependent kinase inhibitor 1c ; igf2 : insulin - like growth factor 2 ; mim : mendelian inheritance in man ; pcna : proliferating cell nuclear antigen. | missense mutations in the imprinted gene that encodes cyclin - dependent kinase inhibitor 1c (cdkn1c, also called p57kip2) result in a rare disorder associated with prenatal growth retardation (image syndrome). loss - of - function mutations in cdkn1c have been previously described in the congenital overgrowth syndrome beckwith - wiedemann syndrome and some cancers. in contrast, a recent study by arboleda. proposes that the cdkn1c mutations associated with image syndrome have a gain - of - function effect. these findings highlight how rare genetic disorders can provide important insights into the regulation of critical processes such as regulation of cell growth. |
having adequate and up - to - date information and knowledge about patients is crucial in order to maintain continuity of care [1, 2 ]. the traditional oral handover, where nurses orally give information and their assessment of the situation and needs of individual patients (supported by some brief written notes), has played a central role in securing that information has been exchanged adequately. the oral handover has become an important part of nursing practice and is strongly embedded in hospital culture. however, it has become a well - known fact that hospitals and other parts of the health care service suffer from communication problems [4, 5 ]. often, such problems are the cause of more serious medical and nursing errors [6, 7 ]. although it clearly has some advantages, such as immediacy, the possibility for interactivity, and its social importance as a meeting point for nurses, the traditional oral handover has been identified as one possible area for miscommunication for instance, because central information is not conveyed. it has also been challenged as being ineffective, focusing on physical aspects of care, and that a majority of information present in the oral handover should be available in formal documentation sources. thus, it is not surprising that many nurses (22%61%) have expressed dissatisfaction with the traditional oral handover. the electronic patient record (epr) has recently become a key factor in the information flow, and it has been suggested that using information technology can be an effective method for improving quality, efficiency, and costs [1416 ]. in any case, it is evident that the introduction of the epr reshapes information management, creates new communication patterns, and enables the development of new practice models in nursing. recently, the development of the profession as well as legal concerns have lead to an increased emphasis on nursing care plans and written nursing documentation. some studies [1821 ] have suggested that the introduction of the written handover procedure has improved nursing documentation as well as communication between nurses. the introduction of the electronic handover is quite recent, and few have examined how nursing staff have responded to this change in routines. moreover, there have been relatively few studies of handover routines in nonacute psychiatric wards. in the present study, we report on the attitudes and perceptions of nursing staff to the electronic handover routine at a ward in a norwegian university hospital. the study was carried out at the psychogeriatric ward at the university hospital of northern norway (unn). this is an inpatient ward with 14 beds that treats patients who are suffering from psychiatric disorders, including depression, psychosis, dementia, and anxiety disorders. the problems facing patients are complex, since most of the patients suffer from somatic illnesses in addition to the psychiatric illness. the patients are subjected to various types of tests (blood work, imaging, physical examination, psychological testing, etc.) and are diagnosed, and appropriate treatment is subsequently initiated. the work at the ward is highly interdisciplinary, and the clinical staff comprises psychiatrists, physicians, psychologists, nurses, assistant nurses, social workers, occupational therapists, physiotherapists, and some unskilled staff. the number of nursing staff on duty at the ward varies. normally, there are 8 on the day shift, 6 on the evening shift, and 3 on the night shift. nurses from all shifts were asked to participate in the study. since the nursing work is based on primary nursing, that is, where a nursing team provides complete care for a small group of patients, the staff gets to know the patients well during the stay. at the study ward, a new handover routine was implemented. instead of presenting all information orally (a 30 minute meeting at each handover covering all patients in the ward), staff would now read the relevant electronic nursing care plans regarding their own patients only. in addition, every morning, there was now a 30 minute meeting for professional discussion, usually covering 2 - 3 patients, but also including discussions not relating to specific patients. the main objective of the morning meeting was to provide an opportunity for discussion and reflection in relation to various nursing topics, and thus ensure the multifunctional aspect to the handover process. during the meeting, the electronic care plan was visualized on the screen wall by a projector to provide an opportunity for collective reading and participation. the topics of the meetings were not prearranged, and staff could suggest topics at the beginning of each meeting. typically, discussions focused on matters related to current nursing challenges in the ward. apart from some items in the questionnaire study reported here, there was no systematic evaluation of the morning meetings, but oral feedback from the participants was used to make minor adjustments to the form and content of the meetings. moreover, to facilitate the introduction of the new handover process and make a gradual transition to the new structure, it was decided to keep the oral handover on monday mornings and friday afternoons and also the oral handover between the evening and night shifts. the remaining traditional oral handovers were meant to supplement the new system where the nurses read the relevant electronic nursing care plans, and the nurses were required to read up on their own patients also when traditional oral handover meetings were arranged. use of the new electronic system also required skills and knowledge of the use of the care plan in general, as well as the use of a standardized language. at the core of the nursing care plan was its shared terminology to describe the patient 's problem (i.e., nursing diagnosis) and link this to one or more interventions. the classification systems of the north american nursing diagnosis association (nanda) and the nursing intervention classification (nic) were embedded in the system and had become an integrated part of the electronic documentation of nursing at the ward. to secure the quality of the electronic nursing care plans during their implementation phase, support and supervision was offered by two specially trained nurses. they were available every day between 12 noon and 1.30 p.m. and could assist the other nursing staff in making sure that the nursing documentation was reliable and updated. the guidance was focused on nursing care plans in general and on use of the electronic records system. a questionnaire was designed in order to examine the attitudes and perceptions of the nursing staff to the new electronic handover practice. the questionnaire focused on issues related to perceived usefulness and perceived ease of use and was inspired by the technology acceptance model (tam). tam is a theory adapted from the theory of reasoned action and was tailored for modeling user acceptance of information technology. according to tam theory, two determinants are particularly important for users ' acceptance : perceived usefulness and perceived ease of use. several developments and extensions to tam have been proposed, typically including several determinants of intention and usage and also moderators of effects [2527 ]. the main idea in tam and its later modifications, that is, the relationship between perceived usefulness and intention to use / actual use, is simple and appears to stand robust. some of the items in the questionnaire used in the present study resembled items on the 6 + 6 item versions of the tam questionnaires originally described by davis, while other items were more specific to the circumstances of the study ward. the appropriateness of the questionnaire - items was discussed in a small group of project members, including clinicians, in order to increase the face validity of the questionnaire. two of the group members (nurses that had working experience from the study ward) made a first version of the questionnaire, and two other group members (a nurse and a doctor, both with clinical and research experience) gave comments and suggestions ; this process was repeated several times until the complete group was satisfied with the final result. the questionnaire was sent to all nursing staff in the form of an e - mail link to a secure web - based form. the researchers did not have access to information regarding who had responded to the questionnaire (and who had not). the questionnaire did not contain information that could be used to identify individual respondents, and all respondents were anonymous. the questionnaire consisted of 22 questions and covered different topics, including questions on satisfaction with the new handover practice and the perceived effectiveness and usefulness of the epr system. there were also questions about how the respondents felt about the new morning meeting and questions relating to guidance and support with respect to the new handover routine as well as the epr in general. three possible options were provided to each question, with the response options low, medium, and high., there was also information about the purpose of the survey, information stating that participation was voluntary, and that anonymity and confidentiality were assured. the study was approved by the head of the hospital division and was in accordance with the regulations of the hospital 's data protection officer. the survey sampled anonymous information about staff attitudes, and no patient information was sampled. in addition, a multiple regression analysis was performed in order to examine predictors of satisfaction with the new handover routine. items entered as predictors in the model included whether the respondents found time and place to read, their trust in written documentation, whether they believed reading saved time, whether they believed other written sources were important, whether they believed morning meetings were important, whether they believed working on handover routines improved nursing standards, whether they felt independent in writing nursing documentation, and if they believed guidance was important in implementing the new routine. the results were analyzed using the statistics software spss 16.0. a significance level of p <.05 was employed. of the 34 that were eligible to participate, 32 responded, giving a response rate of 94%. seventy - five percent were very satisfied or satisfied with the electronic handover (table 1), and 78% also felt the system was satisfactory in securing that they were updated on the patients ' situation and needs. most staff (93.8%) were also satisfied or very satisfied with making their own assessments regarding the need for information about specific patients. in this study, we did not ask which information sources they used, but earlier investigations at the ward have shown that the nursing care module in the epr system has become the single most important element in the documentation work of the nursing staff. nevertheless, there was still a certain amount of skepticism in trusting electronic nursing notes, and only 37.5% felt they could rely unconditionally on the nursing module in the epr. informal sources of information, such as the weekend summary and the message book, still play an important role in the information exchange, and 93.5% responded that such sources of information were very important or important. the other new intervention in the new handover process was the introduction of the morning meeting, and all the respondents felt that the morning meeting was important or very important to professional development, and they were also satisfied or very satisfied with the topics that were discussed during the meetings. interestingly, almost all (96.2%) the respondents considered the daily documentation guidance significant to their professional development. although they had been using epr and the electronic nursing module for several years, there were still many who needed guidance to use the epr, to find nursing concepts, and to make changes in the care plans, and 15.4% felt they needed a lot of guidance. a multiple regression analysis was performed, where a set of variables (see table 2) were entered as predictors and degree of satisfaction with the new handover routine was the outcome variable. in the analysis, we found that staff that were able to find a time and place to read the written (electronic) report, that expressed a high level of trust in written (electronic) reports, that believed written (electronic) reports saved time, that considered other sources of information as less important (i.e., message book, weekend summary, etc.), and that believed that working on handover routines improved nursing standards were significantly more satisfied with the use of the electronic handover than other staff. we also found that being less content with the topics of the morning meeting and having less faith in the importance of the oral handover were related to a higher degree of satisfaction with the new handover routine. believing that one was independent in writing nursing documentation or expressing that guidance in using the nursing documentation was important were not significant predictors (table 2). thus, the results may be taken to suggest that nursing staff that felt they had sufficient opportunity to use the electronic nursing record, and those who trusted the electronic records and felt they saved time were generally more satisfied with the new handover routine. believing that improving routines was important and having less faith in the traditional oral handover the main finding in this study is that the nursing staff was satisfied with the electronic handover routine. they were content with being able to judge which information they needed and believed staff were kept updated on patients ' needs. however, the implementation of the new electronic handover procedure has involved a paradigm shift in the information work at the ward ; the structure of the handover has changed, and the focus has moved from oral to written information. the successful introduction of the new electronic handover is composed by multiple factors that provide new opportunities but also involve new challenges. the study has showed that some nursing staff need guidance in order to make use of the electronic nursing documentation system, including the electronic handover. such a system for guidance should be in place in order to secure a smooth transition from an oral to an electronic handover practice. the study has demonstrated that a gradual implementation of a new handover process has been adopted by the users, and it is likely that one reason for a successful adoption was that local and professional needs were ensured. the use of electronic care plans and nursing classifications has become a major factor in the information work at the ward, and our study shows that a majority of nursing staff rely on the written documentation. reliable and accurate documentation was a prerequisite for changing the handover process. before the implementation of the electronic nursing module, the documentation was incomplete, and the language used in the documentation was inaccurate and often included local (i.e., to the ward) jargon, as has been shown to be the case in other wards. the use of the care plans has increased significantly after the implementation of the nursing module and provides a more complete documentation of both given and planned care. as a main source of information and knowledge of the patients, the use of the electronic nursing module provides a good structure for information exchange between nursing staff and provides a standardized language that contributes to a common understanding of given and planned care. our findings are also in accordance with legal, political, and professional strategies to electronic cooperation in the health and social sector and are consistent with similar studies [1820 ]. however, there are still some that are not satisfied with the new handover routines and some that do not trust the written documentation. this tension between written documentation and face - to - face communication is well described in the nursing literature and in health informatics. clinical judgment in tremendously complex, requires various types of knowledge, and involves both explicit and tacit knowledge. the tacit, embedded know - how is gained from experience and is interplay between theoretical knowledge and practical know - how. this kind of knowledge is difficult to formalize in an electronic care plan, and some kind of face - to - face communication is necessary to ensure all aspects of clinical judgment and reflection. with the introduction of the new morning meeting as the shifts of the nursing staff rotate, all nursing staff get the opportunity to participate (although not all the time). the study also shows that all were satisfied with the topics and the impact of the morning meetings on professional development. typical topics for the meetings were different problems related to care issues, and the meetings provided an opportunity for collective reflection and the development of clinical judgment. it is usually the nurse in charge of professional development that leads the meeting, but the topics are determined in collaboration with nursing staff. often, it is 2 - 3 patient cases that form the basis of the discussion. use of the electronic care plans has also become a key element during the meetings, and they are used as a basis for discussion and reflection. since the care plans are visualized by projecting the pc screen on the wall in the conference room, they provide an opportunity for collective reading and discussion. this interaction provides an opportunity to socialize, share, and learn from each other 's experiences as well as an opportunity for emotional support. particularly interesting was the finding that those who had a high level of trust in written reports, believed these saved time, and had little trouble finding time and a place to read the reports were more positive to the new handover routine. while the small sample of the present study should be taken into consideration when analyzing the results, this finding could suggest that the successful (in terms of the satisfaction of the nursing staff) implementation of electronic nursing routines in part depends on nursing staff being used to written reports. extrapolating from this idea, one can hypothesize / speculate that wards that to a lesser extent have used written (i.e., pen and paper) reports will need more time and effort to successfully implement new electronic routines. although the response rate was quite high, the sample was relatively small and covered one ward only. thus, it is problematic to claim that the findings of the present study reflect nurses ' acceptance of electronic handovers in general. studies with larger samples will be needed to verify the findings of the present study and to make more general claims about nurses ' attitudes to electronic handovers. moreover, several changes in ward routines were simultaneously introduced, that is, the new (electronic) handover routine and the new morning meeting. although we believe it was necessary to make both changes at the same time, we do not know how satisfied the nursing staff would have been with the new handover routine without the new morning meeting. this study contributes to an increased understanding of the nursing handover per se and to the implementation of an e - health solution in clinical practice. further studies should address implications of the electronic handover for issues such as the impact on the quality of patients ' care, patients ' satisfaction, and the impact on how information is shared within and across organizational boundaries. most of the nursing staff was satisfied with the electronic handover procedure although a minority was less trustful of written information. introducing a forum for oral discussion on topics of importance to the staff 's professional development and securing sufficient guidance on how to use the system were perceived as central factors to success in the implementation. | following the implementation of electronic nursing records in a psychogeriatric ward, we examined nursing staff 's attitudes and perceptions to the implementation of an electronic handover routine. a web - based anonymous and secure questionnaire was distributed by e - mail to all nursing staff at a psychogeriatric ward at a university hospital. most respondents were satisfied with the electronic handover, and they believed they managed to keep informed by the new routine. the simultaneous introduction of a morning meeting, to ensure a forum for oral professional discussion, was a success. a minority of staff did not fully trust the information conveyed in the electronic handover, and a significant proportion expressed a need for guidance in using the system. staff that had a high level of trust in written reports believed these saved time, had little trouble finding time and a place to read the reports, and were more positive to the new handover routine. |
bioreactors are widely used in research involving vascular and heart valve substitutes, whether these substitutes are made of biomaterials or constructed using tissue engineering techniques. they are necessary to reproduce physiological conditions, either for the observation of the performance of these substitutes or for the elaboration of new techniques yielding these constructs. a bioreactor designed for this purpose should allow the reproduction of blood flow and arterial pressure, as well as other relevant biochemical and physical parameters such as temperature, ph, co2, and o2 concentrations. difficulties associated with the complexity of the parameters to simulate render an exact reproduction of this environment difficult. the foremost difficulty arises from the dynamic pulsating nature of arterial blood flow and pressure. the strongly nonlinear relation existing between these two quantities is a direct consequence of the nonlinearity of the involved physical phenomena, notably the interaction between the fluid and the visco - elastic tissues. the global physics of the fluid flow produced by a bioreactor is often studied using mathematical models consisting of different assemblies of basic components, and are referred to as windkessel (wk) or lumped models. the basic components used to represent the actual bioreactor and translate it into a mathematical model are a flow source, flow resistances, elastic accumulators, and inertial components representing the mass inertia of blood in arteries. each one of these elements represents a physical component of the bioreactor, which in turn simulates an actual property of the cardiovascular environment. (this parameter is noted r in wk models) mimics the blood flow restrictions caused by arteries, capillaries, and veins. it is responsible for systemic pressure increase when flow is forced into the circulatory system by the pump. the elastic accumulator is obtained by the insertion, in the bioreactor circuit, of a closed volume containing a certain amount of air. during systole, the air is compressed in the accumulator because the fluid is prevented from circulating freely by the flow resistance. the flow accumulator thus simulates the physiological compliance property (c parameter in wk models) associated with tissue proclivity to expand under a blood pressure increase. many studies have reported the importance of an accurate reproduction of compliant effects in bioreactors, especially in tissue engineering applications where strong evidence of a significant relation between the dynamic frequency spectrum of the pulsating flow and tissue compliance has been observed. the work presented in this paper has been carried out in the design process of a new bioreactor dedicated specifically to tissue engineering of heart valves. one of the objectives underlying the project was an optimal simultaneous reproduction of the dynamic waveforms of arterial pressure and blood flow rate. the aim of this article is not to present the bioreactor (which we have reported), but to present experimental data allowing a physical comparison between three much - covered wk architectures. no such systematic, experiment - based comparison is available in the literature, although a very large number of articles report wk - related studies. previous bioreactors producing physiological flow and pressure waveforms were either explicitly based on wk models, such as those designed by dumont., hildebrand., and knierbein., or entail the appropriate elements structured in an adequate manner to simulate the hemodynamic environment, like those developed by warnock., konduri., and narita. generally, macro - physiological properties of the cardiovascular system, such as the aortic and arterial compliance, resistance, and inertial effects, are modeled using few components (from two to four elements). it can be noticed in several articles presenting mathematical analyses of different arrangements that the complexity of the differential equation describing the system rapidly increases with the number of elements constituting the model. some papers propose a dynamic response / transfer function type analysis, while others directly solve the differential equation by numerical methods. stergiopulos. studied the differences between three- and four - component architectures and compared their theoretical responses to physiological data, but not to experimental data obtained with actual bioreactors. more complex models of the arterial tree have also been proposed and combined to numerical resolution algorithms, providing another comprehensive tool for the study of cardiovascular behavior. conlon. published a model composed of 14 elements whereas another model, developed by liang. the usefulness of the wk approach is also enhanced in numerous experimental studies addressing physiological data analysis of human subjects. in particular, it has been demonstrated that frequency content analysis of arterial pressure waveforms and wk modeling could advantageously support pathology identification techniques, notably for hypertension. the literature review of papers presenting the wk approach permits one to appreciate the depth of the concept as well as its ability to model the dynamics of the cardiovascular system, especially in cases of models that include more than two components. although many papers outline the assets of wk architectures more complex than the classic two - component model, the vast majority of bioreactors found in publications are actually developed according to that basic configuration. others present sophisticated bioreactor configurations but without a systematic theoretical analysis to support and justify the design. in the present article, we propose a bridge between wk modeling, theoretical study, and the actual construction of corresponding bioreactors. we will present a complete and systematic analysis of the three most common wk architectures : rc, rrc, and rrcl. in these designations, r and c represent the resistive and compliant components, while l represents inertia. a recent article by westerhof. presents a very interesting review of theoretical aspects related to these three wk models. in the work presented here theoretical results will be compared to experimental data obtained from three actual bioreactors that were built according to these architectures. this will establish the benefit of bioreactor architecture complexity in providing an accurate simultaneous reproduction of both the flow and the pressure waveforms. previous bioreactors producing physiological flow and pressure waveforms were either explicitly based on wk models, such as those designed by dumont., hildebrand., and knierbein., or entail the appropriate elements structured in an adequate manner to simulate the hemodynamic environment, like those developed by warnock., konduri., and narita. generally, macro - physiological properties of the cardiovascular system, such as the aortic and arterial compliance, resistance, and inertial effects, are modeled using few components (from two to four elements). it can be noticed in several articles presenting mathematical analyses of different arrangements that the complexity of the differential equation describing the system rapidly increases with the number of elements constituting the model. some papers propose a dynamic response / transfer function type analysis, while others directly solve the differential equation by numerical methods. studied the differences between three- and four - component architectures and compared their theoretical responses to physiological data, but not to experimental data obtained with actual bioreactors. more complex models of the arterial tree have also been proposed and combined to numerical resolution algorithms, providing another comprehensive tool for the study of cardiovascular behavior. conlon. published a model composed of 14 elements whereas another model, developed by liang. the usefulness of the wk approach is also enhanced in numerous experimental studies addressing physiological data analysis of human subjects. in particular, it has been demonstrated that frequency content analysis of arterial pressure waveforms and wk modeling could advantageously support pathology identification techniques, notably for hypertension. the literature review of papers presenting the wk approach permits one to appreciate the depth of the concept as well as its ability to model the dynamics of the cardiovascular system, especially in cases of models that include more than two components. although many papers outline the assets of wk architectures more complex than the classic two - component model, the vast majority of bioreactors found in publications are actually developed according to that basic configuration. others present sophisticated bioreactor configurations but without a systematic theoretical analysis to support and justify the design. in the present article, we propose a bridge between wk modeling, theoretical study, and the actual construction of corresponding bioreactors. we will present a complete and systematic analysis of the three most common wk architectures : rc, rrc, and rrcl. in these designations, r and c represent the resistive and compliant components, while l represents inertia. a recent article by westerhof. presents a very interesting review of theoretical aspects related to these three wk models. in the work presented here theoretical results will be compared to experimental data obtained from three actual bioreactors that were built according to these architectures. this will establish the benefit of bioreactor architecture complexity in providing an accurate simultaneous reproduction of both the flow and the pressure waveforms. schematic representations of the first bioreactor configuration (rc model) are presented in figure 1. the pump drives the flow in the circuit, composed of a compliance chamber c followed by a resistance r and a return to the pump. the test section is located immediately after the pump, so that the conditions felt by the heart valve are a flow rate q and a pressure p. indices indicate in which component a physical parameter is evaluated. a corresponding electrical circuit is also presented in figure 1, as this is the simplified representation generally encountered in the literature related to wk models, and also because of the physical analogy between the two systems. tissue compliance, simulated by the compliance chamber (and analog to an electric capacitance) is defined as the ratio between the difference in volume induced by a pressure variation and that pressure variation : c = dvdpc where v is the volume and pc the pressure in the compliance chamber. deriving numerator and denominator with respect to time, a direct relation is obtained between the flow and the pressure gradient : c = dvdtdpcdt = qcdpcdt or qc = cdpcdt figure 1(a) rc bioreactor configuration ; (b) corresponding electric circuit. (a) rc bioreactor configuration ; (b) corresponding electric circuit. assuming a negligible pressure drop between the pump and the compliance chamber (very short length of tube linking these two elements), we have p = pc and qc = cdpdt the analysis is based on the assumption that the flow is laminar (which is generally true for physiological flows), and the relation between qr and the pressure difference pr across the resistance can be expressed as : qr= cst pr where cst is a constant. defining the hydraulic resistance as the ratio of the pressure increase to the imposed flow, we have r=1cst=prqr qr=prr assuming that the pressure difference between the exit of the resistance and the entrance of the pump is negligible, we have : pr = pc = p then qr = pr noting that q = qc + qr, the following differential equation is obtained for the rc bioreactor : q = pr+cdpdt an actual bioreactor based on this two - component rc configuration was built. the flow is produced by a custom design diaphragm pump, driven by a computer - controlled proportional servo - valve (festo mpye-5-, mississauga, on, usa). the pump allows a pulsating frequency range of 60180 bpm and a stroke volume up to 160 ml. from the exit of the pump, the fluid flows through a flexible 25-mm internal diameter tube (tygon, us plastic corp., lima, oh, usa), onto which is mounted a noninvasive ultrasonic flow sensor probe (transonics systems inc. immediately downstream, a valve holder maintains the aortic valve while exposing it to a surrounding geometry reproducing the complex tridimensional sinuses forming the aortic root, thus providing an accurate reproduction of physiological flow dynamics. fluid pressure upstream and downstream of the valve is monitored with 07 psi dynamic pressure sensors (argon medical devices, athens, tx, usa). a compliance chamber (custom - made glass reservoir) the compliance value can be adjusted between 0 and 1 ml / mmhg, while the resistance range is 0.51.2 mmhg.s.ml. all components and sensors mentioned in this section are interfaced to a usb national instruments i / o board, with control and data acquisition tasks performed through a custom - built labview application program. the difference between this arrangement (figure 2) and the previous one is that a second resistance rc has been added between the pump and the compliance chamber. although rc presents the units of a resistance, it corresponds to the characteristic (or total) impedance of the aorta, a combination of its local compliance and the inertance of the blood volume. according to the continuity equation, the relation between the different flow rates is : q = qrc = qc+qrp figure 2(a) rrc bioreactor configuration ; (b) corresponding electric circuit. flow qrc can be expressed by the ratio of the pressure difference across the resistance rc, and the resistance rc itself : qrc=prcrc replacing terms qc, qrc, and qrp in equation by their corresponding expressions yields the following differential equation : q=prcrc = cdpcdt+prprp assuming that the pressure difference between the exit of the resistance and the entrance of the pump is negligible, that is : pc=prp the following differential equation is obtained for the rrc bioreactor : prp+cdpdt=(1+rcrp)q+rccdqdt to obtain the rrc arrangement with the actual bioreactor, a second resistance was added between the valve holder and the compliance chamber, while other components remained unchanged. schematic representations of the rrcl bioreactor are presented in figure 3. here a tube with a certain length l and a cross - section a is added in parallel to the resistance rc. the flow restriction in this tube is low compared to that of the resistance rc. however, the fluid mass it contains presents inertia opposed to large accelerations. as outlined by stergiopulos., the frequency response of this arrangement is more likely to produce a physiological pressure waveform corresponding to the one normally observed in vivo ; that is, the natural response to a physiological flow rate input. for low frequencies (near steady flow conditions), the fluid flows freely in the tube as its resistance is lower than that of rc. on the other hand, higher frequency components of the flow rate can not circulate freely because fluid inertia opposes sudden accelerations : the higher frequencies therefore flow through the resistance rc and are attenuated in amplitude. the added tubing thus acts like an electric inductance l. according to the continuity equation, the relation between the different flow rates is : pc=prp figure 3(a) rrcl bioreactor configuration ; (b) corresponding electric circuit. flow ql is defined using the relation existing between pressure and flow modulated by inertial effects (inertance l) of the moving mass of fluid : ql=1lpldt this relation between flow and pressure is a direct result from the fact that a pressure difference pl is required to produce acceleration. considering inertial effects as widely predominant with respect to viscous friction at the wall, we can express this relation with newton s second law : f = ma which can be easily expressed in terms of the flow parameters : apl=al(1adqldt) where is the fluid s density, and the tube s cross section a is assumed to be constant over time. rearranging equation, we obtain an expression for the flow rate ql : pl=(la)dqldtql=(al)pldt=(1l)pldt, l=(la) replacing the terms qc, qrc, qrp and ql in equation by their corresponding expressions [equations 3, 11, and 16 ] yields the following differential equation : q=prcrc+1lpldt=prprp+cdpcdt and noting that prc=pl and prp=pc the following differential equation is obtained for the rrcl bioreactor : rcrpcld2qdt2+l(rc+rp)dqdt+rcrpq = rpcld2pdt2+(l+rcrpc)dpdt+rcp to obtain the rrcl arrangement with the actual bioreactor, flow dividers were inserted before and after the resistance rc, and a certain length of tubing was connected in parallel to that resistance. to test the three configurations presented in the previous section, reference waveforms corresponding to clinical measurements of arterial flow rate and pressure performed simultaneously on a healthy individual were used. calculations with these models were widely used in the design process, with a constant retroaction between theory and design to dimension bioreactor components properly. the first step was to perform, for each model, a parametric study enhancing the influence of the r, c, and l parameters on the system response. a matlab program was developed to solve the differential equations, and corresponding to the three models and perform these parametric studies. for this the flow rate (dotted line in figure 4a) was used as the input of the system, and the corresponding pressure response was calculated as a function of r, c, and l. optimal parameter values were obtained by implementing the conjugate gradients method in the differential equation solver program to determine, from a theoretical point of view, which parameters were more likely to produce the reference pressure waveform when the reference flow rate waveform was used as the input. then the dimensions of the bioreactor were calculated from these results to physically achieve these optimal parameter values. the bioreactor was constructed and the three arrangements were tested and compared, first to the predictions of their corresponding mathematical model, then to each other. to compare the waveforms and obtain an evaluation of the correlation between two curves, a coefficient of correlation r was calculated : r=(pi, exppexp)(pi, refpref)(pi, exppexp)2(pi, refpref)2 where pi, exp represents discrete experimental data, pexp is the mean value of the experimental data, pi, ref represents discrete reference data, and pref is the mean value of the reference data. the coefficient of correlation r ranges from 01, the upper limit corresponding to a perfect fit. schematic representations of the first bioreactor configuration (rc model) are presented in figure 1. the pump drives the flow in the circuit, composed of a compliance chamber c followed by a resistance r and a return to the pump. the test section is located immediately after the pump, so that the conditions felt by the heart valve are a flow rate q and a pressure p. indices indicate in which component a physical parameter is evaluated. a corresponding electrical circuit is also presented in figure 1, as this is the simplified representation generally encountered in the literature related to wk models, and also because of the physical analogy between the two systems. tissue compliance, simulated by the compliance chamber (and analog to an electric capacitance) is defined as the ratio between the difference in volume induced by a pressure variation and that pressure variation : c = dvdpc where v is the volume and pc the pressure in the compliance chamber. deriving numerator and denominator with respect to time, a direct relation is obtained between the flow and the pressure gradient : c = dvdtdpcdt = qcdpcdt or qc = cdpcdt figure 1(a) rc bioreactor configuration ; (b) corresponding electric circuit. (a) rc bioreactor configuration ; (b) corresponding electric circuit. assuming a negligible pressure drop between the pump and the compliance chamber (very short length of tube linking these two elements), we have p = pc and qc = cdpdt the analysis is based on the assumption that the flow is laminar (which is generally true for physiological flows), and the relation between qr and the pressure difference pr across the resistance can be expressed as : qr= cst pr where cst is a constant. defining the hydraulic resistance as the ratio of the pressure increase to the imposed flow, we have r=1cst=prqr qr=prr assuming that the pressure difference between the exit of the resistance and the entrance of the pump is negligible, we have : pr = pc = p then qr = pr noting that q = qc + qr, the following differential equation is obtained for the rc bioreactor : q = pr+cdpdt an actual bioreactor based on this two - component rc configuration was built. the flow is produced by a custom design diaphragm pump, driven by a computer - controlled proportional servo - valve (festo mpye-5-, mississauga, on, usa). the pump allows a pulsating frequency range of 60180 bpm and a stroke volume up to 160 ml. from the exit of the pump, the fluid flows through a flexible 25-mm internal diameter tube (tygon, us plastic corp., lima, oh, usa), onto which is mounted a noninvasive ultrasonic flow sensor probe (transonics systems inc. immediately downstream, a valve holder maintains the aortic valve while exposing it to a surrounding geometry reproducing the complex tridimensional sinuses forming the aortic root, thus providing an accurate reproduction of physiological flow dynamics. fluid pressure upstream and downstream of the valve is monitored with 07 psi dynamic pressure sensors (argon medical devices, athens, tx, usa). a compliance chamber (custom - made glass reservoir) the compliance value can be adjusted between 0 and 1 ml / mmhg, while the resistance range is 0.51.2 mmhg.s.ml. all components and sensors mentioned in this section are interfaced to a usb national instruments i / o board, with control and data acquisition tasks performed through a custom - built labview application program. the difference between this arrangement (figure 2) and the previous one is that a second resistance rc has been added between the pump and the compliance chamber. although rc presents the units of a resistance, it corresponds to the characteristic (or total) impedance of the aorta, a combination of its local compliance and the inertance of the blood volume. according to the continuity equation, the relation between the different flow rates is : q = qrc = qc+qrp figure 2(a) rrc bioreactor configuration ; (b) corresponding electric circuit. flow qrc can be expressed by the ratio of the pressure difference across the resistance rc, and the resistance rc itself : qrc=prcrc replacing terms qc, qrc, and qrp in equation by their corresponding expressions yields the following differential equation : q=prcrc = cdpcdt+prprp assuming that the pressure difference between the exit of the resistance and the entrance of the pump is negligible, that is : pc=prp the following differential equation is obtained for the rrc bioreactor : prp+cdpdt=(1+rcrp)q+rccdqdt to obtain the rrc arrangement with the actual bioreactor, a second resistance was added between the valve holder and the compliance chamber, while other components remained unchanged. schematic representations of the rrcl bioreactor are presented in figure 3. here a tube with a certain length l and a cross - section a is added in parallel to the resistance rc. the flow restriction in this tube is low compared to that of the resistance rc. however, the fluid mass it contains presents inertia opposed to large accelerations. as outlined by stergiopulos., the frequency response of this arrangement is more likely to produce a physiological pressure waveform corresponding to the one normally observed in vivo ; that is, the natural response to a physiological flow rate input. for low frequencies (near steady flow conditions), the fluid flows freely in the tube as its resistance is lower than that of rc. on the other hand, higher frequency components of the flow rate can not circulate freely because fluid inertia opposes sudden accelerations : the higher frequencies therefore flow through the resistance rc and are attenuated in amplitude. the added tubing thus acts like an electric inductance l. according to the continuity equation, the relation between the different flow rates is : pc=prp figure 3(a) rrcl bioreactor configuration ; (b) corresponding electric circuit. flow ql is defined using the relation existing between pressure and flow modulated by inertial effects (inertance l) of the moving mass of fluid : ql=1lpldt this relation between flow and pressure is a direct result from the fact that a pressure difference pl is required to produce acceleration. considering inertial effects as widely predominant with respect to viscous friction at the wall, we can express this relation with newton s second law : f = ma which can be easily expressed in terms of the flow parameters : apl=al(1adqldt) where is the fluid s density, and the tube s cross section a is assumed to be constant over time. rearranging equation, we obtain an expression for the flow rate ql : pl=(la)dqldtql=(al)pldt=(1l)pldt, l=(la) replacing the terms qc, qrc, qrp and ql in equation by their corresponding expressions [equations 3, 11, and 16 ] yields the following differential equation : q=prcrc+1lpldt=prprp+cdpcdt and noting that prc=pl and prp=pc the following differential equation is obtained for the rrcl bioreactor : rcrpcld2qdt2+l(rc+rp)dqdt+rcrpq = rpcld2pdt2+(l+rcrpc)dpdt+rcp to obtain the rrcl arrangement with the actual bioreactor, flow dividers were inserted before and after the resistance rc, and a certain length of tubing was connected in parallel to that resistance. to test the three configurations presented in the previous section, reference waveforms corresponding to clinical measurements of arterial flow rate and pressure performed simultaneously on a healthy individual were used. calculations with these models were widely used in the design process, with a constant retroaction between theory and design to dimension bioreactor components properly. the first step was to perform, for each model, a parametric study enhancing the influence of the r, c, and l parameters on the system response. a matlab program was developed to solve the differential equations, and corresponding to the three models and perform these parametric studies. for this the flow rate (dotted line in figure 4a) was used as the input of the system, and the corresponding pressure response was calculated as a function of r, c, and l. optimal parameter values were obtained by implementing the conjugate gradients method in the differential equation solver program to determine, from a theoretical point of view, which parameters were more likely to produce the reference pressure waveform when the reference flow rate waveform was used as the input. then the dimensions of the bioreactor were calculated from these results to physically achieve these optimal parameter values. the bioreactor was constructed and the three arrangements were tested and compared, first to the predictions of their corresponding mathematical model, then to each other. to compare the waveforms and obtain an evaluation of the correlation between two curves, a coefficient of correlation r was calculated : r=(pi, exppexp)(pi, refpref)(pi, exppexp)2(pi, refpref)2 where pi, exp represents discrete experimental data, pexp is the mean value of the experimental data, pi, ref represents discrete reference data, and pref is the mean value of the reference data. the coefficient of correlation r ranges from 01, the upper limit corresponding to a perfect fit. figure 4 presents experimental measurements of the flow rate and pressure waveforms produced by the rc bioreactor. it can be observed that the shape of the measured flow rate (figure 4a) is very similar to the physiological reference waveform, and the coefficient of correlation between the two curves is 0.976. the pressure waveform measured in the bioreactor (figure 4b) is different from the reference pressure, but nevertheless presenting the same general shape. the differences between the waveforms are a certain phase lag as well as a higher amplitude. the delay is related to the fact that air must be compressed in the compliant element before the pressure increases, while the higher amplitude can be attributed to a slightly suboptimal compliance value. the coefficient of correlation between the measured pressure and the reference is 0.335, owed mainly to the delay rather than to the shape of the waveform. the theoretical rc pressure response, calculated by optimizing the model to obtain experimental r and c parameters (r=1.16 mmhg.s.ml and c=0.91 ml.mmhg), is in good agreement with the experimental pressure waveform (coefficient of correlation = 0.960). the higher frequency oscillations of the pressure waveform at minimal and maximal values may be attributed to vibrations of the flexible components of the system (tubing) at the opening and closing of the valve leaflets, and by free surface movement in the compliance chamber. these results show that appropriately calibrated resistance and compliance components produce a good first approximation of the pressure response, and also that the wk model describes the global physics of the actual bioreactor well (excellent correlation between model and measurements) and constitutes an appropriate approach to support its design and dimensioning. in the rrc configuration, the addition of a second resistance to the circuit before the compliance chamber eliminates the phase shift : a direct and simultaneous pressure increase is produced (proportionally) by the passage of the flow through the resistance. this can be appreciated in figure 5, where the experimental pressure curve can be seen in phase with the physiological reference waveform. excellent agreement can be observed between the theoretical rrc model and the experimental results (coefficient of correlation = 0.947). the addition of a fourth component further improves the natural response of the bioreactor (figure 6). the value of the coefficient of correlation is increased to 0.816, and the overall pressure waveform presents a more accurate reproduction of the shape characteristics of the physiological reference. in particular, the inertance l added in parallel to the resistance rc located upstream from the compliance chamber complicates the dynamic interaction between rc, l, and c. it was found that a significant length of tubing was required to achieve the optimal response of the system (l 2.5 m). at the beginning of systole, the fluid must be set in motion. if it flows through the resistance rc, the pressure increases proportionally to the flow. if it flows through the inertance l, mass inertia also produces a pressure increase. the balance between the amount of fluid flowing through rc and l depends on the relative magnitude of the impedance of both branches (resistance and inertance). a large resistance value will force the fluid to flow through the inertance branch if its length l is such that this path is easier. if l is very long and the mass is difficult to set in motion, the fluid may prefer to flow through the resistance. this balance between the two paths changes dynamically because when the fluid starts to flow into the inertance branch, its resistance to motion decreases and requires a lower pressure to maintain the flow. figure 6experimental results for the rrcl configuration. it was observed experimentally that the initial phase of systole produced a pressure increase in good agreement with the physiological reference. then a combination of the two following phenomena occurred : when the fluid gradually gained velocity in the inductance branch, the flow resistance decreased and the pressure decreased accordingly as this path offered less restriction ; then a flow inversion occurred in the rc branch and fed the inductance branch. the momentum was simply transferred quite freely from one branch to another and the consequence on the pressure was a truncation of the systole pressure peak. it was observed that low resistance values enhance backflow occurrence and truncation of the systole pressure peak. to avoid this truncation during the systolic phase, it was found that a high rc resistance value was required. in addition, to avoid fluid bypass of the rc branch, a large inductance value was required. consequently, high settings of both rc and l ensured that sufficient flow resistance was present in both branches, resulting in an adequate pressure increase over the whole systolic phase. these high settings of rc and l also meant that the flow inverting phenomena in the rc branch was less pronounced in amplitude as well as more delayed in time, but nevertheless present. in fact it occurred later, in the decreasing part of the systolic waveforms, and yielded an additional momentum pulse to an otherwise decreasing waveform, thus reproducing the dicrotic pulse. for the third configuration, the correlation between the theoretical model and the experimental results was good but lower than for the two previous architectures (coefficient of correlation = 0.741). this can be attributed to the fact that the differences between the ideal and actual components are more important ; namely, because of the connections geometry and also because of the multiple - nature of rc and l. in reality l is not purely inductive as it also offers a certain resistance, and correspondingly rc is not purely resistive. this emphasizes certain limitations of wk models in providing accurate information about actual physical systems. even considering the fact that the four - component architecture improves the natural, passive response of the bioreactor, we finally decided that future work would be based on the simpler three - component configuration for two main reasons. first, our aim is to reach the maximum level of simplicity, because fewer components mean easier assembly and more efficient sterilization operations, which in turn should help long - term sterility. this is a major issue for a bioreactor designed specifically for the development of tissue engineering construction techniques, which may require up to several weeks of continuous operation. second, the modification of flow conditions with a four - component bioreactor requires changing hardware components, which is obviously more complicated (if not completely impossible without interrupting flow circulation) than just programing a simpler setup that could eventually be equipped with controllable components. in this paper we presented a detailed study of the theoretical response of three wk configurations. we then compared these results to experimental data obtained with three actual bioreactors constructed according to these architectures. these results showed that : i) wk models are good tools to support the design of experimental bioreactors, and ii) the addition of a third and a fourth component to the bioreactor greatly improved the passive reproduction of the pressure waveform to a physiological flow rate. | this paper presents an experimental study of three bioreactor configurations. the bioreactor is intended to be used for the development of tissue - engineered heart valve substitutes. therefore it must be able to reproduce physiological flow and pressure waveforms accurately. a detailed analysis of three bioreactor arrangements is presented using mathematical models based on the windkessel (wk) approach. first, a review of the many applications of this approach in medical studies enhances its fundamental nature and its usefulness. then the models are developed with reference to the actual components of the bioreactor. this study emphasizes different conflicting issues arising in the design process of a bioreactor for biomedical purposes, where an optimization process is essential to reach a compromise satisfying all conditions. two important aspects are the need for a simple system providing ease of use and long - term sterility, opposed to the need for an advanced (thus more complex) architecture capable of a more accurate reproduction of the physiological environment. three classic wk architectures are analyzed, and experimental results enhance the advantages and limitations of each one. |
one of the few aaa+ proteins for which a large number of nucleotide complexes are available at high resolution is the bacterial transcription activator, pspf, a member of clade 6 of aaa+ proteins 13. pspf activates -dependent rna polymerase (rnap) by converting it from a closed (inactive) state to the open (active) conformation that initiates transcription. structural information is available at high resolution for apo, adp, atp and adpnp bound forms of the aaa+ domain of pspf 13. comparison of the adp and atp complexes (figure 2) reveals that the structures superimpose very well with the exception of a few regions that respond to the presence of the -phosphate. although most of the active site residues alter little, there is a substantial movement of the glutamate residue of the dexx motif. in the adp complex, the glutamate side chain is found in a position similar to that seen in most other atpase active sites (e.g. f1 atpase -subunit 14) (figure 2). by contrast, in the atp complex, the glutamate is rotated approximately 100 from the conformation in the adp complex and forms a hydrogen bond with an asparagine residue that is located on an adjacent -strand (figure 2). the effect of this rotation is to move the glutamate from a position in which it would be able to activate the in - coming attacking water molecule, to one in which it would not. consequently, binding of atp has the surprising effect of inactivating the atpase activity by switching the active site from an active configuration to an inactive one. this glutamate switch converts the enzyme from a low energy ground state to a higher energy form that is now trapped in an inactive state. this conformation of the protein is stabilised by a set of water - mediated interactions that involve the -phosphate of the bound atp as well as the glutamate and asparagine side chains and the magnesium ion. consistent with these observations, the glutamate - asparagine pair is also present in the atp - bound complex of the related activator zrar 15 but is not formed in the adp bound complex of another regulator, ntrc1 16. the second area of movement within the pspf complexes is remote from the active site and involves two loops (l1 and l2) that interact with -rnap 17. however, there is a direct peptide linkage between the e - n pair and these loops, providing communication between the atpase and ligand - binding sites. although there is no high resolution structure of the complex between pspf and -rnap, it is known that the complex is most stable when formed with the transition state analogue adp - alfx rather than adp or atp 18. the corollary of this observation is that the interaction between -rnap and pspf must stabilise the transition state and hence stimulate atpase activity, most likely by releasing the glutamate in the atpase active site from the inactive to the active configuration. however, confirmation of this proposal will require high resolution structural information of the pspf//rnap complex. analysis of the available crystal structures reveals that a glutamate switch operates in a similar manner in other aaa+ protein clades. in addition to pspf (clade 6), other examples include dna clamp loaders such as rfc (clade 1) 19,20, dna replication initiators such as orc (clade 2) 21,22, and the molecular chaperone p97 (clade 3) 23. the hsluv / clp family proteases and ruvb - like helicases (clade 5) have an interesting variation in which the glutamate contacts a conserved threonine (or serine) residue that is placed two residues further along -strand 2 24 - 26. of course, similar pairings may be present in other systems for which there is insufficient structural information at present or for which the resolution of the structural data is too low for detailed analysis. in fact, sequence analysis reveals that examples of a conserved glutamate switch pair can be found in members of six of the seven aaa+ clades (figure 3a). due to its key role in promoting atp hydrolysis, the glutamate residue is almost invariant across all aaa+ proteins but in rare cases can be an aspartate. although also highly conserved, the asparagine can be replaced in some proteins by serine, threonine, or lysine, all residues that could make an interaction with a glutamate. we analysed 50 side chain conformations of the glu residue from aaa+ protein structures in the database with a resolution better than 3.5. the side chain torsion angles (chi1 & chi2) show a clear bimodal distribution of chi2 angles (figure 3b), with approximately 100 degrees difference between the two clusters. this is in perfect agreement with what was observed in pspf between the adp and atp states described above. interestingly, the active conformation, represented by the adp state of pspf, shows a tighter clustering of chi2 angles compared to that of the inactive conformation represented by the atp state of pspf, consistent with the geometric constraints for positioning the glu residue correctly for polarizing the attacking water molecule. furthermore, where it has been identified, the binding site for target ligands on the aaa+ domain involves the same region of the fold in all clades (figure 4) suggesting a conservation of the linkage between atpase and ligand binding sites. interestingly, for type ii aaa+ proteins such as p97, the second aaa+ domain (d2), which is not directly involved in substrate binding, interacts with the first aaa+ domain (d1) via similar regions in d2 (figure 4) 27. similar cooperativity between the two domains has also been proposed for other type ii aaa+ proteins 28,29. the formation of the switch pair when atp is bound offers a means to suppress the atpase which can be alleviated upon ligand binding. however, in principle, there is no reason why this linkage might not be reversed. indeed, this is the case for clade 2 dna initiator proteins, such as orc and dnaa, the only clade for which there is also a crystal structure with the target ligand bound. in these proteins, the glutamate (aspartate in dnaa) remains in the active configuration in both the adp and atp analogue complexes in the absence of dna 4,30,31 (figure 2). however, this family of proteins is unusual in that ligand binding (in this case dna) inhibits the atpase activity rather than stimulating it 9. recent structures of archaeal orc proteins bound to their dna targets 21,22 reveals that the e - n pair is formed in these complexes (figure 2), precisely what would be expected for a system in which ligand binding inhibits, rather than stimulates, atpase activity. consequently, the glutamate switch can function in either direction, to stimulate or inhibit atp hydrolysis at different stages within a complex assembly process. for example, atpase activity of hslu is stimulated by binding of both hslv and substrate albeit by only 3 - 4 fold in each case 32. the structures show the glutamate is locked in the inactive configuration in the adp complex but is free in the atp bound form. in this case, the small stimulation compared to other systems may reflect a difference in equilibrium between on and off states. interestingly, in sv40 large t antigen (ltag), which catalyses unwinding of long stretches of dna during virus replication 33, the glutamate also remains in the active configuration in both adp and atp analogue complexes 34. however, full length ltag also contains a domain that binds to the sv40 replication origin. it is likely that the helicase activity needs to be restrained prior to origin firing and this may explain the presence of the e - n pair and its potential role as a switch upon replication origin binding. however, after origin firing, the enzyme needs to be a processive helicase and the switch is no longer required. consistent with this suggestion, the atpase activity of human papilloma virus e1 helicase (which is closely related to ltag and also has a conserved switch pair) is inhibited by the e2 origin binding protein35. although the glutamate switch is widespread across aaa+ families, there are a few exceptions notably several proteins in clade 7, a group with a wide variety of unrelated functions and for which there are few representative crystal structures 4. the key role for the glutamate residue has been demonstrated for aaa+ proteins of every clade as well as for atpases of many other families. mutations at this position typically reduce atpase activity by at least 1 - 2 orders of magnitude. however, more significantly, the link between atpase activity and ligand binding should be broken in mutants lacking either the glutamate or the asparagine. although there are very few cases where this has been properly explored, this is indeed the case for the archaeal clamp loader complex rfc for which it has been shown that the glutamate to alanine mutant not only has a substantially reduced atpase activity but this basal activity of the complex is no longer stimulated by dna 8. although the effects of mutating the asparagine in rfc have not been reported, biochemical data for a number of substitutions at this residue in pspf have been reported recently 36. mutating the asparagine in pspf abolishes the inhibitory effects of its negative regulator, pspa, consistent with the hypothesis that the e - n pair communicates ligand binding to atpase activity. pspf hexamers are stabilised by atp binding and mutants in which the switch pair can no longer form are defective in hexamerisation. consequently, analysis of the data is complicated by these defects in hexamerisation (also required for maximal atpase activity) and hence limits the information that can be drawn from these mutations. the only other protein in which the asparagine has been mutated is the clade 2 protein, cdc6, which has a role in the initiation of dna replication in eukaryotes. yeast cell lines expressing a mutant cdc6 protein in which the asparagine, and the residues either side of it, were replaced with alanine residues showed a temperature - sensitive phenotype despite showing a wildtype level of expression of the mutant protein 37. the mutant cell line was defective in the rate of dna replication and cells halted growth in s phase. many atpases utilise the free energy of hydrolysis of atp to drive reactions, either by transferring chemical energy from one form to another or converting it into mechanical energy. by contrast, the sorts of reactions catalysed by aaa+ enzymes are frequently different in a subtle way. inhibition of atpase activity is found in systems with complex assembly pathways and multiple intermediate steps. typically, atp hydrolysis is suppressed (e.g. through atp binding in rfc or ligand binding as in orc) until the system is fully assembled, but atp turnover then allows completion of the reaction and/or recycling of components. many reactions, such as cargo delivery or loading, involve single events on a particular macromolecular substrate requiring a series of steps that need to occur in a precise pathway. in these systems, atp is used to control the directionality of the reaction rather than simply in a catalytic role, usually by regulating the assembly of a multi - component system. the role of atp is to ensure the correct assembly of the components rather than as a means to lower the activation energy of the reaction. as the system begins to assemble, there are several well characterised examples of these principles across many clades of aaa+ proteins. one of these is synaptic vesicle fusion in which the complex between nsf, snaps and snares is stabilised on atp binding but disassembles when atp is hydrolysed38. the loading of pcna rings onto dna primer junctions by rfc is another example (figure 5). in this case, atp binding is sufficient to stabilise the rfc / pcna complex and even to support loading of the clamp around dna but dna - stimulated hydrolysis of atp releases the pcna onto the dna and recycles the rfc components to allow them to pick up another pcna ring8. yet another example is the activation of rna polymerase/ by transcriptional activators such as pspf or ntrc39 (figure 5). rnap/ binds to the promoter site and forms a stable closed complex that is unable to initiate transcription without being remodelled by activators. these activators bind to a specific activation sequence, located 80 - 150 bp upstream from the transcriptional starting site. atp binding is required for stable hexamer formation of the activators and their interactions with rnap/ through dna looping, a process facilitated by dna bending proteins such as integrative host factor (ihf). atp hydrolysis releases the activators from the activated rnap/ allowing transcription to proceed. in all of these examples, atp hydrolysis serves as a switch to control the process and recycle the components after completion of the reaction. the coupling to atp hydrolysis allows control of the process and explains the requirement for atp in processes that, formally, are energetically favourable or neutral. for example, some ring helicases can load themsleves onto dna but the involvement of atp - dependent helicase loaders (such as orc and dnac) provides controlled loading at specific sites and times during the cell cycle rather than at random sites over the genome. similarly, although cleavage of the peptide bond is favourable, controlled proteolysis by aaa+ proteases, such as clpxp and hsluv, is a much more discriminating process that only digests proteins that are selectively delivered to the protease by unfolding them in situ. the small energetic cost of utilising atp as a switch is outweighed by the advantages of assembling the systems correctly and thereby controlling reactions that might otherwise have drastic consequences for the cell. | aaa+ proteins carry out diverse functions in cells. in most cases, their atpase activity is very tightly regulated by protein partners and target ligands but the mechanism for this control has remained unclear. we have identified a conserved link between the ligand binding and atpase sites in aaa+ proteins. this link, which we call the glutamate switch, regulates atpase activity directly in response to binding of target ligands by controlling the orientation of the conserved glutamate residue in the dexx motif, switching it between active and inactive conformations. the reasons for this level of control of the atpase activity are discussed in the context of the biological processes catalysed by aaa+ proteins. |
the main pathophysiologic abnormalities in type 2 diabetes (t2d) are impaired tissue sensitivity to insulin action (i.e., insulin resistance) and impaired -cell insulin secretion (1). autopsy studies have observed a relative -cell mass reduction of 40% from normal by the time impaired fasting glucose develops (110 mg / dl) and > 60% reduction with overt t2d (2). this decline in -cell mass has been associated with increased -cell apoptosis (2), and an emerging role of defective autophagy - associated cell death is linked with the onset of -cell dysfunction (3,4). functional -cell mass is best estimated in vivo as the -cell secretory capacity derived from glucose potentiation of arginine - induced insulin secretion (5). consistent with the autopsy data, metabolic studies have reported a relative -cell secretory capacity reduction of > 50% from normal as the fasting glucose increases over 110 mg / dl (1,5). the preservation of functional -cell mass in t2d remains a major focus of research in hopes of stabilizing or reversing disease progression (6). agents that enhance glp-1 action are purported to hold promise for the preservation of -cell mass in t2d. glp-1 is an incretin hormone secreted by l cells of the intestine in response to nutrient ingestion, enhances insulin production and secretion, and inhibits -cell glucagon secretion in a glucose - dependent manner (7). the biologically active glp-1736 amide is rapidly inactivated by the ubiquitous protease dipeptidyl peptidase iv (dpp4). raising glp-1 to supraphysiologic levels current strategies to enhance glp-1 effects in t2d include the use of injectable glp-1 analogues that resist inactivation by dpp4 and oral inhibitors of dpp4 that effectively increase endogenous glp-1 levels. in rodent models, glp-1 stimulates -cell proliferation and exerts antiapoptotic effects, which, together with increased insulin production, is expected to augment functional -cell mass in vivo (9,10). whether glp-1 can increase functional -cell mass in human diabetes in vivo remains to be elucidated. current investigations suggest the acute improvement in -cell sensitivity to glucose observed with enhancing glp-1 effects in human t2d (8) may not extend to long - term effects on functional -cell mass. (11,12) demonstrated that 1-year treatment with exenatide in t2d significantly improved -cell secretory capacity while on the drug ; however, this benefit was not sustained 1 month after discontinuation. similarly, there was a significant increase in -cell secretory capacity in drug - naive t2d subjects treated with the dpp4 inhibitor vildagliptin for 1 year that again was not maintained after a 3-month washout period (13). these studies indicate an acute effect of glp-1 analogues or dpp4 inhibitors to increase -cell secretion but do not demonstrate a modifying effect of either drug on functional -cell mass. these conflicting glp-1 effects in rodents versus human t2d may be due to the lengthy duration of the washout period, during which glycemic control worsened in both clinical studies such that glucotoxicity may have obviated any previous improvement in -cell secretory capacity (1113). the purpose of this investigation was to address if increasing glp-1 effects early in the course of t2d would preserve or increase functional -cell mass as measured by -cell secretory capacity derived from the glucose - potentiated arginine (gpa) test. in this study, we present the results of a randomized controlled trial comparing the effects of exenatide or sitagliptin with glimepiride as an active comparator insulin secretogogue on -cell secretory capacity before and after 6 months of treatment 5 days off drug in subjects with impaired fasting glucose or early t2d (fasting glucose 110 but 12-h overnight fast performed off any antidiabetogenic agent for 2 weeks (6 weeks for thiazolinediones) and of stable body weight (5%) for at least 2 weeks. exclusion criteria included any prior exposure to glp-1 analogues or dpp4 inhibitors and active cardiovascular, liver, or kidney disease and are provided in full detail under clinicaltrials.gov identification number nct00775684. the study protocol was approved by the university of pennsylvania institutional review board, and all subjects provided written informed consent. one hundred seventy subjects underwent the screening process, out of which 50 subjects were enrolled (supplementary fig. randomization was performed with stratification designed to balance sex and tiers of age (1844 and 4570 years), fasting glucose level (110126 and 127159 mg / dl), and bmi (10 half - lives ; see supplementary data) prior to completing a final gpa test. the washout period was used to obviate any acute effect of exenatide, sitagliptin, or glimepiride on -cell insulin or -cell glucagon secretion during testing. all subjects arrived at the university of pennsylvania clinical and translational research center on the morning of testing having fasted overnight after 2000 h for 12 h. by 0700 h, one catheter was placed in an antecubital vein for infusions, and one catheter was placed in a contralateral hand or forearm vein, retrograde when possible, for blood sampling, with the hand or arm warmed by a heating pad to promote arterialization of venous blood (21). after baseline blood sampling at 5 and 1 min, 75 g of anhydrous glucose in solution was ingested over a 5-min period. blood samples were collected at 15, 30, 60, 90, and 120 min postingestion. at the 5-month visit, morning study medication was held until the completion of testing. after baseline blood sampling at 5 and 1 min, 5 g arginine hydrochloride (10% solution) blood samples were collected at 2, 3, 4, and 5 min postinjection. after this baseline arginine stimulation test (ast), a hyperglycemic clamp technique (22) utilizing a variable rate of a 20% glucose solution was performed to achieve a plasma glucose level of 230 mg / dl. blood samples were taken every 5 min and measured at the bedside by an automated glucose analyzer (ysi 2300 ; yellow springs instruments, yellow springs, oh) and used to adjust the infusion rate to achieve the desired level. it has been demonstrated that the first administration of arginine has no effect on the subsequent response to arginine using this protocol (23). then, after a 2-h period without glucose infusion, a hyperglycemic clamp was performed to achieve a plasma glucose level of 340 mg / dl. forty - five minutes after initiation of the glucose infusion, another ast was performed. samples were collected on ice into tubes containing edta and protease inhibitor cocktail (and for ogtt samples, dpp4 inhibitor ; sigma - aldrich, st. louis, mo), centrifuged at 4c, separated, and frozen at 80c for subsequent analysis. plasma glucose was determined in duplicate by an automated glucose analyzer (ysi 2300 ; yellow springs instruments). plasma insulin, proinsulin, and glucagon were measured in duplicate by double - antibody radioimmunoassays and glp-1736 amide by elisa (millipore, billerica, ma). each immunoassay for all time points in a given subject was conducted simultaneously, with representative subjects from each group included with each assay run. plasma glucose, insulin, and glp-1 responses during the ogtt were evaluated by the incremental area under the curve (auc) calculated by the trapezoidal rule with the mean of the baseline values subtracted using the computer software origin (northampton, ma). the gpa test enables characterization of glucose - dependent insulin secretion from the glucose dose - response curve for acute insulin response to arginine (airarg) performed at fasting, 230 mg / dl, and 340 mg / dl glucose levels. the ast measures first - phase insulin release to a maximally stimulating dose of the nonglucose secretagogue arginine as the airarg. the airarg was determined as the mean of the 2-, 3-, 4-, and 5-min insulin levels minus the mean of the baseline values (8,24,25). the airarg performed during the 230 mg / dl glucose clamp enables determination of glucose potentiation of arginine - induced insulin release (airpot). the airarg performed during the 340 mg / dl glucose clamp allows for determination of the -cell secretory capacity (airmax) since the airarg is maximal at plasma glucose concentrations > 315 mg / dl (26,27). between 60 and 250 mg / dl, the magnitude of airarg is a linear function of the plasma glucose level, so the difference in airarg at fasting and 230 mg / dl glucose levels divided by the difference in plasma glucose (airarg/pg) gives the glucose - potentiation slope (gps) (8,2426). using the y - intercept (b) from the line created by these two points, the plasma glucose level at which half - maximal insulin secretion is achieved (pg50) is derived from solving the equation 1/2 (airmax) = (gps pg50) + b, and provides a measure of -cell sensitivity to glucose (8,2426). insulin sensitivity (m / i) was determined by dividing the mean glucose infusion rate required during the 230 mg / dl glucose clamp (m) by the mean prestimulus insulin level (i) between 40 and 45 min of the glucose infusion (28). the proinsulin - to - insulin (pi / i) ratio was calculated as the molar concentration of proinsulin divided by the molar concentration of insulin 100. estimation of the pi / i ratio within the secretory granules of the -cell is most reliable after acute stimulation of release (29) ; therefore, we examined the proinsulin secretory ratio in response to each injection of arginine from the respective acute pi / i responses to arginine by dividing the acute proinsulin responses by the acute insulin responses. the acute glucagon response to arginine (agrarg), glucose inhibition of arginine - induced glucagon release (agrinh), and minimum arginine - induced glucagon secretion (agrmin) were calculated similarly as the mean of the postarginine values minus the mean of the prestimulus values under the fasting, 230 mg / dl, and 340 mg / dl glucose clamp conditions (26). the primary outcome was to determine if increasing glp-1 effects by exenatide or sitagliptin preserved or increased functional -cell mass measured as -cell secretory capacity compared with glimepiride in subjects with early t2d. during year 4 of the study, a prespecified interim analysis was performed after 30 subjects had completed the study (10 subjects in each study group). this interim analysis demonstrated that the change in -cell secretory capacity (airmax) was less in exenatide (p 12-h overnight fast performed off any antidiabetogenic agent for 2 weeks (6 weeks for thiazolinediones) and of stable body weight (5%) for at least 2 weeks. exclusion criteria included any prior exposure to glp-1 analogues or dpp4 inhibitors and active cardiovascular, liver, or kidney disease and are provided in full detail under clinicaltrials.gov identification number nct00775684. the study protocol was approved by the university of pennsylvania institutional review board, and all subjects provided written informed consent. one hundred seventy subjects underwent the screening process, out of which 50 subjects were enrolled (supplementary fig. randomization was performed with stratification designed to balance sex and tiers of age (1844 and 4570 years), fasting glucose level (110126 and 127159 mg / dl), and bmi (10 half - lives ; see supplementary data) prior to completing a final gpa test. the washout period was used to obviate any acute effect of exenatide, sitagliptin, or glimepiride on -cell insulin or -cell glucagon secretion during testing. all subjects arrived at the university of pennsylvania clinical and translational research center on the morning of testing having fasted overnight after 2000 h for 12 h. by 0700 h, one catheter was placed in an antecubital vein for infusions, and one catheter was placed in a contralateral hand or forearm vein, retrograde when possible, for blood sampling, with the hand or arm warmed by a heating pad to promote arterialization of venous blood (21). after baseline blood sampling at 5 and 1 min, 75 g of anhydrous glucose in solution was ingested over a 5-min period. blood samples were collected at 15, 30, 60, 90, and 120 min postingestion. at the 5-month visit, after baseline blood sampling at 5 and 1 min, 5 g arginine hydrochloride (10% solution) was injected over a 1-min period. blood samples were collected at 2, 3, 4, and 5 min postinjection. after this baseline arginine stimulation test (ast), a hyperglycemic clamp technique (22) utilizing a variable rate of a 20% glucose solution was performed to achieve a plasma glucose level of 230 mg / dl. blood samples were taken every 5 min and measured at the bedside by an automated glucose analyzer (ysi 2300 ; yellow springs instruments, yellow springs, oh) and used to adjust the infusion rate to achieve the desired level. it has been demonstrated that the first administration of arginine has no effect on the subsequent response to arginine using this protocol (23). then, after a 2-h period without glucose infusion, a hyperglycemic clamp was performed to achieve a plasma glucose level of 340 mg / dl. forty - five minutes after initiation of the glucose infusion, another ast was performed. samples were collected on ice into tubes containing edta and protease inhibitor cocktail (and for ogtt samples, dpp4 inhibitor ; sigma - aldrich, st. louis, mo), centrifuged at 4c, separated, and frozen at 80c for subsequent analysis. plasma glucose was determined in duplicate by an automated glucose analyzer (ysi 2300 ; yellow springs instruments). plasma insulin, proinsulin, and glucagon were measured in duplicate by double - antibody radioimmunoassays and glp-1736 amide by elisa (millipore, billerica, ma). each immunoassay for all time points in a given subject was conducted simultaneously, with representative subjects from each group included with each assay run. plasma glucose, insulin, and glp-1 responses during the ogtt were evaluated by the incremental area under the curve (auc) calculated by the trapezoidal rule with the mean of the baseline values subtracted using the computer software origin (northampton, ma). the gpa test enables characterization of glucose - dependent insulin secretion from the glucose dose - response curve for acute insulin response to arginine (airarg) performed at fasting, 230 mg / dl, and 340 mg / dl glucose levels. the ast measures first - phase insulin release to a maximally stimulating dose of the nonglucose secretagogue arginine as the airarg. the airarg was determined as the mean of the 2-, 3-, 4-, and 5-min insulin levels minus the mean of the baseline values (8,24,25). the airarg performed during the 230 mg / dl glucose clamp enables determination of glucose potentiation of arginine - induced insulin release (airpot). the airarg performed during the 340 mg / dl glucose clamp allows for determination of the -cell secretory capacity (airmax) since the airarg is maximal at plasma glucose concentrations > 315 mg / dl (26,27). between 60 and 250 mg / dl, the magnitude of airarg is a linear function of the plasma glucose level, so the difference in airarg at fasting and 230 mg / dl glucose levels divided by the difference in plasma glucose (airarg/pg) gives the glucose - potentiation slope (gps) (8,2426). using the y - intercept (b) from the line created by these two points, the plasma glucose level at which half - maximal insulin secretion is achieved (pg50) is derived from solving the equation 1/2 (airmax) = (gps pg50) + b, and provides a measure of -cell sensitivity to glucose (8,2426). insulin sensitivity (m / i) was determined by dividing the mean glucose infusion rate required during the 230 mg / dl glucose clamp (m) by the mean prestimulus insulin level (i) between 40 and 45 min of the glucose infusion (28). the proinsulin - to - insulin (pi / i) ratio was calculated as the molar concentration of proinsulin divided by the molar concentration of insulin 100. estimation of the pi / i ratio within the secretory granules of the -cell is most reliable after acute stimulation of release (29) ; therefore, we examined the proinsulin secretory ratio in response to each injection of arginine from the respective acute pi / i responses to arginine by dividing the acute proinsulin responses by the acute insulin responses. the acute glucagon response to arginine (agrarg), glucose inhibition of arginine - induced glucagon release (agrinh), and minimum arginine - induced glucagon secretion (agrmin) were calculated similarly as the mean of the postarginine values minus the mean of the prestimulus values under the fasting, 230 mg / dl, and 340 mg / dl glucose clamp conditions (26). the primary outcome was to determine if increasing glp-1 effects by exenatide or sitagliptin preserved or increased functional -cell mass measured as -cell secretory capacity compared with glimepiride in subjects with early t2d. during year 4 of the study, a prespecified interim analysis was performed after 30 subjects had completed the study (10 subjects in each study group). this interim analysis demonstrated that the change in -cell secretory capacity (airmax) was less in exenatide (p < 0.05) and not different in sitagliptin compared with the glimepiride group, an effect driven by an increase in -cell secretory capacity in the glimepiride group (p < 0.05). these results effectively refuted our initial hypothesis such that no further subjects were enrolled, although those already active in the study completed the clinical trial to yield the final number of subjects reported. subjects who did not complete the final gpa test were not included in the primary analysis. this was a two - factor experimental design with one repeated measure (baseline and final) and one nonrepeated measure (exenatide, sitagliptin, and glimepiride). to determine if exenatide or sitagliptin induced significant changes from baseline, the change for each measure (= final baseline) for each group was compared with the change in the glimepiride group using independent student t tests or the mann - whitney u test for nonparametric data. one - way anova was used to compare baseline measures across all three groups. repeated - measures anova was used to compare changes in weight, capillary blood glucose, and hba1c over time between groups. when a statistical trend (p < 0.1) was evident with at least one of the between - group differences with glimepiride, within - group changes from baseline to final measures were compared with dependent student t tests or the wilcoxon matched - pairs test as appropriate. of the 50 subjects enrolled, 47 subjects underwent randomization and completed baseline gpa testing (supplementary fig. 1) with 17 subjects randomized to exenatide, 13 to sitagliptin, and 17 to glimepiride. there were 7 dropouts, such that 14 subjects in the exenatide, 12 in the sitagliptin, and 14 in the glimepiride group completed the study for a total of 40 subjects used in the analysis. reasons for discontinuation included noncompliance with study medication and/or procedures and adverse events (supplementary fig. baseline demographic measures were similar across all three groups (table 1). at 6 months, change in weight or bmi were not significantly different across all three groups. within the exenatide group, subjects had a decrease in weight over the 6-month course of study (2.6 0.8 ; p < 0.05 ; fig. 1) that was also reflected by a decrease in bmi (p < 0.05 ; table 1). fasting plasma glucose, insulin, and glucagon were similar at baseline and 6 months in all three groups (table 1). the change in average capillary blood glucose over time was significantly different across all three groups (p < 0.01 ; fig. 1). the glimepiride group had a greater reduction in capillary blood glucose than the sitagliptin group (p < 0.01) and the exenatide group by trend (p < 0.1). the lower glucose levels were achieved by 3 months when the average dose of glimepiride was 3 mg. change in hba1c over time was different by trend across all three groups (p = 0.07). the change in hba1c in the exenatide group was not different from that in the glimepiride group, but the change in hba1c in the sitagliptin group was less from that in the glimepiride group (p < 0.05 ; fig. hba1c trended lower within the exenatide group (p = 0.09) at the end of 6 months, whereas it decreased significantly with glimepiride (p < 0.05). lipid profiles were similar across all three groups at baseline, but at 6 months, there was an increase in hdl cholesterol with exenatide, but not sitagliptin treatment, that was significantly greater than that seen with glimepiride treatment (p < 0.05 ; table 1). subject demographics at baseline and final (after 6 months of therapy) visits, change from baseline to 6 months with each value. final visits after 6 months of therapy were conducted following a 5- to 7-day drug washout. to convert to mmol / mol, multiply by 10.93 and subtract 23.50. means se of weight, fasting capillary glucose as determined by glucometer readings, and hba1c in each group. changes in weight over time were not significantly different across the three groups [f(12, 222) = 1.1013 ; p = 0.4 ]. average capillary glucose was significantly different [f(12, 204) = 2.53 ; p < 0.01 ] when comparing across all three groups. hba1c was different by trend [f(4, 74) = 2.28 ; p < 0.1 ] when comparing across all three groups. p < 0.05 when comparing from baseline within each group at each time point. at 5 months, the changes in plasma glucose (supplementary fig. 2, middle) responses were not significantly different with either exenatide or sitagliptin when compared with glimepiride treatment. the change in endogenous glp-1 response was increased with sitagliptin compared with glimepiride treatment (auc 284 70 vs. 31 24 pmol 2, bottom). there were no significant differences in the change in airarg after 6 months of exenatide or sitagliptin treatment compared with glimepiride (supplementary fig. 3 and table 2), whereas the change in airpot was significantly lower in the exenatide group, but not in the sitagliptin group, when compared with the glimepiride group (p < 0.05 for exenatide vs. glimepiride ; table 2), and a similar trend was also evident for the change in airmax (p = 0.1 for exenatide vs. glimepiride ; table 2). in fact, within each group, -cell secretory capacity (airmax) increased only in the glimepiride group at 6 months compared with baseline (p < 0.05 ; table 2). -cell sensitivity to glucose (pg50) was not different at baseline and remained unchanged following any treatment (table 2). the glucose infusion rates (m), second - phase insulin levels (i), and the resulting estimate of insulin sensitivity (m / i) were not different at baseline and after 6 months across all three groups (table 2). measures of -cell secretory capacity, -cell sensitivity to glucose, insulin sensitivity, and glucagon secretion derived from the gpa test, change from baseline to 6 months with each value. final visits after 6 months of therapy were conducted following a 5- to 7-day drug washout. p 0.1 (statistical trend) when comparing between exenatide and glimepiride groups. fasting proinsulin and aprs were not different across the groups at baseline or in response to treatment. pi / i ratios and proinsulin secretory ratios were unchanged from baseline to 6 months with no significant differences between the exenatide or sitagliptin and glimepiride groups (data not shown). there were no significant differences in the change in agrarg and agrinh with exenatide or sitagliptin treatment after 6 months compared with glimepiride ; however, there was a statistical trend when comparing the change in agrmin with exenatide, but not sitagliptin, to glimepiride (p = 0.06 for exenatide vs. glimepiride ; table 2). within each group, glucagon secretion was increased only in the glimepiride group at 6 months compared with baseline for agrmin (p < 0.05 ; table 2). of the 50 subjects enrolled, 47 subjects underwent randomization and completed baseline gpa testing (supplementary fig. 1) with 17 subjects randomized to exenatide, 13 to sitagliptin, and 17 to glimepiride. there were 7 dropouts, such that 14 subjects in the exenatide, 12 in the sitagliptin, and 14 in the glimepiride group completed the study for a total of 40 subjects used in the analysis. reasons for discontinuation included noncompliance with study medication and/or procedures and adverse events (supplementary fig. baseline demographic measures were similar across all three groups (table 1). at 6 months, change in weight or bmi were not significantly different across all three groups. within the exenatide group, subjects had a decrease in weight over the 6-month course of study (2.6 0.8 ; p < 0.05 ; fig. 1) that was also reflected by a decrease in bmi (p < 0.05 ; table 1). fasting plasma glucose, insulin, and glucagon were similar at baseline and 6 months in all three groups (table 1). the change in average capillary blood glucose over time was significantly different across all three groups (p < 0.01 ; fig. 1). the glimepiride group had a greater reduction in capillary blood glucose than the sitagliptin group (p < 0.01) and the exenatide group by trend (p < 0.1). the lower glucose levels were achieved by 3 months when the average dose of glimepiride was 3 mg. change in hba1c over time was different by trend across all three groups (p = 0.07). the change in hba1c in the exenatide group was not different from that in the glimepiride group, but the change in hba1c in the sitagliptin group was less from that in the glimepiride group (p < 0.05 ; fig. hba1c trended lower within the exenatide group (p = 0.09) at the end of 6 months, whereas it decreased significantly with glimepiride (p < 0.05). lipid profiles were similar across all three groups at baseline, but at 6 months, there was an increase in hdl cholesterol with exenatide, but not sitagliptin treatment, that was significantly greater than that seen with glimepiride treatment (p < 0.05 ; table 1). subject demographics at baseline and final (after 6 months of therapy) visits, change from baseline to 6 months with each value. final visits after 6 months of therapy were conducted following a 5- to 7-day drug washout. to convert to mmol / mol, multiply by 10.93 and subtract 23.50. means se of weight, fasting capillary glucose as determined by glucometer readings, and hba1c in each group. changes in weight over time were not significantly different across the three groups [f(12, 222) = 1.1013 ; p = 0.4 ]. average capillary glucose was significantly different [f(12, 204) = 2.53 ; p < 0.01 ] when comparing across all three groups. hba1c was different by trend [f(4, 74) = 2.28 ; p < 0.1 ] when comparing across all three groups. p < 0.05 when comparing from baseline within each group at each time point. 2, middle) responses were not significantly different with either exenatide or sitagliptin when compared with glimepiride treatment. the change in endogenous glp-1 response was increased with sitagliptin compared with glimepiride treatment (auc 284 70 vs. 31 24 pmol there were no significant differences in the change in airarg after 6 months of exenatide or sitagliptin treatment compared with glimepiride (supplementary fig. 3 and table 2), whereas the change in airpot was significantly lower in the exenatide group, but not in the sitagliptin group, when compared with the glimepiride group (p < 0.05 for exenatide vs. glimepiride ; table 2), and a similar trend was also evident for the change in airmax (p = 0.1 for exenatide vs. glimepiride ; table 2). in fact, within each group, -cell secretory capacity (airmax) increased only in the glimepiride group at 6 months compared with baseline (p < 0.05 ; table 2). -cell sensitivity to glucose (pg50) was not different at baseline and remained unchanged following any treatment (table 2). the glucose infusion rates (m), second - phase insulin levels (i), and the resulting estimate of insulin sensitivity (m / i) were not different at baseline and after 6 months across all three groups (table 2). measures of -cell secretory capacity, -cell sensitivity to glucose, insulin sensitivity, and glucagon secretion derived from the gpa test, change from baseline to 6 months with each value. final visits after 6 months of therapy were conducted following a 5- to 7-day drug washout. p 0.1 (statistical trend) when comparing between exenatide and glimepiride groups. fasting proinsulin and aprs were not different across the groups at baseline or in response to treatment. pi / i ratios and proinsulin secretory ratios were unchanged from baseline to 6 months with no significant differences between the exenatide or sitagliptin and glimepiride groups (data not shown). there were no significant differences in the change in agrarg and agrinh with exenatide or sitagliptin treatment after 6 months compared with glimepiride ; however, there was a statistical trend when comparing the change in agrmin with exenatide, but not sitagliptin, to glimepiride (p = 0.06 for exenatide vs. glimepiride ; table 2). within each group, glucagon secretion was increased only in the glimepiride group at 6 months compared with baseline for agrmin (p < 0.05 ; table 2). this study evaluated functional -cell mass as determined by the -cell secretory capacity in subjects with early t2d treated with exenatide, a glp-1 analogue, or sitagliptin, a dpp4 inhibitor, compared against an active comparator sulfonylurea, glimepiride. our results demonstrate that in early t2d, 6-month treatment with a glp-1 analogue or dpp4 inhibitor does not increase functional -cell mass relative to treatment with a sulfonylurea. while these data were contrary to our hypothesis, they are consistent with previous reports demonstrating no sustained effects of glp-1 on -cell secretory capacity (1113). unlike previous studies in which the drug washout period was 1 month, during which any potential beneficial effects could be reversed by documented worsening glycemic control, our results were obtained 5 days after discontinuation of the study medication to ensure effective drug washout while avoiding development of hyperglycemia. indeed, fasting plasma glucose was controlled within each group at the 6-month visit compared with baseline. thus, these are the first data to demonstrate a lack of improvement in -cell secretory capacity with a glp-1 analogue or a dpp4 inhibitor off drug and in the absence of overt hyperglycemia, while demonstrating a remarkable increase in -cell secretory capacity with sulfonylurea treatment. exenatide - treated subjects experienced a decrease in weight and consequently bmi, although this was not statistically different from a neutral weight effect seen in the glimepiride group. in the exenatide - treated subjects, there was also an increase in plasma hdl cholesterol that has previously been demonstrated (30). sitagliptin treatment was effective in increasing the endogenous glp-1 response 2.3-fold to oral glucose in our study that is consistent with previous reports (31). in contrast to previous studies, there was no significant effect of exenatide or sitagliptin on hba1c. this is likely attributable to the early t2d in our subjects who were at the threshold of overt diabetes (average hba1c of 6.5% [48 mmol / mol ]), while most outcomes studies have included subjects with more advanced t2d (average hba1c 8.5% [69 mmol / mol ]) (32,33). our study is limited by its small sample size and short duration and thus does not allow us to determine whether prolonged treatment with exenatide or sitagliptin early in the course of t2d may prevent deterioration in glycemic control, perhaps through mechanisms other than affecting the -cell secretory capacity. six months of treatment with glimepiride was effective in decreasing capillary blood glucose and lowering hba1c without producing hypoglycemic episodes or weight gain with careful dose titration. postmarketing reports and clinical trials have demonstrated significant increases in weight with glimepiride treatment but these observations were notably with higher concentrations of glimepiride (48 mg) titrated rapidly over a 14-week period (34). similar to our finding of an increase in -cell secretory capacity evident after 6 months, karunakaran. (35) demonstrate a significant improvement in -cell function with glicazide treatment for 1 year while demonstrating lower fasting plasma glucose and hba1c without adversely effecting weight. interpretation of our study s results with glimepiride must be made cautiously, however, as glimepiride was included as an active comparator without a placebo group for comparison. while the increase in -cell secretory capacity after 6 months of treatment with glimepiride observed in this study requires confirmation and further assessment of durability, there are a few speculative mechanisms to explain such an effect. compared with the exenatide and sitagliptin groups, the glimepiride - treated subjects experienced a reduction in capillary glucose. another possibility may be a more specific effect of sulfonylureas against autophagy - associated cell death. autophagy is a self - digestive mechanism that regulates protein turnover, and current evidence links impaired autophagy with accumulation of autophagic vacuoles in the -cells of t2d when compared with nondiabetic islets (4). altered autophagic mechanisms are evident when there is a mismatch between insulin production and secretion, as may occur in t2d, in which there is impaired -cell sensitivity to glucose (36). as sulfonylureas such as glimepiride have no effect on insulin production but stimulate insulin secretion (36), these agents may correct a synthetic mismatch and protect against autophagy - associated cell death. whether such effects may be associated with increases in functional -cell mass as measured by -cell secretory capacity warrants further study. the effect of sulfonylureas on -cells remains unclear at present due to conflicting results that may be attributed to variation in model systems, with intact islets in which paracrine signaling remains intact versus diseased islets in which paracrine signaling is disrupted versus isolated -cells (37,38). for example, cheng - xue. (37) demonstrated a glucagontrophic effect of tolbutamide when paracrine signaling by somatostatin was disrupted. these authors postulate that glucagon secretion is controlled by two mechanisms, one that is direct from the closure of katp channels and one that is indirect via control from paracrine signaling (37). thus, glimepiride may induce depolarization of the -cell, and under normal, nondiseased conditions, glucose, insulin, and/or somatostatin via paracrine action can inhibit glucagon release. however, in t2d, there may be an uncoupling phenomenon in which glucagon secretion can become independent of these paracrine inhibitory signals, and the stimulatory effect of sulfonylureas at the -cells predominates such that 6-month treatment may be glucagontrophic, as reported in this study. whether long - term benefits of sulfonylureas that may enhance functional -cell mass outweigh any adverse consequence of the increased glucagon secretion on glycemic control remains to be determined. in conclusion, the implication of the current study is that a 6-month treatment with the glp-1 analogue exenatide or dpp4 inhibitor sitagliptin does not increase -cell secretory capacity in human t2d as purported in rodent models. furthermore, our study indicates that the sulfonylurea glimepiride may be effective in at least short - term improvement in -cell secretory capacity associated with improved glycemic control early in the course of t2d. clinically, these findings support t2d treatment algorithms that place sulfonylureas ahead of incretin - based approaches (39), with special consideration for third - generation sulfonylureas such as glimepiride, in which careful dose titration as conducted in this study may avoid weight gain and hypoglycemia. | objectiveagents that augment glp-1 effects enhance glucose - dependent -cell insulin production and secretion and thus are hoped to prevent progressive impairment in insulin secretion characteristic of type 2 diabetes (t2d). the purpose of this study was to evaluate glp-1 effects on -cell secretory capacity, an in vivo measure of functional -cell mass, early in the course of t2d.research design and methodswe conducted a randomized controlled trial in 40 subjects with early t2d who received the glp-1 analog exenatide (n = 14), the dipeptidyl peptidase iv inhibitor sitagliptin (n = 12), or the sulfonylurea glimepiride (n = 14) as an active comparator insulin secretagogue for 6 months. acute insulin responses to arginine (airarg) were measured at baseline and after 6 months of treatment with 5 days of drug washout under fasting, 230 mg / dl (glucose potentiation of arginine - induced insulin release [airpot ]), and 340 mg / dl (maximum arginine - induced insulin release [airmax ]) hyperglycemic clamp conditions, in which airmax provides the -cell secretory capacity.resultsthe change in airpot was significantly greater with glimepiride versus exenatide treatment (p < 0.05), and a similar trend was notable for the change in airmax (p = 0.1). within each group, the primary outcome measure, airmax, was unchanged after 6 months of treatment with exenatide or sitagliptin compared with baseline but was increased with glimepiride (p < 0.05). -cell glucagon secretion (agrmin) was also increased with glimepiride treatment (p < 0.05), and the change in agrmin trended higher with glimepiride than with exenatide (p = 0.06).conclusionsafter 6 months of treatment, exenatide or sitagliptin had no significant effect on functional -cell mass as measured by -cell secretory capacity, whereas glimepiride appeared to enhance - and -cell secretion. |
mesial temporal lobe epilepsy (mtle) is possibly the most frequent form of epilepsy and is associated with overt or subtle structural abnormalities in the hippocampus, parahippocampal gyrus, and amygdala. recent investigations have suggested that functional and structural abnormalities in mtle may extend beyond the temporal lobes. volume reduction has been observed in extratemporal areas such as thalamus, caudate nuclei, lenticular nuclei, corpus callosum, and frontal lobes (for a review, see). alterations of frontotemporal white matter tracts and of neurotransmitter systems have also been reported. metabolic changes have been observed not only in mesial and lateral temporal areas, but also in the prefrontal cortex and in subcortical structures [7, 8 ]. thalamus, basal ganglia, and frontal lobes are part of corticosubcortical circuits that are involved in the regulation of motor functions, behavior, and cognitive functions such as set - shifting, planning, and inhibitory control [9, 10 ]. as increasing evidence points to functional and structural alterations of these corticosubcortical circuits the term executive functions refers to high - order cognitive functions which allow adaptation to nonroutine situations such as novel, conflicting, or complex tasks [11, 12 ]. planning, set - shifting, strategic behavior, response initiation, and response inhibition are some of the cognitive functions that may be listed under this umbrella term. executive function deficits are often found in association with frontal lobe damage [13, 14 ]. however, recent neuropsychological investigations as well as neuroimaging studies have pointed to a distributed brain network, which encompasses frontal areas as well as posterior areas (e.g., parietal association areas : ; cerebellum :) and subcortical structures (e.g., basal ganglia : ; thalamus :). executive function deficits may be found in a large number of neurological pathologies, in diffuse brain damage, and in focal brain damage [11, 12 ]. single - case analyses of brain - damaged patients have shown that executive function deficits may have different clinical manifestations and that performance on executive function tests may be dissociated with deficits observed on some tests and not on others [11, 12 ]. executive function deficits may have important consequences on the patient 's autonomy and severely compromise the quality of life. given the multicomponent aspect of executive functioning, its systematic assessment needs to cover several cognitive processes. chronic mtle may affect cognitive and emotional processing [1921 ]. compared with healthy controls, mtle patients often show reduced episodic memory and difficulties in learning new information [22, 23 ]. some studies have reported reduced performance for mtle patients as compared with controls [2628 ] ; others have found no relevant differences [22, 24 ]. as, in most studies, only a single test or a very short test battery has been used to assess executive functioning in mtle, it may be possible that deficits have been overlooked in some cases. also, studies that have used the wisconsin card sorting task (wcst) have found significant differences between mtle patients and controls, pointing to abstraction, categorization, and set - shifting deficits in mtle (e.g., [24, 2628 ] ; see also). studies that have screened for deficits in working memory [22, 24 ], inhibitory control [24, 28 ], verbal fluency [22, 28 ], or cognitive flexibility [22, 24 ] have obtained heterogeneous findings, making it difficult to conclude whether and to what extent mtle patients present executive function deficits. giovagnoli found that patients with left - sided mtle are more impaired than patients with right - sided mtle on the wcst, whereas corcoran and upton [28, 44 ] reported the opposite pattern of results. other investigations found no significant effect of seizure lateralization on executive functioning [22, 45 ]. this study aimed at investigating executive functioning in a group of chronic, unilateral mtle patients. differently from previous studies, we used an extensive battery of neuropsychological tests and assessed different aspects of executive functioning (verbal attention, verbal working memory, psychomotor speed, cognitive flexibility, verbal fluency, set - shifting, categorization, inhibition, and planning). in this study, we compared performance of mtle patients with that of healthy peers as well as with normative data. in separate analyses, we also controlled for possible effects of seizure lateralization (left - sided mtle versus right - sided mtle), structural abnormality (hippocampus versus amygdala), and antiepileptic drug therapy (aed), which has been documented to significantly affect cognitive performance [4649 ]. a correlation analysis also examined the association of executive function performance with age at seizure onset and epilepsy duration. following previous reports on deficits of tle patients in single executive function tests, we expected the mtle patients to perform poorly relative to the healthy controls in the more comprehensive neuropsychological test battery. as epilepsy in the dominant hemisphere may have detrimental effects on language processing, we also expected the left - sided mtle patients to perform poorly relative to the right - sided mtle patients on verbal fluency tests. finally, we hypothesized that executive function deficits are enhanced in the patients with the earlier onset epilepsy and the longer disease duration. (patients also performed tasks of decision making ; these results are described in.) patients had pharmacoresistant, focal epilepsy and were candidates for epilepsy surgery (age at seizure onset : mean 21.5 14.5 years ; disease duration : mean 18.2 15.2 years). patients underwent a detailed clinical and neurological examination including prolonged video - eeg monitoring, high - resolution mri, interictal spect or pet, and a neuropsychological examination. twenty - six patients were right hander ; two were ambidextrous (only right - hander patients were taken into consideration in the analysis of the effect of seizure lateralization). patients underwent a high - resolution 1.5-tesla mri with t1-weighted spin echo and gradient echo 3d multiplanar reconstruction images (with and without intravenous contrast application), axial and coronal t2-weighted turbospin echo, t1 inversion recovery, t2-weighted fast fluid attenuated inversion recovery (flair), and diffusion - weighted sequences. axial images were acquired with an angle parallel to the long axis of the hippocampus. all but three mri scans were visually evaluated by two independent raters (g. k., f. k.) who were familiar with the radiological analysis of the temporal lobe structures and were blinded to diagnosis. for these three patients, we obtained a detailed written description of the mesial temporal structures. agreement was achieved in all but two cases (agreement : 93%). a third rater (e.t.) who was blinded to diagnosis mri revealed a clear unilateral abnormality in the left mesial temporal lobe in 17 patients (lmtle) and a clear unilateral abnormality in the right mesial temporal lobe in 11 patients (rmtle). hippocampal abnormality was found in 10 patients (hippocampus group = mtle - hc), amygdala abnormality in 11 patients (corpus amygdaloideum group = mtle - ca), and abnormalities affecting both structures in 7 patients (see figure 1 for examples of mri features for the three groups). the mtle - hc group included patients with mri features of hippocampal sclerosis (atrophic hippocampus with missing internal three - layer architecture and increased signal in t2 and flair sequences) and hippocampal dysplasia (hippocampus with distorted anatomy, with blurring of white / grey matter interface, and increased signal in t2 and flair sequences). amygdala was regarded as dysplastic if it was larger than the contralateral side, if increased signal in t2 and flair sequences was observed, or both. fifteen patients received antiepileptic drug monotherapy (carbamazepine, oxcarbazepine, lamotrigine, and levetiracetam) ; 13 patients were on polytherapy (carbamazepine, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, pregabalin, primidone, topiramate, valproate, zonisamide ; aed daily doses and serum levels are reported for each patient in table 4). the serum level was measured within one day from the neuropsychological examination in 24/28 patients (86%) ; in the remaining cases, the serum level refers to the closest previous measurement (deviation from the day of the neuropsychological examination : 17 to 44 days). the patient with the serum level for one antiepileptic drug slightly above the normal range did not manifest signs of intoxication. also, neuropsychological performance did not differ between this patient and the patient group. exclusion criteria for participation were history of stroke, head trauma, psychiatric disorder, substance abuse, or neurological disorders which may compromise cognition. healthy participants and mtle patients were comparable in age (controls : mean 36.0 13.0 years ; mtle patients : mean 40.3 11.3 years) and education (controls : mean 11.4 1.6 years ; mtle patients : mean 10.6 1.7 years). mtle patients completed tests of verbal memory (learning, free recall, and recognition mnchner gedchtnistest, mgt), nonverbal memory (subtest of the visueller und verbaler merkfhigkeitstest, vvm), vocabulary, numeracy [30, 31 ], mental complex calculation, and executive functions (verbal attention and verbal working memory digit span tests of the wechsler memory scale - revised, wms - r ; psychomotor speed and cognitive flexibility trail making test - a and b, tmt - a and b ; categorical verbal fluency, phonological verbal fluency, and verbal fluency with alternating categories subtests of the regensburger wortflssigkeitstest, rwt ; planning and problem solving planungstest ; abstraction and categorization wcst ; inhibitory control go - nogo task adapted from). mtle patients also responded to a questionnaire on anxiety and depression (hospital anxiety and depression scale - deutsche version, hads - d). controls were tested on vocabulary, numeracy, mental complex calculation, and executive functions. the numeracy test assesses basic probability and mathematical concept understanding [30, 31 ]. therefore, group differences were analyzed through nonparametric methods (kruskal - wallis h - test, mann - whitney u - test). a spearman - rank correlation analysis was carried out for the mtle group between executive function scores, age at seizure onset, and epilepsy duration. the following executive function variables were entered into the analysis : completed categories (wcst), perseverative errors (wcst), digit span backward (wms - r), correct nogo trials (go - nogo task), sec in the tmt - b, total number of moves (planungstest), categorical verbal fluency (rwt), and verbal fluency with alternating categories (set - shifting, rwt). applying bonferroni correction for multiple correlations, median scores of healthy controls were in the average range of normative data in all tests (tables 1 and 2). the mtle patients ' median score in the numeracy test was below cut - off. in other tests, the mtle patients ' median scores were in the average range of normative data. the evaluation of individual scores revealed several patients with performance below cut - off or below the 10th percentile of normative data (454% ; tables 1 and 2). regarding the executive function tests, 7 patients (25%) performed below the 10th percentile of normative data in 5-to-8 measures ; 19 patients (68%) performed below the 10th percentile in 1-to-3 measures. performance of mtle patients was compared with that of healthy controls by means of mann - whitney u - test. mtle patients obtained slightly lower vocabulary scores, numeracy scores, and mental complex calculation scores than controls (table 1). relative to controls, mtle patients also performed poorly in tests of verbal working memory, cognitive flexibility, categorical verbal fluency, set - shifting, categorization, and planning (table 1). mtle patients responded less accurately than controls to go trials ; groups did not differ from each other in nogo trials. there were no significant group differences in tests of verbal attention, psychomotor speed, and phonological verbal fluency (table 1). rmtle patients had a median score slightly below cut off in the numeracy test and a median score slightly below the 10th percentile in the wcst (table 2). lmtle patients had a median score slightly below cut off in the numeracy test and a median score slightly below the 10th percentile in the verbal memory test (immediate free recall) and the phonological verbal fluency test (table 2). in other tests, the median scores of both groups were in the average range of normative data. as indicated in table 2, results were significant in tests of numeracy, mental complex calculation, verbal working memory, cognitive flexibility, phonological verbal fluency, set - shifting, categorization, and planning. two - by - two comparisons were carried out by means of mann - whitney u - tests. lmtle patients obtained lower scores than rmtle patients in the phonological verbal fluency test (z = 2.06, p =.041) ; other differences between the patient groups were not significant. relative to healthy controls, lmtle patients performed poorly in tests of cognitive flexibility (z = 2.68, p =.007), phonological verbal fluency (z = 2.81, p =.004), set - shifting (z = 3.01, p =.002), categorization (completed categories : z = 2.35, p =.019 ; perseverative errors : z = 2.46, p =.014), and planning (z = 2.16, p =.033). rmtle patients obtained lower scores than healthy controls in tests of numeracy (z = 2.86, p =.005), mental complex calculation (z = 2.30, p =.019), verbal working memory (z = 2.33, p =.022), categorization (completed categories : z = 2.15, p =.035), and planning (z = 2.24, p =.028). mann - whitney u - tests indicated that rmtle patients and lmtle patients were comparable in terms of age at seizure onset and epilepsy duration. there were also no significant group differences in tests of verbal memory, nonverbal memory, anxiety, and depression. mtle - ca patients scored slightly below cut - off in the numeracy test (table 3). mtle - hc patients scored below cut - off in the numeracy test and below the 10th percentile in tests of verbal memory, categorization, and inhibitory control (table 3). other median scores were in the average range of normative data for both patient groups. as the mtle - hc patients were older and had less education than both the healthy controls and the mtle - ca patients, we residualized age and education on the dependent variables and then used these measures in the analysis. there were significant group differences in tests of vocabulary, verbal working memory, and categorization (table 3). mtle - hc patients obtained lower scores than mtle - ca patients in the wcst (completed categories : z = 1.97, p =.051). relative to healthy controls, mtle - hc patients performed poorly in tests of vocabulary (z = 1.98, p =.005), verbal working memory (z = 2.68, p =.006), and categorization (completed categories : z = 2.75, p =.005 ; perseverative errors : z = 2.48, p =.012). differences between mtle - ca patients and healthy controls were not significant. as indicated by mann - whitney u - test, mtle - hc patients and mtle - ca patients had comparable age at seizure onset. the mtle - hc group had longer epilepsy duration than the mtle - ca group (z = 2.44, p =.012). differences between mtle - hc patients and mtle - ca patients in tests of verbal memory, nonverbal memory, anxiety, and depression were not significant when residualized scores were submitted to mann - whitney u - tests. patients on monotherapy and patients on polytherapy had comparable scores in all demographic, clinical, and neuropsychological measures (mann - whitney u - tests, all p >.05). the comparison of individual scores with the patient group distribution indicated that five patients had an outlier performance that is, below 2.5 sd from the group mean in some memory and executive function measures. outlier cases were evenly distributed between the monotherapy group and the polytherapy group (see table 4). there was a significant correlation between epilepsy duration and performance on the wcst (completed categories, r =.581, p =.002). no significant correlation was found between age at seizure onset and performance on executive function tests. (an exploratory analysis indicated that performance on nonexecutive function tests (vocabulary, memory, and complex numerical processing) directly correlated with performance on executive function tests. neither performance on executive function tests nor performance on nonexecutive function tests correlated with anxiety and depression scores.) this study used an extensive neuropsychological test battery and assessed several aspects of executive functioning in chronic, unilateral mtle patients. performance of a carefully selected group of pharmacoresistant mtle patients, who were candidates for epilepsy surgery, was compared with that of healthy peers as well as with normative data. possible effects of seizure lateralization, structural abnormality, antiepileptic drug therapy, age at seizure onset, and epilepsy duration were also analyzed. results indicated that both the mtle group and the healthy control group had median scores in the average range of normative data. relative to healthy peers, mtle patients performed poorly in tests of verbal working memory, cognitive flexibility, categorical verbal fluency, set - shifting, categorization, planning, and inhibitory control. they performed comparably to healthy controls in tests of verbal attention, psychomotor speed, and phonological verbal fluency. several patients had scores below cut - off or below the 10th percentile of normative data. also, almost one - third of the patients had impairments in 5-to-8 measures of executive functioning ; two thirds had impairments in 1-to-3 measures. these findings raise an important methodological issue as they suggest that executive function deficits in chronic mtle may be individually variable and that their assessment needs to include different tests. we suggest the use of an extensive neuropsychological test battery which assesses different aspects of executive functioning (e.g., working memory, cognitive flexibility, categorization, inhibitory control, and planning). as shown by this study, the use of a single task (e.g., go - nogo) is not sufficient for the detection of executive function deficits which may be highly specific. as a correlation analysis indicated, performance of mtle patients on the wcst inversely correlated with epilepsy duration. similar results were described by kim., whereas martin. found no association between performance on the wcst and epilepsy duration or age at seizure onset. tested working memory and executive functions in a large group of tle patients (the majority of patients had mesial temporal lobe epilepsy). results showed that age at seizure onset and seizure frequency, which significantly correlated with epilepsy duration, were significant predictors of the patients ' impairments. in this study, mtle patients had intractable epilepsy, and epilepsy duration ranged from few months to several years. as the brain suffers for longer time from the epileptic discharges, it seems likely that the cognitive deficits were more pronounced in the mtle patients with the longer disease duration. the negative effect of epilepsy duration on cognition may be related not only to the effect of the epileptic seizures themselves, but also to other factors including the long - term antiepileptic drug therapy. all commonly used antiepileptic drugs seem to have some behavioral and cognitive effects, with the patients on polytherapy being on average more affected than the patients on monotherapy [4649 ]. for example, phenytoin, phenobarbital, and valproate have been related to decline in attention and psychomotor speed (for a review, see) ; also, patients taking topiramate have been found to perform poorly on verbal fluency and categorization. compared with older antiepileptic drugs, some of the new antiepileptic drugs have fewer negative effects on cognition [46, 48 ]. in this study, we found that only few patients had an outlier performance relative to performance of the whole patient group. also, there were no differences between patients on monotherapy and patients on polytherapy in any memory or executive function measure. although we can not exclude the possibility that small group differences between patients on monotherapy and patients on polytherapy have been missed because of the small sample group, these findings may suggest that the antiepileptic drug therapy had no relevant effect on the executive function performance of mtle patients in this study. results of the comparison of left - sided mtle patients with right - sided mtle patients showed no relevant group differences with the only exception of a verbal fluency measure. relative to right - sided mtle patients, left - sided mtle patients performed poorly in a phonological verbal fluency test. these results are in line with other investigations, which suggest no severe effect of seizure lateralization on executive functioning [22, 43, 45 ]. for example, tudesco. tested mtle patients with unilateral hippocampal sclerosis. comparably to this study, they found that left - sided mtle patients differed from right - sided mtle patients in verbal fluency tests, but not in working memory and set - shifting tests. there are competing hypotheses in the literature as to the underlying mechanisms of executive function deficits in mtle. a first hypothesis (nociferous cortex hypothesis) proposes that executive function deficits in mtle result from the propagation of the epileptic discharges from the temporal lobe epileptic focus to the frontal lobes. there are white matter tracts connecting the temporal lobes with the frontal lobes [54, 55 ], and the epileptic discharges may propagate through these projections. according to a second hypothesis (hippocampal contribution hypothesis), executive function deficits in mtle specifically, as the hippocampus is involved in the retrieval of information from short - term memory, in the formation of memory associations, and in learning new information, the mtle patients ' impairments in working memory tasks as well as in other executive function tasks, such as the wcst, that rely on such processes would result from dysfunction of the hippocampus. finally, increasing evidence points to the implication of extratemporal functional and structural brain abnormalities for example, damage to frontosubcortical circuits, volume loss, or metabolic changes in extratemporal regions in the executive function disorders of tle patients. our study design does not allow us to precisely define the impact of these different factors on executive function performance. although an exploratory analysis indicated that patients with hippocampal abnormalities (mtle - hc) scored lower than patients with amygdala abnormalities (mtle - ca) and healthy controls in the wcst, we may not exclude the possibility that different factors propagation of epileptic discharges, dysfunction of mesial temporal structures, and extratemporal brain modifications contributed to the executive function disorders of mtle patients in this study. other investigation methods for example, functional magnetic resonance imaging, voxel - based morphometry, or diffusion tensor imaging have to be employed, together with neuropsychological testing, to address this question. one could ask whether there is a specific pattern in the executive function deficits of mtle patients. miyake. used a latent variable analysis to investigate how executive functions relate to each other. results indicated that functions such as updating, shifting, and inhibition are clearly separable, but that they are not completely independent from each other and seem to share some common processes. proposed that the central executive component of working memory is a feasible candidate to account for some of the commonality underlying different executive functions [56, 58 ]. the central executive is an attentional control system, which allocates resources and coordinates and monitors short - term storage processes as well as more general processes. several executive function tests clearly load on central executive resources, as they require the maintenance of goal and content information, the manipulation of memory representations, or the suppression of irrelevant information. in this study, mtle patients had difficulties in particular in the tests that put high demands on attentional control resources (e.g., wcst, planning test, and verbal fluency test with alternating categories), whereas they performed relatively well in the less demanding tests (e.g., digit span forward, tmt - a). in sum, this study indicates that deficits in chronic mtle are not limited to temporal lobe functions, such as memory, but may extend to extratemporal cognitive domains. patients with chronic, pharmacoresistant mtle have executive function deficits, and these deficits are individually variable. executive functions such as cognitive flexibility, planning, and inhibitory control are essential in everyday life. it is thus possible that mtle patients experience difficulties in everyday functioning, in particular in those situations that are unknown and require rapid adaptation or flexible problem solving. this study adds to the characterization of cognitive deficits in mtle and suggests the use of a comprehensive assessment battery in the individual neuropsychological diagnosis. | there is no consensus as to whether mesial temporal lobe epilepsy (mtle) leads to executive function deficits. in this study, we adopted an extensive neuropsychological test battery and assessed different executive functions in chronic, unilateral mtle. performance of mtle patients was compared with that of healthy peers and with normative data. several mtle patients had scores below cut - off or below the 10th percentile of normative data. scores of the whole patient group were overall in the average range of normative data. relative to controls, mtle patients performed poorly in tests of working memory, cognitive flexibility, categorical verbal fluency, set - shifting, categorization, and planning. these findings raise an important methodological issue as they suggest that executive function deficits in chronic mtle may be individually variable and that their assessment should include different tests. deficits in chronic mtle are not limited to temporal lobe functions, such as memory, but may extend to extra temporal cognitive domains, such as executive functions. |
dementia is a massive and growing global health - economic challenge with an estimated prevalence of 35.6 million people affected worldwide in 2010. it is therefore of great importance to identify patients who suffer from cognitive impairment due to a neurodegenerative disease in order to implement proper treatment and care early in the course of the disease, as these patients will continue to deteriorate. it is equally important to identify in which cases alzheimer disease (ad) is the cause of the cognitive impairment, as this patient group is by far the largest to whom symptomatic treatment can be offered. likewise, an identification of patients with a high probability of progression early in the course of the disease is becoming increasingly important for clinical trials of disease - modifying agents. treatment should preferably be initiated early in the course of the disease, when neuronal damage is still limited. frequently, patients referred for cognitive evaluation may still remain diagnostically unresolved with regard to the etiology of their cognitive dysfunction even after an extensive diagnostic workup including cerebrospinal fluid (csf) biomarker analysis. these diagnostically unresolved patients may be diagnosed with either mild cognitive impairment (mci) or as demented with unknown etiology. the critical issue is to differentiate the underlying etiology in order to predict progression of the condition. not every case of mci will progress to a dementia disorder ; however, 10 - 15% of patients with mci will develop ad within 1 year, and even 40 - 60% will develop ad within 5 years. the concept of dementia of unknown etiology (due) has been used to describe demented patients who do not meet the criteria for any well - defined degenerative dementia such as ad, lewy body dementia, frontotemporal dementia, or other causes of secondary dementia (e.g. vitamin b12 deficiency or head trauma). the diagnosis of dementia is currently based on different sets of clinical diagnostic criteria, of which the nia - aa criteria and icd-10 criteria are generally accepted. various biomarkers, in particular csf biomarkers, have proven useful in increasing the diagnostic certainty in vivo, and findings from biomarker analyses have been incorporated into different research criteria for ad but are also gaining increasing attention in clinical practice. potential biomarkers of cognitive decline and dementia need to exhibit a high diagnostic accuracy and should reflect the biochemical and pathological hallmarks of the disease. at present, csf analysis may to a large extent support the diagnosis if ad is the underlying pathology, but ad biomarkers (i.e. a42, t - tau, and p - tau) can not definitely confirm the diagnosis even in the presence of typical findings, since these alterations may also be present in cognitively healthy persons as well as in patients with other neurodegenerative disorders. however, decreased levels of csf a42 and increased levels of t - tau and p - tau confer a high likelihood of ad being the cause of the cognitive dysfunction in both dementia and mci. there is an increasing interest in the involvement of neuroinflammation in the development and progression of dementia. evidence of this involvement derives from pathophysiologic studies, which have revealed a number of inflammatory mediators to be upregulated in the ad brain. other studies have suggested that the presence of amyloid plaques and neurofibrillary tangles has the ability to initiate and sustain a chronic inflammatory response via the complement system during the disease. the csf / serum albumin ratio, as an indicator of blood - brain barrier (bbb) integrity, has a limited differential diagnostic power, as it may be affected in both neuroinflammatory diseases as well as in cerebrovascular lesions ; however, it is generally found to be a reliable sign of an active process. elevated csf / serum albumin ratio, white cell count, and intrathecal igg synthesis may therefore be potential markers of neuroinflammatory processes, which implies that analysis of theses markers in csf can help to improve diagnostic accuracy and predict prognosis in patients referred for dementia evaluation. in our study, we aimed to investigate the role of csf neuroinflammation and ad biomarkers in the diagnostic workup for a group of diagnostically unresolved patients in a typical memory clinical setting. in this group, despite an extensive evaluation program including csf biomarker analysis, experienced clinicians were not able to establish whether a neurodegenerative disease was the cause of the cognitive impairment or not. hence, we investigated the biomarkers ' ability to predict clinical progression, and thereby their ability to potentially differentiate between neurodegenerative and nonneurodegenerative disorders as causes of cognitive impairment in diagnostically unresolved patients. furthermore, we compared the diagnostically unresolved patients to a control group of ad patients as well as a group of healthy elders to elucidate similarities. data were retrospectively collected from the danish dementia biobank on patients consecutively referred for cognitive evaluation and diagnosed with either mci, due, or ad at 2 multidisciplinary memory clinics (regional dementia research centre, department of neurology, zealand university hospital, and copenhagen memory clinic, department of neurology, university hospital copenhagen, rigshospitalet) between june 2009 and june 2013. the study population also included a healthy control (hc) group recruited solely for research purposes. as part of the diagnostic workup, at baseline all patients underwent a standardized diagnostic assessment including physical and neurological examinations, routine blood analysis, lumbar puncture with csf analysis, brain ct or mri, as well as cognitive screening [i.e. mini - mental state examination (mmse) and addenbrooke 's cognitive examination (ace) ]. if it was thought diagnostically relevant, the patients also had a full neuropsychological evaluation undertaken by a neuropsychologist. diagnoses were settled by consensus in an experienced multidisciplinary team based on all the available examination results, including the results from the csf analysis. patients with ad were diagnosed according to the nincds - adrda criteria (2009 - 2011), subsequently according to the nia - aa criteria. patients with due were those diagnosed as being demented according to the icd-10 and nia - aa criteria but who did not fulfill the diagnostic criteria for any subtype of dementia or secondary dementia because the underlying etiology could not be identified. all patients were clinically evaluated - including repeated mmse testing and information from caregivers - on an ongoing basis with at least 1-year intervals as part of the normal clinical setting in the 2 multidisciplinary memory clinics. the diagnoses were continuously reevaluated at the follow - up visits. based on clinical progression, to fulfill the criteria for ad or other well - defined dementia disease, they were all without memory complaints or other cognitive symptoms as well as signs of major neurological, psychiatric, or physical diseases that could potentially elicit cognitive impairment. the healthy subjects all underwent the same standardized diagnostic assessment in one of the 2 memory clinics, including physical and neurologic examinations, routine blood analysis, brain ct or mri, cognitive screening, as well as blood sample screening, ecg, and lumbar puncture at inclusion. csf was obtained by puncture in the l3-l4 or l4-l5 intervertebral space and collected in polypropylene tubes. the csf analyses included white cell count, erythrocytes, total protein, glucose, and albumin as well as igg index and oligoclonal bands and the core ad biomarkers (i.e. a42, t - tau, and p - tau). the csf / serum albumin ratio was used as a marker of the integrity of the bbb, due to the fact that albumin is exclusively synthesized in the liver. the csf / serum albumin ratio is therefore a dimensionless csf protein concentration, independent of blood variations. the igg index was used to determine igg synthesis, and along with the csf white cell count was used as an indicator of active neuroinflammation as well as a marker of bbb integrity. for the purposes of this study, patients initially diagnosed with mci or due were combined into a group of patients defined as diagnostically unresolved. based on the ongoing clinical evaluation, the diagnostically unresolved patients were subsequently divided into a group of patients who progressed during follow - up to eventually fulfill the criteria for a well - defined dementia disease and a group of stable patients who did not progress or who were diagnosed with a nonneurodegenerative disorder. the study was reported and approved by the data protection agency authorities. as the study is a retrospective study exclusively analyzing data from the danish dementia biobank, comparisons between groups were performed using the nonparametric kruskal - wallis test followed by the mann - whitney u test for pairwise comparisons. to correct for multiple comparisons, receiver operating characteristic (roc) curves of each csf parameter and biomarker individually as well as in combination were drawn as a plot of the true - positive value (sensitivity) against the false - positive value (1 - specificity) for different possible cutoff values of the respective markers. the roc curves illustrate the ability of each biomarker or combination of biomarkers to correctly classify progressive and stable patients. the area under the curve (auc) was calculated as a measure of a biomarker 's overall accuracy. optimized cutoff levels for ad biomarkers individually and in combination were derived from the roc curves by the cutoff that maximized the sum of sensitivity and specificity. previous studies have found a clear correlation between age and t - tau level, which makes it necessary to determine separate cutoff values for different age categories. in our study, only 4 patients were under the age of 45 years at baseline ; hence, age stratification was not performed. we also used previously reported reference values for csf a42, t - tau, and p - tau to evaluate how they performed in predicting clinical progression in a population of diagnostically unresolved patients (table 3). the statistical analysis was carried out using spss version 22 and the r environment for statistical computing version 3.1.2. they were either diagnosed with mci (n = 60), due (n = 83), or ad (n = 172) or as hc (n = 33). the group of diagnostically unresolved patients (the mci and due groups) included 143 patients. seventy - two of the 143 diagnostically unresolved patients remained clinically stable during the follow - up period (mean follow - up time 21.4 months, range 1 - 59), and 71 patients progressed to fulfill the criteria for a well - defined dementia disease. seven patients in the stable group had a follow - up period of less than 1 year due to the identification of nonneurodegenerative conditions during clinical follow - up (e.g. depression, cerebral infarction) or full recovery. the descriptive data on diagnosis, age, gender, baseline mmse scores, and follow - up period are presented in table 1. as expected, the baseline mmse scores were significantly higher in the hc group than in all the other groups (p < 0.01). the ad group had significantly lower baseline mmse scores than the clinically progressive group (p < 0.01), but not than the clinically stable group (p = 0.016). no significant differences in mmse score were observed between the progressive and the stable group. table 2 illustrates the baseline csf inflammation and ad biomarker levels and ratios among the diagnostic groups. the concentration of csf a42 was significantly lower in patients who clinically progressed during the follow - up period than in the clinically stable patients and the hc, but it was not significantly different from that of the ad group. the clinically stable group had a significantly lower baseline csf a42 level than the hc, but a higher level than the ad group. as expected, the hc group had a significantly higher baseline csf a42 level than all the other groups. with regard to tau protein, the clinically progressive group had significantly higher levels of both csf t - tau and p - tau protein than the stable group, but it did not significantly differ in either t - tau or p - tau levels from the ad or the hc group. the stable group had significantly lower levels of csf t - tau and p - tau protein than the ad group, but no significant differences in either level were found compared to the hc group. the hc group had only a significantly lower csf p - tau level, but not csf t - tau level, than the ad group. the csf inflammation biomarkers (i.e. white cell count, igg index, and csf / serum albumin ratio) did not show any significant differences when comparing the clinically progressive, the clinically stable, and the hc group. only a significantly higher csf white cell count was found in the ad group when compared to the clinically progressive group. this can be attributed to a few outlier patients with highly elevated csf white cell counts only in the ad group. our data showed great variability in the few patients with an elevated white cell count and may therefore not be representative of the different patient groups. the csf a42/p - tau ratio differed significantly between all groups when comparing the groups pairwise. the ratio was significantly lower in both patients who progressed clinically and patients with ad when compared to clinically stable patients and hc. the csf a42/p - tau ratio was moreover significantly higher in the progressive group than in the ad group. similarly, a higher csf a42/p - tau ratio was seen in the hc than in the clinically stable patients, as illustrated in figure 1 and table 2. we calculated the csf t - tau p - tau / a42 ratio for each diagnostic group. the ratio was significantly higher in the clinically progressive group than in both the clinically stable group and the hc. similarly, a higher ratio was found in the ad group than in the clinically stable patients and hc. no significant difference was observed between the clinically stable group and the hc, as illustrated in figure 2 and table 2. individual aucs for the different csf ad biomarkers as predictors of future progression ranged from 0.69 for t - tau and p - tau to 0.77 for a42, indicating that csf a42 had the highest predictive value of the 3 ad biomarkers. a better performance in predicting future progression in our group of diagnostically unresolved patients was found for csf ad biomarkers in combination : a42/p - tau ratio (auc = 0.82) and t - tau p - tau / a42 ratio (auc = 0.80). the roc analysis revealed a low prognostic accuracy for csf inflammation biomarkers (i.e. csf white cell count : auc = 0.56 ; csf / serum albumin ratio : auc = 0.56 ; csf igg index : auc = 0.51). the main objective of our study was to investigate the role of csf inflammation and ad biomarkers in the diagnostic workup in a group of diagnostically unresolved patients in a routine clinical setting. this is highly relevant to clinicians in a normal clinical setting, as they frequently meet these unresolved patients. in particular, we aimed to investigate how csf inflammation and ad biomarkers performed as well as to study their ability to predict clinical progression - and thereby their ability to identify neurodegenerative versus nonneurodegenerative disorders as causes of cognitive impairment in diagnostically unresolved patients. in our study, we found that those diagnostically unresolved patients who clinically progressed to fulfill the criteria for a well - defined dementia due to a neurodegenerative disease had a biomarker profile characteristic of ad at baseline which reflected the neuropathological hallmarks of ad with progressive accumulation of a plaques, neurofibrillary tangles, and neuronal degeneration. in contrast, a similar biomarker profile was not seen in the stable diagnostically unresolved patients. therefore, it is very possible that the majority of the progressive diagnostically unresolved patients in our group in fact represent patients with underlying ad, but with initially atypical or subtle signs and symptoms. some may, however, also suffer from another neurodegenerative dementia, since the csf biomarkers ' performance in distinguishing between different forms of dementia is less solid. previous studies have demonstrated that ad biomarkers have the ability to differentiate between ad patients and healthy elders with high sensitivity and specificity (80 - 90%) ; also, an abnormal biomarker pattern in mci may predict progression to ad in 90% of mci cases within 9.2 years. the ad biomarkers assessed in our group of diagnostically unresolved patients exhibited a slightly lower ability to accurately predict future progression, with diagnostic accuracies ranging from 0.69 to 0.7 (fig. this may partly be explained by the fact that we exclusively analyzed whether cognitively impaired patients progressed to neurodegenerative dementia or not, which included all degenerative disorders and not only ad. the csf a42 level had the highest predictive ability of the 3 well - established ad biomarkers, which supports the theory of amyloid positivity being a risk factor for developing ad as well as the notion that ad biomarkers become abnormal in a temporally ordered manner in which t - tau and p - tau protein are usually not abnormal until later in the course of ad. the notion of combining 2 aspects of ad pathology - i.e. plaques (a42) and neurodegeneration (p - tau) - as a biomarker of continuous cognitive decline and clinical progression to ad in mci patients has shown promising results. in particular, the csf a42/p - tau ratio exhibited a great capacity to predict progression in mci patients younger than 70 years, probably due to the lower importance of comorbidities when compared to older groups. however, to our knowledge, similar findings have not previously been described for a mixed group of diagnostically unresolved patients. our findings with regard to the csf a42/p - tau ratio suggest that it may also be a useful predictor of future clinical progression in a heterogeneous group of diagnostically unresolved patients in a normal clinical setting. roc analysis did indeed reveal that the ratio outperformed the prevalent biomarkers individually (fig. it is noteworthy though that the mean age in all groups of our study was in fact below 70 years, which could partly explain the considerable potential of the a42/p - tau ratio as a prognostic indicator. furthermore, of all the biomarkers, only the a42/p - tau ratio had a significant discriminative power to separate all the groups individually. however, supposing the a42/p - tau ratio were to be incorporated as a useful diagnostic tool into a normal clinical setting with a heterogeneous group of cognitively impaired patients, generally accepted cutoff values for demented populations would need to be established. the optimized cutoff values derived from the roc curves for the individual ad biomarkers were similar to well - established reference values (table 3). our optimized cutoff value for the a42/p - tau ratio may therefore potentially be representative of a heterogeneous group of diagnostically unresolved patients, even though cutoff levels established within a population sample tend to overestimate their true diagnostic accuracy. however, buchhave. reported a somewhat lower optimized cutoff value with regard to the csf a42/p - tau ratio of < 6.16 (with a sensitivity of 0.88 and a specificity of 0.90) for predicting development of ad within 9.2 years in mci patients. the observed difference may partly be due to the shorter follow - up period in our study, since some of the clinically stable patients might still progress during a longer follow - up period. this discrepancy is probably partly due to methodological variability and the absence of validated technical standard processes for csf analysis. adding the t - tau protein as a biomarker of nonspecific neural damage did not add any discriminatory power over the a42/p - tau ratio for separating clinically progressive from clinically stable patients (fig. csf neuroinflammation biomarkers are traditionally used in diagnostic workup primarily to exclude nonneurodegenerative disorders as causes of dementia, especially inflammatory diseases. in our population, only 1 patient growing evidence indicates that unspecific neuroinflammation contributes to the pathophysiology of several neurodegenerative diseases including ad and related dementias. neuropathological findings suggest that cerebral inflammation plays a key role in the toxicity of amyloid and thus is part of an essential mechanism that determines which patients with intracerebral tau and amyloid pathology will become demented or stay nondemented. other studies suggested that amyloid plaques and neurofibrillary tangles have the ability to initiate and sustain a chronic intracerebral inflammatory response. in our study the fact that no increased signs of neuroinflammation or bbb dysfunction were found in clinically progressive diagnostically unresolved patients - and even in ad patients - compared to clinically stable patients was unexpected in light of the mounting evidence of the role of neuroinflammation in neurodegenerative dementias. this is quite in contrast to a recent study by ngga., which reported neuroinflammation to be independently linked to both rapidly progressive disease and early death in patients with ad however, it is important to note that these authors found neuroinflammation to be independently linked to rapidly progressive disease and early death in patients with ad, whereas the less aggressive courses of the disease may not exhibit same level of neuroinflammation. furthermore, ngga. found an elevated csf / serum albumin ratio to be a predictor of death in ad patients. it has limited differential diagnostic power ; nevertheless, it is a reliable sign of an active process. previous studies have reported conflicting evidence regarding findings of bbb dysfunction in neurodegenerative dementias, often attributing the presence of altered bbb integrity to concurrent neurovascular diseases. however, evidence of altered bbb integrity in both neurodegenerative and vascular dementias has been established - even if bbb dysfunction was still found to be more pronounced in vascular dementia than in ad. there were no compelling signs of bbb dysfunction (i.e. abnormal csf / serum albumin ratio or igg index) in any of the groups of our study. likewise, the roc curves revealed a poor performance of all csf neuroinflammation parameters when used to predict future cognitive decline and clinical progression in our population of diagnostically unresolved patients. the strengths of our study are, first of all, its clinical setting, since we used a consecutively recruited, heterogeneous memory clinic population. second, all diagnoses were established by consensus among an experienced multidisciplinary team based upon all the available examination results, and the diagnoses were reevaluated on an ongoing basis. third, all lumbar punctures were performed by experienced physicians at 2 memory clinics, with established standard procedures for the lumbar puncture and subsequent sample handling. finally, the csf ad biomarker analyses were all carried out at one central laboratory. the lumbar punctures and csf analyses were performed in a clinical setting during the diagnostic workup, and the results were used in the diagnostic consensus - building process. this may have influenced diagnoses both at baseline and during the ongoing clinical evaluation, but not with regard to whether patients clinically progressed during follow - up. this may particularly be an issue with regard to the clinically stable group, as it is well established that time to conversion varies within a wide range, and it is likely that some of the patients labelled clinically stable in our study will eventually convert to having a neurodegenerative disease. the variability in follow - up periods is partly due to the fact that shortly after their initial diagnosis, a few patients were reevaluated and found without cognitive complaints or to suffer from nondegenerative diseases, and thus were lost to follow - up. the strengths of our study are, first of all, its clinical setting, since we used a consecutively recruited, heterogeneous memory clinic population. second, all diagnoses were established by consensus among an experienced multidisciplinary team based upon all the available examination results, and the diagnoses were reevaluated on an ongoing basis. third, all lumbar punctures were performed by experienced physicians at 2 memory clinics, with established standard procedures for the lumbar puncture and subsequent sample handling. finally, the csf ad biomarker analyses were all carried out at one central laboratory. the lumbar punctures and csf analyses were performed in a clinical setting during the diagnostic workup, and the results were used in the diagnostic consensus - building process. this may have influenced diagnoses both at baseline and during the ongoing clinical evaluation, but not with regard to whether patients clinically progressed during follow - up. this may particularly be an issue with regard to the clinically stable group, as it is well established that time to conversion varies within a wide range, and it is likely that some of the patients labelled clinically stable in our study will eventually convert to having a neurodegenerative disease. the variability in follow - up periods is partly due to the fact that shortly after their initial diagnosis, a few patients were reevaluated and found without cognitive complaints or to suffer from nondegenerative diseases, and thus were lost to follow - up. we investigated a group of patients with cognitive impairment who, even after an extensive diagnostic assessment involving csf biomarkers, remained diagnostically unresolved as to the underlying cause of their impairment. we found that baseline csf ad biomarkers - but not csf neuroinflammation parameters - had the potential to predict clinical progression, regardless of the underlying condition. furthermore, the csf a42/p - tau ratio showed both high discriminative power in separating progressive from stable diagnostically unresolved patients and a great ability to predict ad as the underlying neurodegenerative disease. this implies that the csf a42/p - tau ratio might potentially be superior to the csf ad biomarkers individually in predicting continuous cognitive decline and clinical progression in a group of initially diagnostically unresolved patients. our findings also suggest that a large proportion of initially diagnostically unresolved patients might in fact present with early ad with atypical or subtle symptoms and signs. our findings may be clinically relevant in improving prognostic measures for initially diagnostically unresolved patients, but they are also relevant in identifying suitable patients for clinical trials of disease - modifying agents, which should be administered very early in the clinical course. more longitudinal studies of the diagnostic and predictive performance of csf biomarkers in diagnostically unresolved patients with longer follow - up periods are needed to support our findings. | backgrounddespite an extensive evaluation program, patients may remain diagnostically unresolved with regard to the etiology of their cognitive dysfunction. cerebrospinal fluid neuroinflammation and alzheimer disease (ad) biomarkers may act as indicators of neurodegenerative disorders in diagnostically unresolved patients.methodsdata on 348 patients were retrospectively evaluated. all participants had a standardized diagnostic workup and follow - up in a memory clinic.resultsa42 levels and a42/p - tau ratios were reduced and levels of t - tau and p - tau as well as the t - tau p - tau / a42 ratio were elevated in diagnostically unresolved patients who clinically progressed, compared to a stable group. no differences in neuroinflammatory parameters were found.conclusionad biomarkers - in particular the a42/p - tau ratio, but not neuroinflammatory parameters - predicted clinical progression, regardless of etiology. |
the nduja of spilinga protected geographical indication (pgi) is a spreadable salami produced in calabria region (southern italy), on the plateau of monte poro. it is produced especially in a little town called spilinga (vibo valencia province), from which its name is derived. it is obtained using only lean and fat of pork, red pepper and sodium chloride. the lean and fat used is derived from lard, bacon, cheek lard and throat, trimming of lean, obtained exclusively from pigs slaughtered in italy. the mixture is made by using 50% fat, 25% lean meat and the remaining 25% red pepper (sweet and spicy) dried and shredded. the red pepper, according to the product specification, must be exclusively produced in calabria region.. then it is stored at refrigeration temperature for 12 h, followed by stuffing into natural casings (large intestine or caecum of pig) (giuffrida and callipo, 2010). the process continues with a phase of draining and drying at controlled temperature and humidity, and finally ripening, characterised by intense phenomena of proteolysis and lipolysis. lipolysis is related to a relevant mycotic flora, predominantly represented by yeasts, which are characterised by lipolytic activity (sorensen, 1997). in nduja, the yeast at the end of seasoning reach values slightly below 6 log colony forming units (cfu)/g, representing the predominant flora (giarratana., 2011), contrary to other italian cured sausages (comi and cantoni,1980), which are characterised by value of 3 log cfu / g. due the need to enhance and protect the traditional local products, it seemed interesting to carry out a characterisation of yeasts isolated from the nduja of spilinga pgi. the present study was carried out on a total of 127 strains of yeast isolated from samples of nduja of spilinga pgi. seventy - nine strains were from 15 samples analysed in a previous study (giarratana., 2011) at different days of curing, while the remaining 48 strains were isolated from 15 nduja of spilinga pgi collected at detail. twenty - five grams of each sample were collected and transferred into a sterile stomacher bag, where 225 ml of saline - peptone water was added in a ratio of 1:10, and then the mix were homogenised, for 120 s at 230 rpm, with a stomacher (stomacher 400 circulator ; international pbi s.p.a., serial dilutions were made, and each dilution was inoculated in duplicate on plates of dichloran rose bengal chloramphenicol agar (drbc) (oxoid ltd., basingstoke, uk) and of malt extract agar (mea) (oxoid) and incubated at 25c for 3 - 5 days. for each sample single pure colonies obtained on mea, were subject to the following evaluation : i) morphological characterisation [colony morphology on mea, cellular morphology by scanning electron microscope (sem) (phenom g2 prox), and ascospores production on modified gorodkowa agar were evaluated ] ; ii) hydrolysis of urea (streaking on christensen s urea agar incubated at 25c for 2 days, and on rapid urea broth, at 37c for 4 h) ; iii) lipolytic activity [assayed by measuring the halo all around the colonies grown on butter agar at 25c for 7 days. g / l) in distilled water and the ph was adjusted to 7 and sterilised at 121c for 15 min. successively, butter fat previously melted at 50c and pasteurised (100c for 5 min) was added to the media with a concentration of 5%. all the strains were finally identified with api 20c aux, i d 32c systems (biomrieux, marci letoile, france) and with simplified identification system (dek and beuchat, 1996). one hundred twenty - three (96.8%) strains isolated on mea, appeared as spherical whitish colonies, raised, with a shiny surface. microscopically cells were spherical or slightly oval, organised as single, in pairs or in short chains. these strains were ascribable to phylum ascomycetes, resulting also urease negative. the remaining 4 strains (3.2%), instead, belonged to the phylum of basidiomycetes, being urease positive. were characterised by a typical pinky color on mea, and the remaining (criptococcus spp.) appeared translucent. in all cases the cells showed elliptical shaped, single or in pairs and debaryomyces hansenii and its anamorfa shape, candida famata, represented the most prevalent species, reaching values of 61.42 and 17.32% respectively, followed by candida glabrata (8.66%) and pichia (candida) guilliermondii (5.17%) (table 1). all the strains of candida zeylanoides and 10 (7.8%) of debaryomyces hansenii showed lipolytic activity. the yeasts isolated from samples of commerce had charges of 60.3 log cfu / g. figures 1 and 2 show the identifications of the strains isolated from the samples during the production and from commerce. in both type of samples, the species isolated with greater frequency were represented by debaryomyces hansenii and its anamorfa shape, candida famata. eleven strains (22.92%) of candida glabrata were isolated from samples of commerce (figure 2). the yeasts identified in nduja of spilinga are similar to those reported in italian and european meat products. the main species isolated in this study was d. hansenii with a prevalence of 84%. d. hansenii is also frequently isolated from different italian, spanish and danish meat products (cocolin., 2006 ; gardini., 2001 ; nielsen, 2008 ; nunez., 1996 ; simoncini., 2007). in this regard d. hansenii was identified in 52% of the strains isolates from typical lucan sausages (gardini., 2001). a slightly lower prevalence (30%) of d. hansenii was reported in parma ham, followed by c. zeylanoides (23%) and debaryomyces maramus (21%) (simoncini., 2007). candida famata, c. catenulata, c. guilliermondii, hyphopichia burtonii, yarrowia spp., rhodotorula spp. and cryptococcus spp. d. hansenii is a yeast with nacl tolerance (up to concentrations of 24%), characterised by a very rapid aerobic metabolism. it is frequently isolated in cheeses, refrigerated foods, from brines for the production of cheese or meat products. it is able to metabolise lactic acid and citric acid and seems to have a good lipolytic and proteolytic activity. the lipolytic activity is performed not only by d. hansenii, but also by candida zeylanoides, as observed in strains isolated from nduja of spilinga. further investigation must be carried out on lipolytic activity of yeast isolated from nduja of spilinga, due that it is made by 50% of fat. these studies, supported by the use of molecular techniques, could significantly contribute to improve the aroma and taste of this product. yeasts and moulds traditionally play, in fact, an important role in sausage fermentation and contribute to the formation of characteristic flavours and the surface appearance. in this regard the yeasts, notably debaryomyces hansenii, contribute to nitrate reduction and colour formation (grazia., 1989 ; lucke and heckelmann, 1987), and seem to influence the aromatic profile by an appreciable production of volatile esters (sorensen, 1997). the eventual selection of autochthonous strains that could be used for the creation of specific starter, certainly could contribute to an improvement of the process of characterisation of nduja of spilinga, but also to guarantee a greater safety of the product. several yeasts show, in fact, competitive action against different pathogens or spoilage bacteria, such as enterobacteriaceae, as well as against moulds producing mycotoxins (mbadi., 2004). | the nduja of spilinga protected geographical indication (pgi) is a spreadable italian salami, obtained by using fat (50%), lean of pork (25%), chili pepper (25%) and nacl, stuffed into natural pork casing. its predominant flora is represented by yeasts, reaching at the end of seasoning values of 6 log cfu / g. considering the need to enhance and protect traditional local products, it seemed interesting to carry out a characterisation of yeasts of the nduja of spilinga pgi. a total of 127 strains of yeast isolated from samples of nduja of spilinga pgi (79 strains from samples at different days of curing and 48 from samples of commerce) was subjected to morphological identification, hydrolysis of urea, lipolytic activity and identification with api 20c aux, i d 32c and simplified identification systems. one hundred twenty three (96.8%) strains were attributable to the phylum ascomycetes (urease - negative), the remaining 4 strains (3.2%) were basidiomycetes (urease - positive). debaryomyces hansenii and its anamorph shape, candida famata, represented the most prevalent species (61.42 and 17.32% respectively), followed by candida glabrata (8.66%), pichia (candida) guilliermondii (5.17%), candida parapsilosis and rhodotorula glutinis (1.57%). candida catenulata, criptococcus uniguttulatus, rhodotorula minuta, candida zeylanoides and candida utilis were observed with 0.79%. the lipolytic activity was observed only in 10 strains of d. hansenii and in one of c. zeylanoides. further investigation will contribute to the selection of indigenous strains that could be used for the creation of specific starter, useful to improve the process of characterisation of the nduja of spilinga and also to guarantee its safety. |
mucopolysaccharidosis type ii (mps ii, hunter disease, mim 309900) is an x - linked lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase (ids). clinical manifestations are coarse facial features, hepatosplenomegaly, dysostosis multiplex, heart disease, obstructive upper respiratory disease, and frequent ear infections. the human ids gene is located in chromosome xq28, spans approximately 24 kb and contains nine exons. a full length cdna clone of the gene showed an open reading frame of 1650 bp long predicting a protein with 550 aminoacids. an ids pseudogene (idsp1) has been detected 20 kb distal to the telomeric side from the functional gene, whose sequences are highly homologous to exons 2 and 3 and introns 2,3 and 7 of the ids gene. to date, over 500 different mutations associated with mps ii have been identified [human gene mutation database (hgmd) ; http://www.hgmd.org/ ]. we report the results of genotype and phenotype characterization of 49 hunter patients of 40 families from argentina. forty nine hunter patients and their mothers (only 28 of mother samples where available) from 40 families were included in this study. parents of the patients gave their informed consent previous to the participation in the study. mps ii diagnosis was confirmed by clinical examination and deficient ids activity (activity of arilsulfatase b was normal in all cases, ruling out multiple sulfatase deficiency). phenotype of mpsii patients has been classified into attenuated (non - neuronopathic) or severe (neuronopathic) according to the clinical evidence of cognitive or developmental involvement. genomic dna was extracted from edta blood using a qiaamp dna blood mini kit (qiagen, valencia, ca, usa). the 9 exons of the ids gene including exon / intron boundaries were amplified by pcr using specific primers as described. sequencing was carried out in both directions in a megabase 1000 sequencer (amersham biosciences, sunnyvale, ca, usa). recombination between ids and idsp1 leading to any inversion mutation was analyzed by a pcr amplification assay as described. total cellular rna was isolated from blood leukocytes by means of a total rna isolation system (macherey nagel, duren, germany). the isolated rna samples were reverse transcribed using a specific primer, according to the method of jonsson and then amplified in 2 overlapping fragments and sequenced. forty nine hunter patients and their mothers (only 28 of mother samples where available) from 40 families were included in this study. parents of the patients gave their informed consent previous to the participation in the study. mps ii diagnosis was confirmed by clinical examination and deficient ids activity (activity of arilsulfatase b was normal in all cases, ruling out multiple sulfatase deficiency). phenotype of mpsii patients has been classified into attenuated (non - neuronopathic) or severe (neuronopathic) according to the clinical evidence of cognitive or developmental involvement. genomic dna was extracted from edta blood using a qiaamp dna blood mini kit (qiagen, valencia, ca, usa). the 9 exons of the ids gene including exon / intron boundaries were amplified by pcr using specific primers as described. sequencing was carried out in both directions in a megabase 1000 sequencer (amersham biosciences, sunnyvale, ca, usa). recombination between ids and idsp1 leading to any inversion mutation was analyzed by a pcr amplification assay as described. total cellular rna was isolated from blood leukocytes by means of a total rna isolation system (macherey nagel, duren, germany). the isolated rna samples were reverse transcribed using a specific primer, according to the method of jonsson and then amplified in 2 overlapping fragments and sequenced. until now, we have found the mutation in all the patients analyzed from argentina. thirty different mps ii alleles have been identified in a cohort of 49 argentinean patients from 40 families : 18 missense (60%), 5 splicing (17%), 3 short deletions (10%), 2 nonsense mutations (7%), 1 complex rearrangement (a gene pseudogene recombination leading to inversion) (3%) as well as 1 gross gene deletion (3%) (table 1). of the mutations in this cohort, 17 out of 30 different alleles (57%) were novel (missense : p.pro228gln, p.lys295ile, p.asp198asn, p.trp345arg, p.ser142tyr, p.gly134glu, p.gly312asp, p.thr214met, p.gln465leu, p.pro157ser ; splicing : c.508 - 1 g > a, c.241 - 5a > t, c.708 + 1 g > a ; nonsense : p.glu274x ; and short deletions : c.908_909delct, c.411delt, c.22_37del16pb). they are all predicted to be probably damaging, as analyzed by the prediction tool polyphen, and are localized in conserved aminoacids between human and murine ids but found in patients with both attenuated and severe phenotypes. although the change in the structure of these highly conserved aminoacids is high, both patients display an attenuated phenotype. the mutations p.pro228gln and p.pro157ser in severe patients result from the replacement of a rigid nonpolar aminoacid by a polar one. the rest of the novel missense mutations are not localized in conserved aminoacids, however the effect in the phenotype is more devastating. previously described mutations detected by us were p.arg468gln, p.ala85thr, p.ser333leu, p.pro120his, p.ser61pro, p.leu339pro, p.asp478gly, p.gln465x, p.arg443x, the splice mutations c.1122c > t, c.1181 - 1 g > a, and the recombination gene pseudogene. the mutation c.508 - 1 g > a was found in a patient with an attenuated phenotype. at the molecular level, the mutation produced a predominant alternative transcript and a minor quantity of the correct transcript. the mutant transcript results from the disruption of the 3 original splice - site leading to the deletion of exon 5. the presence of the correct mrna, although in minor quantity, would explain the less severe clinical manifestations observed in this patient. on the other side, the splicing mutations c.241 - 5a > t and c.708 + 1 g > a, each generate a unique mrna product, whose sequence corresponds to the complete deletion of exons 3 and 5, respectively, and are associated with a severe phenotype. in one patient, two different neighbor mutations (p.arg468gln + p.gln465leu) were found. the mother was a carrier of both mutations, but not the grandmother and the 4 maternal aunts. the finding of these two close mutations in the patient and his mother but not in the grandmother suggests that they are de novo mutations in a mutation - prone region and reinforces the idea that they arose simultaneously. none of the flanking markers tested were detected, suggesting that the deletion spans more than 20 kb upstream and 7 kb downstream of the ids locus, and includes the idsp1 locus. no atypical symptoms of hunter syndrome were found in these patients, indicating that the deletion did not compromise contiguous genes. the same phenotype was observed for the three of the patients who exhibited a recombination between gene and pseudogene. the absence of the probands ' mutations in their mother suggests that in the three cases, the mutations occurred de novo (10%). however, the possibility of germinal mosaicism could not be ruled out. the theoretical rate calculated by the bayesian methods for this condition would be 33%, so the value found in our cohort is much less. in the cohort reviewed by froissart., they found values of de novo mutations close to the expected in any x chromosomal disease in which male reproductive fitness is extremely low. the low value found in our group could be because much of the patients from argentina were diagnosed because of a previous family history of mps ii, meaning there are still many undiagnosed patients in argentina. we also found a low rate of the polymorphism in exon 4 c.438c > t, only in 4 families (10%) as compared to the frequency reported in normal controls of around 30%. the percentage of private mutations in our series was 75%, a high proportion as usually seen in other series. although genotype phenotype correlation is difficult to establish, it is worth mentioning that both missense (p.ser61pro, p.ala85thr, p.gly134glu, p.gly312asp, p.asp478gly) and splice mutations (c.1122c > t, c.508 - 1 g > a) were associated with non - neuronopathic (attenuated) phenotype. from our series of 30 alleles, 7 (23%) three of the mutations associated with the non - neurological phenotype, are new (c.508 - 1 g > a, p.gly134glu, p.gly312asp). our results provide further evidence of mutational heterogeneity of the ids gene observed in patients with mps ii. this work was financially supported by a grant from agencia nacional de promocin cientfica y tecnolgica, argentina (pict 01070) to pr, however the funding source had no involvement in study design ; in the collection, analysis and interpretation of data ; in the writing of the report nor in the decision to submit the article for publication. | mucopolysaccharidosis type ii (mpsii) is an x - linked lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase (ids). the human ids gene is located in chromosome xq28. this is the first report of genotype and phenotype characterization of 49 hunter patients from 40 families of argentina. thirty different alleles have been identified, and 57% were novel. the frequency of de novo mutations was 10%. overall, the percentage of private mutations in our series was 75%. |
obesity is not only a problem of appearance, but also a main cause of various lifestyle diseases such as hyperlipidemia, hypertension, cardiovascular diseases, and diabetes. although obesity has emerged as a cause of these complications, in reality, there are lack of efforts to prevent and manage it, thu, the prevalence of obesity is steadily increasing. obesity is mainly caused by lack of regular physical exercise, westernized diet, genetic factors, and excessive and unbalanced nutrition. especially, the workers have high vulnerability to obesity and lifestyle diseases by unhealthy life habits such as lack of physical exercise due to busy work, stress, eating - out, and alcohol consumption. impact of intestinal microbes on obesity and health is a new topic started to get attention recently. in 2009, an experimental research was introduced to prove the hypothesis, " gastrointestinal bacteria contribute to obesity. " four atypical human twins (pair of one lean and one obese twin) were recruited. the observed results have shown that mice received gastrointestinal bacteria from the lean twin maintained their weight whereas mice transplanted with gut microbiota from obese twin showed a rapid weight gain. after the researchers confirmed " the effects of gastrointestinal bacterial transplantation on obesity ", they assessed two mice received bacteria from one pair of twins by an additional experiment to verify the exchange of two different bacteria. it confirmed the unidirectionality of bacterial survival, in which the gut microbiota from obese mice ceases to survive in the gastrointestinal tract of lean mice whereas bacteria from lean mice survived in the obese mice. bacteria in the gastrointestinal tract of lean mice did not tolerate high proportion of saturated fat ' human ' diet with less fruits and vegetables but transformed into the bacteria in the fat twin. follow - up studies have confirmed that diet determines the composition of a bacteria, and gastrointestinal bacteria has an impact on obesity. considering to the global concern and research trends, there are few research on the association between intestinal bacteria and body weight change in korean. in this report, one voluntary case (female) will be introduced to show the change in gut microbiota and weight by diet intervention. the 27-year - old korean woman with no underlying disease or medical history volunteered in this research to improve obesity. she visited outpatient department of family medicine on october 1, 2013. according to anthropometric measurements, she was 1.73 meters tall with body mass index (bmi) of 23.2 kg / m with weight of 69.5 kilograms. the who regional office for the asia pacific region recommends defines obesity by a bmi 25 kg / m and overweight by bmi above 23 kg / m and below 25 in asians. the korean society for the study of obesity also adopted the who - recommended definition to study the cutoff of bmi for obesity - related disease. now, korean government organizations officially use this definition when defining and implementing health policies regarding obesity in korea. after verifying obesity test results, she was explained about the research in detail and asked to compose a meal record for three days prior to the test. the result from the hospital 's dietician showed the causes of overweight are frequent drinking and meat consumption at late evening hours. to determine the diet responsible for the weight loss, change in the intakes of total calorie, carbohydrate, and fat (especially animal fat) the result showed that overall intakes of total calories, carbohydrate, and protein were evenly decreased after the diet intervention, and the reduction in large amount of animal fat was observed by cutting down the consumption of fish and meat. the amount of alcohol she consumes at once is approximately total of 95 grams ; from 30 grams in 4.5 glasses of soju and 65 grams in 9 cups of beer. blood and urine tests were performed to determine whether comorbid conditions associated with drinking habit. the results confirmed normal except for mild anemia (hemoglobin, hb 11.6g / dl) and mild increases in aspartate aminotransferase (ast ; 58iu / l). in detail, frequent intake of carbohydrate= and excessive consumption of fish, meat, bread, ice cream, etc. at late evening hours however, after the diet intervention, she put efforts to reduce food intake and select vegetable side dishes and fruit as a replacement for snacks, thus, average consumption of calories, carbohydrates, protein, and animal fat decreased (table 1). additionally, decrease in frequency of food intake with high in sodium such as soup or stew was observed. changed behavior in alcohol consumption can not be determined by the short study period ; however, a consultation confirmed that she managed to reduce her alcohol intake by approximately 50 percent. for 47 days, she was administered an improved diet with breakfast and dinner with less meat and reduction of alcohol consumption and frequency by 50% and once a week, respectively. at the second follow - up the request on case 's fecal sample analysis was submitted to chunlab, inc. prior to participating in diet improvement research. after the 47 days of research, the case 's fecal sample was again collected on november 19, 2013 to compare the distribution of intestinal micro flora with the first sample. bacterial dna was extracted from the fecal samples of subjects before and after the treatment using a fastdna spin extraction kit (mp biomedicals, santa ana, ca, usa). gut microbiota were compared before and after the treatment using the high - throughput sequencing technique. the compositions of phyla and genera were compared between before and after the diet correction. firmicutes was predominant phylum (75.7% of total microbiota) before the diet correction, whereas firmicutes (47.3%) and bacteroidetes (47.7%) were dominant phyla after the diet correction. in sample collected before the diet correction, genera of faecalibacterium (14.9% of total microbiota), roseburia (14.8%), blautia (9.5%) and lactobacillus (9.1%) within firmicutes, and prevotella (15.0%) within bacteroidetes after the diet correction, the relative abundance of prevotella (42.1% of total microbiota) and megasphaera (10.7%) within firmicutes was observed, while those of faecalibacterium (8.6%), roseburia (1.2%), blautia (3.0%), and lactobacillus (1.7%) were decreased. the proportion of bacteroides (4.4%) within bacteroidetes was also increased after sample (figure 1). the 27-year - old korean woman with no underlying disease or medical history volunteered in this research to improve obesity. she visited outpatient department of family medicine on october 1, 2013. according to anthropometric measurements, she was 1.73 meters tall with body mass index (bmi) of 23.2 kg / m with weight of 69.5 kilograms. the who regional office for the asia pacific region recommends defines obesity by a bmi 25 kg / m and overweight by bmi above 23 kg / m and below 25 in asians. the korean society for the study of obesity also adopted the who - recommended definition to study the cutoff of bmi for obesity - related disease. now, korean government organizations officially use this definition when defining and implementing health policies regarding obesity in korea. after verifying obesity test results, she was explained about the research in detail and asked to compose a meal record for three days prior to the test. the result from the hospital 's dietician showed the causes of overweight are frequent drinking and meat consumption at late evening hours. to determine the diet responsible for the weight loss, change in the intakes of total calorie, carbohydrate, and fat (especially animal fat) were analyzed based on the diet history of the case. the result showed that overall intakes of total calories, carbohydrate, and protein were evenly decreased after the diet intervention, and the reduction in large amount of animal fat was observed by cutting down the consumption of fish and meat. the amount of alcohol she consumes at once is approximately total of 95 grams ; from 30 grams in 4.5 glasses of soju and 65 grams in 9 cups of beer. blood and urine tests were performed to determine whether comorbid conditions associated with drinking habit. the results confirmed normal except for mild anemia (hemoglobin, hb 11.6g / dl) and mild increases in aspartate aminotransferase (ast ; 58iu / l). in detail, frequent intake of carbohydrate= and excessive consumption of fish, meat, bread, ice cream, etc. at late evening hours however, after the diet intervention, she put efforts to reduce food intake and select vegetable side dishes and fruit as a replacement for snacks, thus, average consumption of calories, carbohydrates, protein, and animal fat decreased (table 1). additionally, decrease in frequency of food intake with high in sodium such as soup or stew was observed. changed behavior in alcohol consumption can not be determined by the short study period ; however, a consultation confirmed that she managed to reduce her alcohol intake by approximately 50 percent. for 47 days, she was administered an improved diet with breakfast and dinner with less meat and reduction of alcohol consumption and frequency by 50% and once a week, respectively. at the second follow - up the request on case 's fecal sample analysis was submitted to chunlab, inc. prior to participating in diet improvement research. after the 47 days of research, the case 's fecal sample was again collected on november 19, 2013 to compare the distribution of intestinal micro flora with the first sample. the seoul national university - boramae hospital institutional review board approved this study bacterial dna was extracted from the fecal samples of subjects before and after the treatment using a fastdna spin extraction kit (mp biomedicals, santa ana, ca, usa). gut microbiota were compared before and after the treatment using the high - throughput sequencing technique. the compositions of phyla and genera were compared between before and after the diet correction. firmicutes was predominant phylum (75.7% of total microbiota) before the diet correction, whereas firmicutes (47.3%) and bacteroidetes (47.7%) were dominant phyla after the diet correction. in sample collected before the diet correction, genera of faecalibacterium (14.9% of total microbiota), roseburia (14.8%), blautia (9.5%) and lactobacillus (9.1%) within firmicutes, and prevotella (15.0%) within bacteroidetes after the diet correction, the relative abundance of prevotella (42.1% of total microbiota) and megasphaera (10.7%) within firmicutes was observed, while those of faecalibacterium (8.6%), roseburia (1.2%), blautia (3.0%), and lactobacillus (1.7%) were decreased. the proportion of bacteroides (4.4%) within bacteroidetes was also increased after sample (figure 1). this observation, confirmed the association between the changes in body weight, diet and gut microbiota. in particular, 3 kilograms of body weight was reduced by diet correction, which includes limiting the overall intakes of total calories, fat, protein, and carbohydrate, as well as reducing salt intake and alcohol consumption frequency. considering previous studies with various method of short - term dietary interventions, it is observed that a person with diet intervention showed 2 - 4% additional weight loss than general attempts. during the weight loss, this change was consistent with previous studies which determined that the ratio of firmicutes / bacteroidetes was higher in an obese person than a lean person. although the impact on gut microbiota depends on genetic, and environmental factors, and obesity, obese people showed a change of microbiota on the phylum levels and rapid decrease of microbiota diversity compared to healthy people. it is meaningful to confirm the changes in gut microbiota and weight loss through the diet intervention. further study on this subject is needed to investigate the effect of dietary intervention on body weight loss and gut microbiota change. | impact of intestinal microbes on obesity and health is a new topic recently started to get attention. comparing to the global concern and research trends, there are few research on the association between intestinal bacteria and life style disease in korean. one voluntary case (female) was reported to show the change in gut microbiota and weight by diet intervention. she was overweight (bmi 23.2 kg / m2) and has abnormal liver function, and the causes of overweight were frequent drinking and meat consumption at the late evening hours. for 47 days, she was administered an improved diet on breakfast and dinner with reduction of meat consumption frequency by 50%. alcohol consumption was reduced to once a week. as a result, she lost 3 kilograms of body weight. her fecal sample was collected before and after the intervention, and gut microbiota change was compared using a high - throughput sequencing technique. after diet correction, the shift of gut microbiota was clearly observed with decreased proportion of firmicutes (from 75.7% to 47.3% in total microbiota) but increased proportion of bacteroidetes upto 47.7%. after incorporating the diet intervention, it is meaningful to confirm the changes in dominant gut microbiota and weight loss. |
samples collection and processing : tissue samples were taken during post mortem examinations from the intestine of 12 healthy laboratory beagle dogs (8 males/4 females, age 1013 years), after finishing another non - related study which was approved by the finnish national animal experiment board (study license no. the dogs participated in dietary studies and were kept under the same environmental and nutritional conditions before they needed to be euthanized. they were housed according to european union guidelines in groups in indoor pens with possibilities to use outdoor runs. the dogs were exposed to both natural and artificial light (from 07:00 to 16:00). they were fed with a standard commercial diet and were determined to be healthy based on history, physical examination, complete blood count, serum biochemistry and fecal examination. immediately after euthanasia, full - thickness tissue samples were taken from four different parts of the intestine (duodenum, jejunum, ileum and colon) and were snap frozen in liquid nitrogen and stored at 80c until histological and zymographic analyses. for histologic evaluation, parts of the frozen intestinal tissue samples were later slowly thawed and fixed in 4% formaldehyde solution in phosphate buffered saline (pbs) at 8c under permanent automatic rotation of the sample tube. then, the samples were trimmed and paraffin wax embedded. sections (35 m) were prepared and stained with hematoxylin and eosin for histological examination. assessment of intestinal health : all dogs were clinically healthy without any signs of gastrointestinal diseases based on history, physical examination and laboratory examination of feces and blood. histological assessment of the intestinal samples was performed using the guide lines of the world small animal veterinary association (wsava) gastrointestinal standardization group. in all small bowel samples, 5 morphological features (villous stunting, epithelial injury, crypt distention, lacteal dilation and mucosal fibrosis) and 3 types of infiltrated leukocytes (intraepithelial lymphocytes, lamina propria lymphocytes and lamina propria neutrophils) were selected and scored from 0 to 3 according to the wsava standardization guidelines. in the colonic samples, crypt hyperplasia, dilatation and distortion were additionally evaluated. the total scores were classified as insignificant (scores 04), mild (scores 59), moderate (scores 1014), severe (scores 1519) or very severe (scores 20). sample preparation for zymography : mucosa samples (50 mg) were taken from the snap frozen intestinal tissues and homogenized for 2 50 sec with 5,000 g in ice - cold extraction buffer (20 l / mg tissue) containing 50 mm tris base, 150 mm nacl, 10 mm cacl2, 0.2 mm nan3 and 0.01% triton x-100 (ph 7.6) in the presence of edta - free protease cocktail tablets (complete edta - free tablets, roche, basel, switzerland) using precellys 24 ceramic beads (bertin technologies, paris, france). after homogenization, samples were centrifuged at 13,000 g at 4c for 10 min, and the supernatants were collected and stored at 80c for measurement of mmp-2 and mmp-9. protein concentrations of the supernatants were measured with bicinchoninic acid protein assay reagents (pierce, rockford, il, u.s.a.). zymography : gelatinolytic activities of mmp-2 and mmp-9 in supernatant were measured by gelatin zymography in mini - gels as previously described in detail. supernatants were separated by electrophoresis in 11% polyacrylamide gel impregnated with 0.7 mg / ml of gelatin as a substrate (porcine skin gelatin, g-8150, sigma, st. each lane of an 11% sds - polyacrylamide gel was loaded with 20 l of supernatants containing either 10 g or 25 g of total protein mixed with a 10 l aliquot of loading buffer. 8122) (bdh), 20% glycerol and 6% sodium dodecyl sulphate (sds, prod. electrophoresis was performed by using a mini - protean tetra cell electrophoresis system (bio- rad laboratories, hercules, ca, u.s.a.) under a constant current of 60 ma for 10 min and then 30 ma until the bromophenol blue reaches the bottom of the gel. after electrophoresis, the gels were washed in distilled water and then soaked (2 30 min) in renaturing buffer (2.5% triton x-100) with gentle shaking at room temperature in order to remove the sodium dodecyl sulfate. then, the gels were soaked in zymogram developing buffer (50 mm tris base, ph 7.5 containing 200 mm nacl, 5 mm cacl22h2o and 0.02% brilj-35) for 30 min at room temperature, then replaced with fresh developing buffer and incubated for another 18 hr at 37c. after washing the gels with distilled water 3 times for 10 min, they were stained with pageblue protein staining solution (fermentas) and stained with gentle agitation for 5 hr. the areas of proteinase activity were visualized as clear bands by washing the gels with distilled water. as a control, each gel was loaded with a diluted (1:600) recombinant human mmp-2 (9 l) and -9 (2 l) (r&d systems, minneapolis, mn, u.s.a.), respectively, to determine whether mmps in canine intestinal tissue are equivalent in molecular size and activity to pro - mmp-2 (72 kda gelatinase) and pro - mmp-9 (92 kda gelatinase). for quantification of gelatin degradation, gels were scanned and were assessed by densitometer analysis method creating an arbitrary unit (au) for each band by calculating the integrated area under each peak (alpha - imager densitometer, alpha innotech, san leandro, ca, u.s.a.). the activity levels of pro- and active mmp-2 and -9 for each sample were expressed in au related to the level of pro - mmp-2 of the positive - control standard loaded on each gel. each band s activity was reported as the mean of two different measurements of the same sample. statistical analysis : the differences between the separate parts of the intestine (duodenum, jejunum, ileum and colon) in their proportions of samples with pro - mmp-2 and -9 activities were analyzed with exact mcnemar s test. data are presented as number (%) or median (range) as appropriate. all statistical analyses were performed using the sas 9.3 statistical software (sas institute inc., histological examination : based on the histological examination of the intestine, the median total wsava score of all samples was 0 (range 03) classifying all findings as insignificant. there were no histological abnormalities in the submucosa, the muscularis externa and the serosa in any of the samples. zymography : zymography was performed with 48 samples from four different intestinal areas of 12 healthy dogs. when positive, it showed gelatinolytic activity at the same level as the positive control bands of pro - mmp-2 and -9 and was therefore considered to represent canine pro - mmp-2 and pro - mmp-9 (fig. 1fig.1.representative zymogram of protein (25 g) from intestinal mucosa samples { pro - mmp-2 and -9 positive samples (lanes 36) and samples with no activity (lanes 7 and 8) } and recombinant human pro - mmp-2 (lane 2) and -9 (lane 1).). since the majority of samples containing 10 g of total protein showed no activity (pro - mmp-2 : 40/48, 83% ; pro - mmp-9 : 33/48, 69%), only the results for samples containing 25 g of total protein are presented in this paper. pro - mmp-2 and -9 activities were found in 17/48 (35%) and 25/48 (52%) of the samples, respectively. none of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active mmp-2 and mmp-9. representative zymogram of protein (25 g) from intestinal mucosa samples { pro - mmp-2 and -9 positive samples (lanes 36) and samples with no activity (lanes 7 and 8) } and recombinant human pro - mmp-2 (lane 2) and -9 (lane 1). results of the mcnemar test for statistical analysis showed no significant difference of pro - mmp-2 and -9 activities between the four separate parts of the intestine (duodenum, jejunum, ileum and colon) (p>0.05). however, for pro - mmp-2, the number (and percentage) of positive samples in four different parts of the intestine of 12 dogs were from the highest to the lowest as follows : ileum, 7/12 (58.3%) ; jejunum, 5/12 (41.7%) ; duodenum, 3/12 (25%) ; and colon, 2/12 (16.7%). for pro - mmp-9, ileum had the highest positivity rate (8/12 ; 66.7%), followed by jejunum (6/12 ; 50%), duodenum (6/12 ; 50%) and colon (5/12 ; 41.7%). from all 48 canine intestinal mucosa samples examined with zymography, no pro - mmp-2 and -9 activity was found in 31/48 (65%) and 23/48 (48%), respectively. the enzyme activities ranged for pro - mmp-2 between 0.015 and 6.449 au and for pro - mmp-9 between 0.018 and 5.680 au. in the examined four different parts of the intestine, the median (range) pro - mmp-2 activity was from the highest to the lowest : ileum, 0.175 (03.337) au ; jejunum, 0 (06.449) au ; duodenum, 0 (04.301) au ; and colon, 0 (00.146) au. the highest pro - mmp-9 activity (median and range) was found in the ileum (0.086 (01.443) au), followed by colon (0.018 (01.652) au), jejunum (0.011 (02.810) au) and duodenum (0 (05.680) au). in the majority of positive samples (36/42 ; 86%), either pro - mmp-2 or -9 activities were lower than 2 au throughout the whole intestine (table 1table 1.distribution of pro - mmp-2 and -9 gelatinolytic activities in mucosa samples from duodenum, jejunum, ileum and colon of 12 healthy dogs au : arbitrary units, pro : pro - enzymeintestinal partvariabledog s no123456789101112duodenumpro - mmp-2 (au)00004.300000.511.4600pro - mmp-9 (au)0.2300000.04005.685.420.620jejunumpro - mmp-200006.450.581.530.230.23000pro - mmp-90.02000001.090.182.810.0200.73ileumpro- mmp-20.150.290000.5603.340.200.5001.88pro - mmp-90.5901.440.0600.1000.620.180.080.090colonpro - mmp-20.1500000000000.03pro - mmp-91.6500.140.0900000.040.0500.04). comparably high activities (au 2) of pro - mmp-2 were recorded in two mucosa samples from duodenum and jejunum and one ileum. two duodenal mucosa samples and one jejunum sample had the highest activities of pro - mmp-9 (table 1). this study aimed to detect the presence of mmp-2 and -9 in four different parts of the intestinal mucosa of clinically healthy beagle dogs by using gelatin zymography. the method has been used earlier to study mmp-2 and -9 in intestinal samples from humans [2, 10, 17 ], but to the best of our knowledge, not yet in samples from canine intestinal mucosa. in the current study, we used intestinal mucosa samples from 12 clinically healthy beagle dogs. to avoid the unnecessary use of laboratory animals for experimental studies, we sampled intestinal mucosa during necropsy from all 12 dogs after they were euthanized when finishing another non - related study. this approach complies with the principles of replacement, reduction and refinement of animal experiments. it seems unlikely that the previous studies could have interfered with our results since all dogs had the same living and nutritional conditions and were clinically healthy at the time of euthanasia. in addition, the histological examination of the intestinal mucosa was a part of our study to investigate the possibility of subclinical intestinal inflammation which might have influence on the interpretation of the study results. mmp-2 and -9 were extracted from canine intestinal mucosa according to castaneda.. however, in the current study, precellys device (bertin technologies) with tubes containing ceramic beads was used for grinding the mucosa samples. to prevent temperature rising during the lysis process and to protect sensitive molecules from degradation, cold air (50c) was sprayed by cryolys device (bertin technologies) beside the tubes so that temperature during homogenization remained at approximately 4c. gelatin zymography is a highly sensitive technique and is still widely used in the determination of mmp-2 and -9 in tissue samples. this technique can detect even small amounts of mmp-2 and -9 from tissue - homogenization supernatant. the gelatin zymography used here was described by rajamki. selecting both, 10 and 25 g of total protein in the extracted samples. the amount of 10 g protein is often used in human ibd research for determination of mmps by gelatin zymography. however, the majority of our samples containing 10 g of total protein showed no activity. it is likely that the levels of mmp-2 and -9 activities in healthy dogs are too low to be detected in 10 g protein samples in comparison to 25 g protein samples. therefore, only the results for samples containing 25 g of total protein are presented in this paper for the characterization of mmp-2 and -9 in the intestinal mucosa of healthy dogs. to assess whether higher protein amounts lead to mmp detection in negative samples, we used also larger protein amounts of 100 g leading also to negative results. in our canine samples, the zymography method produced distinct bands on the gel which corresponded to the standards with human pro - mmp-2 and -9. we did not detect any substantial difference between the migration of these enzymes from human standards and intestinal mucosa samples obtained from healthy dogs, which is in concordance with mmp-2 and -9 enzymes being highly conserved among species. gelatin zymography appears to be a useful method for the evaluation of intestinal mucosa samples, because it requires only a small piece of intestinal tissue sample (5 mg mucosa) which is the weight of about 12 endoscopically taken biopsies. in the present study, 65% and 48% of all samples showed no activity for pro - mmp-2 and -9, respectively, and the colon showed the lowest activity. one possible reason for the high amount of undetectable activity might be that the level of gelatinolytic activity of both mmps was less than the detection limit of the gelatin zymography assay. in addition, the extraction of mmps from tissues has some limitations for the following reasons : (1) mmps, such as mmp-1, mmp-2 and mmp-9, are tightly bound to the ecm ; (2) it is difficult to ascertain whether all mmps are extracted ; (3) specific mmps localized in a relatively small part of the tissue may not be detected due to dilution in the entire tissue extract [41, 44 ]. therefore, difficulties in the extraction of mmps from tissues might also be another reason for samples without mmp-2 and -9 activities. similar results were reported for normal human colon mucosa samples. in the study by baugh., pro - mmp-2 and -9 were not detected in 20% and 56% of their samples, respectively, using gelatin zymography when examining colon mucosa from nine healthy human subjects. in our study, none of the intestinal samples showed gelatinolytic activity corresponded to the control bands of active mmp-2 and mmp-9. did not detect active forms of mmp-2 and -9 in any of the colonic mucosal samples in healthy human subjects. reported the mean (sem) activities of pro - mmp-2 and pro - mmp-9 in normal colonic mucosa of humans to be 2 au for which we have not found any explanation so far. thirty - five percent of the samples were positive for pro - mmp-2 activity in the present study. in healthy dogs, pro - mmp-2 activity was mostly seen in the small intestine. this might be due to high levels of epithelial cell turnover in the small intestine. mmp-2 is commonly expressed in normal intestinal tissues and is believed to participate in the maintenance of collagen homeostasis and the intestinal tissue remodeling [10, 39 ]. in the current study, pro - mmp-9 activity was detected within 25/48 (52%) of the samples. under normal conditions, mmps including mmp-9 are expressed at low levels, usually in the pro - form, and when activated, they play a role in normal tissue ecm turnover, including intestinal tissue. in humans, mmp-2 and mmp-9 seem to be actively involved in the inflammatory and remodeling processes in ibd [2, 10, 16 ]. in a mouse model of ibd, mucosa - derived mmp-2 serves to protect against tissue damage possibly by regulation of epithelial barrier function [11, 12, 37 ]. in human ibd, it participates in the ecm remodeling and degradation of the basal membrane type iv collagen leading to intestinal ulceration, epithelial damage and/or fistula formation [10, 28, 29, 42 ]. the exact role of mmp-2 in mucosal injury of ibd remains to be fully clarified, however. mmp-9 has a crucial role in the induction of intestinal tissue inflammation through defective re - epithelialization, increased paracellular permeability and reduction in adhesion complex integrity, resulting in impaired wound healing, especially in the acute phase [11, 12, 37 ]. in human ibd, several studies have investigated the role of mmps and their inhibitors in the differentiating subtypes of ibd [25,26,27, 32, 33, 36, 38, 42 ]., mmp-7 has been suggested to aid in differentiation between crohn s disease and ulcerative colitis, while mmp-10 and timp-3 may be markers of ibd etiology in inflammatory processes of the gut. mmp - profiles in operated inflammatory disease (ibd) patients have also been looked at to evaluate the etiology of inflammatory flares - ibd or other - to properly treat them [25, 42 ]. to our knowledge, mmp profiles are not yet used for therapeutic purposes, but efforts to characterize ibd and to examine how treatment affects mmp expression have been made [26, 27, 32, 33 ]. mmp expression may also predict precancerous potential and long - term remission. while treatment for ibd subtypes share similarities, the most notable difference is found in surgical treatment, being more conservative in crohn s disease. mmp expression in the gut or elsewhere in the body, e.g. serum, may in the future help in properly differentiating the disease subtypes and severity and to enable tailored treatment choices for individuals. whether mmp-2 and -9 are also involved in the pathogenesis of canine ibd in future studies, however, immunohistochemistry should be included to also assess the localization of mmps in canine intestinal mucosa and their correlation with intestinal pathologies in dogs. the activity of mmp-2 and mmp-9 in different parts of the intestinal mucosa of healthy dogs has been determined here, laying the groundwork for future studies in canine chronic intestinal diseases, including ibd. pro - mmp-2 and -9 activities were found in the intestinal mucosa of the small and large intestines in healthy dogs, mostly in the ileum. active forms of mmp-2 and -9 were not detected in the intestinal mucosa of healthy dogs. understanding the role of mmp-2 and -9 in the pathogenesis of chronic intestinal inflammation in the future and using them as biomarkers for differentiating canine ibd subtypes might offer a possibility to improve the current treatment approach. the sample processing used for the study led to histological slides of sufficient quality for assessment. a limitation of the study might be that in some occasional samples, it was not possible to interpret epithelial changes due to processing artifact. however, since we used several samples per location for histology, there was enough epithelial layer on the majority of samples from the same intestinal part of every dog to allow a final interpretation. the submucosa, muscularis and serosa were not affected by artifacts. in conclusion, based on the results of the current study and reports from studies in human ibd [2, 10, 41 ], zymography presents as an appropriate method to evaluate mmp-2- and -9 profiles in canine intestinal mucosa. the results of this study may provide valuable information for future studies evaluating the role of mmps in the pathogenesis of canine chronic enteropathies including ibd. | abstract matrix metalloproteinases (mmps) 2 and 9 are zinc - dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. the main objective of this study was to identify the presence of mmp-2 and -9 in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. intestinal mucosa samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were taken from 12 healthy laboratory beagle dogs and examined histologically. based on wsava histology standards, recorded findings of all samples were considered insignificant. pro - mmp-2 and -9 activities were found in 17/48 (35%) and 25/48 (52%) of the samples, respectively. among four different parts of the intestine of 12 dogs, the ileum had the highest positivity rates of 7/12 (58.3%) and 8/12 (66.7%) for pro - mmp-2 and -9 activities, respectively. however, statistical analysis showed no significant difference of pro - mmp-2 and -9 activities between the separate parts of the intestine (p>0.05). none of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active mmp-2 and mmp-9. this study showed that pro - mmp-2 and -9 could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of mmp-2 and -9 in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases. |
ludwig 's angina was coined after the german physician, wilhelm friedrich von ludwig who first described this condition in 1836 as a rapidly and frequently fatal progressive gangrenous cellulitis and edema of the soft tissues of the neck and floor of the mouth. with progressive swelling of the soft tissues and elevation and posterior displacement of the tongue, the most life - threatening complication of ludwig 's angina is airway obstruction. as a result of antibiotic therapy, along with improved imaging modalities and surgical techniques, mortality currently averages approximately 8%. in ludwig 's angina, the submandibular space is the primary site of infection. this space is subdivided by the mylohyoid muscle into the sublingual space superiorly and the submaxillary space inferiorly. the majority of cases of ludwig 's angina are odontogenic in etiology, primarily resulting from infections of the second and third molars. the roots of these teeth penetrate the mylohyoid ridge such that any abscess, or dental infection, has direct access to the submaxillary space. infection can also spread contiguously to involve the pharyngomaxillary and retropharyngeal spaces, thereby encircling the airway. other causes include peritonsillar or parapharangeal abscesses, mandibular fractures, oral lacerations / piercing or submandibular sialadenitis, and oral malignancy. predisposing factors include dental caries, recent dental treatment, systemic illness such as diabetes mellitus, malnutrition, alcoholism, compromised immune system such as aids and organ transplantation.[69 ] without a treatment, it is frequently fatal from the risk of asphyxia with a mortality rate of 50%. the aggressive surgical intervention, the antibiotic introduction, and the improvement of dental care have determined a significant reduction of the mortality rate to less than 10%. a 25-year - old gentleman reported to the department of oral and maxillofacial surgery with a chief complaint of inability to open the mouth, pain, and swelling in relation to the lower jaw and neck since a day. on physical examination, he had respiratory distress and was toxic in appearance and his vital signs were monitored immediately. his temperature was 101.8f with a pulse rate of 106 beats per minute, blood pressure of 140/90 mmhg, and a respiratory rate of 25 breaths per minute. mouth opening was limited to 1.5 cm (interincisal distance) and revealed grossly decayed lower right second molar tooth with drainage of pus. extra - oral swelling was indurated, nonfluctuant with bilateral involvement of the submandibular and sublingual glands [figure 1 ]. an immediate diagnosis of ludwig 's angina was made, and the patient was posted for surgical decompression under general anesthesia. however, elective tracheostomy was planned for airway maintenance with the help of an otolaryngologist. elective tracheostomy was done under local anesthesia, airway secured and general anesthesia was provided. separate stab incisions was made in relation to the submandibular space bilaterally and submental space. a sinus forceps was introduced to open up the tissue spaces and pus was drained. the wound was irrigated with normal saline, and a separate tube drain was placed and secured to the skin with silk sutures [figure 2 ]. intravenous administration of cefotaxime 1 g bd, gentamycin 80 mg bd, metrogyl 500 mg, tid were given for 5 days with a tapering dose of decadran 84 mg bd for first two postoperative days. postoperative irrigation was done through the drain which was removed after 36 h along with the infected tooth. tracheostomy tube care was taken in the postoperative period, and the skin was strapped on the fifth postoperative day after the removal of the tracheostomy tube. preoperative appearance with bilateral involvement of the submandibular, sublingual, and the submental spaces showing brawny induration of the swelling postoperative view showing the tube drains and tracheostomy tube in place ludwig 's angina and deep neck infections are dangerous because of their normal tendency to cause edema, distortion, and obstruction of airway and may arise as a consequence of airway management mishaps. in the early stages of the disease, patients may be managed with observation and intravenous antibiotics. this is complicated by pain, trismus, airway edema, and tongue displacement creating a compromised airway. -hemolytic streptococcus associated with anaerobic germs such as peptostreptococcus and pigmented bacteroides have been described as causative agents. streptococcus viridans (40.9%), staphylococcus aureus (27.3%), and staphylococcus epidermis (22.7%) were isolated from deep neck infections. intravenous penicillin g, clindamycin or metronidazole are the antibiotics recommended for use prior to obtaining culture and antibiogram results. recent case reports advocated the use of intravenous steroids which potentially avoided the need for airway management. if patients present with swelling, pain, elevation of the tongue, malaise, fever, neck swelling, and dysphagia, the submandibular area can be indurated, sometimes with palpable crepitus. inability to swallow saliva and stridor raise concern because of imminent airway compromise. the most feared complication is airway obstruction due to elevation and posterior displacement of the tongue. to reduce the risk of spread of infection airway compromise is always synonymous with the term ludwig 's angina, and it is the leading cause of death. the stage of the disease and comorbid conditions at the time of presentation, physician experience, available resources, and personnel are all crucial factors in the decision making. blind nasotracheal intubation should not be attempted in patients with ludwig 's angina given the potential for bleeding and abscess rupture. flexible nasotracheal intubation requires skill and experience, if not feasible, cricothyrotomy and tracheostomy under local anesthesia are occasionally performed in the emergency department in those with advanced stages of the disease. elective awake tracheostomy is a safer and more logical method of airway management in patients with a fully developed ludwig 's angina. tracheostomy using local anesthesia has been considered the gold standard of airway management in patients with deep neck infections, but it may be difficult or impossible in advanced cases of infection because of the position needed for tracheostomy or because of anatomical distortion of the anterior neck.[1820 ] | ludwig 's angina is a form of severe diffuse cellulitis that presents an acute onset and spreads rapidly, bilaterally affecting the submandibular, sublingual and submental spaces resulting in a state of emergency. early diagnosis and immediate treatment planning could be a life - saving procedure. here we report a case of wide spread odontogenic infection extending to the neck with elevation of the floor of the mouth obstructing the airway which resulted in breathlessness and stridor for which the patient was directed to maintain his airway by elective tracheostomy and subsequent drainage of the potentially involved spaces. late stages of the disease should be addressed immediately and given special importance towards the maintenance of airway followed by surgical decompression under antibiotic coverage. the appropriate use of parenteral antibiotics, airway protection techniques, and formal surgical drainage of the infection remains the standard protocol of treatment in advanced cases of ludwig 's angina. |
objective : this methodological and descriptive investigation was performed to test validity and reliability of a turkish adaptation of an instrument to measure patient perception of quality of nursing care and related hospital services developed in sri lanka. time and place of the investigation : the investigation was performed in the department of surgery and department of internal medicine of istanbul university faculty of medicine hospital between november 2013 and november 2014. study population and sample : study population consisted of all adult patients hospitalized in the department of internal medicine and department of surgery of istanbul university faculty of medicine hospital between november 2013 and november 2014. sample consisted of 200 literate patients without any mental or psychological problems who were hospitalized for at least 3 days in medical or surgical units who volunteered to participate. twenty patients who did not complete questionnaire due to time constraints at discharge were excluded, and the study was completed with 180 patients. in evaluation of scale, rule stipulating that size of sample should be at least 5 times greater than the number of variables was strictly observed. data of the investigation were collected using patient information form and 36-item questionnaire to evaluate patient perception of the quality of nursing care and related hospital services. patient information form : form consisted of 12 questions related to the unit of hospitalization, gender, age, marital status, educational level, profession, place of residence (i.e., metropolitan city or rural area), employment status, income level, previous hospital admissions (if any), and patient description of their illness. patient perception of the quality of nursing care and related hospital services scale : the original scale was created at national hospital of sri lanka, the country s foremost training and research hospital, with patients being discharged after hospitalization for between 3 and 90 days in the medical and surgical units. all items are affirmative expressions that were rated with 5-point likert - type scale as follows : 1. i am not satisfied at all / i do nt agree at all, 2. factor 1 was related to interpersonal care, and concerned the way nurses personally interacted with the patient, asking about such things as communicating respect, courtesy, and concern. factor 2 was efficiency, and included items regarding competence of nurses and their actions to fulfill health needs without delay. factor 3 asked patients about comfort, and included items about quality of privacy and sleep. factor 4 was hygiene, and was related to adequacy and cleanliness of restrooms. factor 5 asked patients individual information about hospital facilities and illness, factor 6 queried them about perception of physical environment, including such items as climate control and cafeterias, and factor 7 was related to competency, and asked patients questions related to knowledge and skills of the nurses. application of data collection tools : data were collected by charge nurses in the clinics during face - to - face interviews. after discharge procedures were completed, a suitable environment was located, the study was explained, and questionnaire was given to patients who volunteered to participate. twenty patients who had time constraints at discharge and did not complete the forms were excluded from the study. evaluation of data : statistical analysis of data was performed with spss software, version 15.0 (ibm corp., armonk, ny, usa). two specialists whose native language is english translated the patient perception of the quality of nursing care and related hospital services scale into turkish. then, opinions of 10 turkish language specialists were requested regarding style of expression used in turkish to test language validity. in line with their views, some expressions used in the questionnaire were altered for better comprehension. next, a translator and interpreter who had perfect command of both languages back - translated the scale from turkish into english. the final version was sent to upul senarath, the corresponding author of the original study. construct validity was performed using explanatory factor analysis. in reliability study, for internal consistency of the scale and sub - dimensions, cronbach s alpha coefficient was used, and for item - total score analysis, pearson correlation analysis was performed. ethical aspect of the investigation : written permission was obtained from upul senarath via e - mail for the validity, and reliability studies of turkish version of patient perception of the quality of nursing care and related hospital services scale. approval for the study itself was obtained from the ethics committee of the istanbul university faculty of medicine, and the principles of the helsinki declaration of human rights were observed. data of the investigation were collected using patient information form and 36-item questionnaire to evaluate patient perception of the quality of nursing care and related hospital services. patient information form : form consisted of 12 questions related to the unit of hospitalization, gender, age, marital status, educational level, profession, place of residence (i.e., metropolitan city or rural area), employment status, income level, previous hospital admissions (if any), and patient description of their illness. patient perception of the quality of nursing care and related hospital services scale : the original scale was created at national hospital of sri lanka, the country s foremost training and research hospital, with patients being discharged after hospitalization for between 3 and 90 days in the medical and surgical units. all items are affirmative expressions that were rated with 5-point likert - type scale as follows : 1. i am not satisfied at all / i do nt agree at all, 2. i am not satisfied / i do nt agree, 3. factor 1 was related to interpersonal care, and concerned the way nurses personally interacted with the patient, asking about such things as communicating respect, courtesy, and concern. factor 2 was efficiency, and included items regarding competence of nurses and their actions to fulfill health needs without delay. factor 3 asked patients about comfort, and included items about quality of privacy and sleep. factor 4 was hygiene, and was related to adequacy and cleanliness of restrooms. factor 5 asked patients individual information about hospital facilities and illness, factor 6 queried them about perception of physical environment, including such items as climate control and cafeterias, and factor 7 was related to competency, and asked patients questions related to knowledge and skills of the nurses. application of data collection tools : data were collected by charge nurses in the clinics during face - to - face interviews. after discharge procedures were completed, a suitable environment was located, the study was explained, and questionnaire was given to patients who volunteered to participate. twenty patients who had time constraints at discharge and did not complete the forms were excluded from the study. evaluation of data : statistical analysis of data was performed with spss software, version 15.0 (ibm corp., two specialists whose native language is english translated the patient perception of the quality of nursing care and related hospital services scale into turkish. then, opinions of 10 turkish language specialists were requested regarding style of expression used in turkish to test language validity. in line with their views next, a translator and interpreter who had perfect command of both languages back - translated the scale from turkish into english. the final version was sent to upul senarath, the corresponding author of the original study. construct validity was performed using explanatory factor analysis. in reliability study, for internal consistency of the scale and sub - dimensions, cronbach s alpha coefficient was used, and for item - total score analysis, pearson correlation analysis was performed. ethical aspect of the investigation : written permission was obtained from upul senarath via e - mail for the validity, and reliability studies of turkish version of patient perception of the quality of nursing care and related hospital services scale. approval for the study itself was obtained from the ethics committee of the istanbul university faculty of medicine, and the principles of the helsinki declaration of human rights were observed. more than half of participants were male (61.3%) and married (77.7%). more than half (69.4%) were not working at the time, and 54.4% of study participants lived in metropolitan city. majority (63.3%) of the patients had been hospitalized before, and had mean hospital stay of 16.70524 days. the final version of the scale was administered to 78 medical and 192 surgical treatment patients. based on results retrieved, high level of positive correlation was found between turkish version and original english - language scale (r=.90 ; p<.001). once equivalency of turkish and english versions of the scale was established, validity and reliability studies were conducted. to assess construct validity, suitability of data for factor analysis was evaluated using kaiser - meyer - olkin and bartlett tests, and suitability of data for factor analysis was confirmed (table 1). principal component analysis : as a result of explanatory factor analysis using varimax rotation with kaiser normalization, 4 factors with eigenvalue over 1 were detected, which explained 82.40% of total variance. therefore, 8 factors in the original scale were consolidated into 4 factors : factor 1 contained items related to nursing care and its applications ; factor 2 was related to efficiency, competency, personal information, and quality of general instructions ; factor 3 was concerned with number, quality, and hygiene of restrooms ; and factor 4 was related to quality and hygiene of beds and bed coverings. cronbach s alpha internal consistency coefficient and item - total score correlation were used to determine reliability level of the scale. item - total correlation of each of 36 items was examined using pearson correlation analysis. correlation reliability coefficient was between r=0.57 and r=0.86, which indicated a strong, positive relationship that was statistically significant (p<0.001 ; table 2). further analysis of the items revealed that cronbach s alpha reliability coefficient for each factor was determined to be 0.98, 0.97, 0.92, and 0.84, respectively. while total cronbach s alpha reliability coefficient of the current scale was calculated to be 0.98. factor composition of the turkish version of the scale, item - total correlation, and cronbach s alpha values (n=180) extraction method : principal component analysis ; rotation method : varimax with kaiser normalization. the final version of the scale was administered to 78 medical and 192 surgical treatment patients. based on results retrieved, high level of positive correlation was found between turkish version and original english - language scale (r=.90 ; p<.001). once equivalency of turkish and english versions of the scale was established, validity and reliability studies were conducted. to assess construct validity, suitability of data for factor analysis was evaluated using kaiser - meyer - olkin and bartlett tests, and suitability of data for factor analysis was confirmed (table 1). principal component analysis : as a result of explanatory factor analysis using varimax rotation with kaiser normalization, 4 factors with eigenvalue over 1 were detected, which explained 82.40% of total variance. therefore, 8 factors in the original scale were consolidated into 4 factors : factor 1 contained items related to nursing care and its applications ; factor 2 was related to efficiency, competency, personal information, and quality of general instructions ; factor 3 was concerned with number, quality, and hygiene of restrooms ; and factor 4 was related to quality and hygiene of beds and bed coverings. cronbach s alpha internal consistency coefficient and item - total score correlation were used to determine reliability level of the scale. item - total correlation of each of 36 items was examined using pearson correlation analysis. correlation reliability coefficient was between r=0.57 and r=0.86, which indicated a strong, positive relationship that was statistically significant (p<0.001 ; table 2). further analysis of the items revealed that cronbach s alpha reliability coefficient for each factor was determined to be 0.98, 0.97, 0.92, and 0.84, respectively. while total cronbach s alpha reliability coefficient of the current scale was calculated to be 0.98. factor composition of the turkish version of the scale, item - total correlation, and cronbach s alpha values (n=180) extraction method : principal component analysis ; rotation method : varimax with kaiser normalization. during the process of adapting the scale, validity and reliability studies were performed to analyze psychometric characteristics. validity is defined as accurate measurement of required characteristics with the aid of measurement tools developed without interference from other characteristic features. reliability is the capability of a test or any measurement tool to yield sensitive, compatible, consistent, and stable results. analysis of correlation between scores of english and turkish versions of the relevant scale revealed high level of consistency (r=.90 ; p<.001). this result is significant in that it shows effective, high quality translation of the scale into turkish. the objective of content / scope validation is to request an expert group to determine if items contained in the assessment tool fully represent the domain to be measured in order to form an integrity. result of cvi test performed to evaluate content validity of the scale found no significant difference among expert opinions. it was concluded that the expressions used were compatible with turkish culture and sufficiently represented all facets of the construct. when using likert - type scale, reliability coefficient should be as close to 1 as possible. in the literature, item - total item correlation scores above 0.25, and cronbach s alpha reliability values greater than 0.5 have been specified as expected limits for internal consistency of scales [19, 20 ]. as a result of explanatory factor analysis, 4 factors had eigenvalues above 1, which explained 82.40% of the variance, and factor loading of 36 items listed under these factors was above. detection of high internal consistency coefficient indicates adequate level of agreement between items used on the scale. if level of reliability for measurement tools to be used in investigations is. 70, then reliability level of all sub - dimensions of the scale can be deemed to be adequate. in the interpretation of item - total correlation, if we consider that items with correlation coefficient of.30 identify individuals much better on the characteristic feature measured, then item - total correlations appear to be adequate. results obtained from validity and reliability tests demonstrated that turkish version of the scale developed in this study is a valid and reliable measurement tool. in conclusion, scale with validity and reliability in terms of content pertaining to nursing and related hospital services provided in turkey was developed. as it was designed for use with turkish population, it may be more applicable in turkey than other available measurement tools. in further studies to be performed, comparison of this scale with similar scales may be analyzed. additional studies should also include patients from other types of hospitals to expand validity and reliability of the tool and add to its utility. | objective : this study aimed to test the validity and reliability of a version of the tool developed in sri lanka in 2011 to assess patient perceptions of the quality of nursing care and related hospital services created for use with turkish patients.methods:this methodological study was conducted between november 2013 and november 2014 after obtaining ethical approval and organizational permission. data was collected during discharge from 180 adult patients who were hospitalized for at least 3 days at a medical school hospital located in istanbul. after language validation, validity and reliability analyses of the scale were conducted. content validity, content validity index (cvi), construct validity, and exploratory factor analysis were assessed and examined, and reliability was tested using the cronbach s alpha coefficient and item - total correlations.results:mean cvi was found to be 0.95, which is above expected value. exploratory factor analysis revealed 4 factors with eigenvalues above 1, which explained 82.4% of total variance in the turkish version of the tool to measure patient perceptions of nursing care and other hospital services. factor loading for each item was.40. cronbach s alpha coefficient of sub - dimensions and total scale were found to be 0.84 - 0.98 and 0.98, respectively. item - total correlations ranged from 0.56 to 0.83 for the entire group, which was above expected values.conclusion:the turkish version of the scale to assess patient perceptions of the quality of nursing care and related hospital services, which comprised 4 sub - dimensions and 36 items, was found to be valid and reliable for use with the turkish population. |
the oral submucous fibrosis (osmf) as defined by pindborg and sirsat as an insidious chronic fibrotic disease that involves the oral mucosa and occasionally the pharynx and upper third of oesophagus. osmf is characterized by a juxtraepithelial inflammatory reaction followed by fibroelastic changes in the submucosa and epithelial atrophy, that leads to stiffness of the oral mucosa causing trismus and inability to eat.1 the etiological factors are excessive consumption of spicy food, nutritional deficiencies like chronic iron and vitamin b complex deficiency, areca nut chewing habits.2 the symptoms and subjective signs observed are burning sensation exacerbated by spicy or acidic foods, pain often referred to temporal region, increased or decreased salivation, reduced mouth opening, difficulty with mastication, difficulty with phonation and deglutition, vesiculation or ulceration of oral mucosa3 (figure 1). hyaluronidase by breaking down hyaluronic acid (the ground substance in connective tissue) lowers the viscosity of intercellular cement substance. better results were observed with respect to trismus and fibrosis.4 acts as an immune suppressive agent by its antagonistic activity on the soluble factors released by the sensitized lymphocytes succeeding the activation by nonspecific antigens.5 it additionally muzzles the inflammatory reaction. thus, fibrosis is prevented by a decrease in fibroblastic proliferation and deposition of collagen. hyaluronidase by breaking down hyaluronic acid (the ground substance in connective tissue) lowers the viscosity of intercellular cement substance. acts as an immune suppressive agent by its antagonistic activity on the soluble factors released by the sensitized lymphocytes succeeding the activation by nonspecific antigens.5 it additionally muzzles the inflammatory reaction. thus, fibrosis is prevented by a decrease in fibroblastic proliferation and deposition of collagen. the study was conducted on 28 patients with osmf who attended as outpatients in the department of oral medicine and radiology and department of oral and maxillofacial surgery, with grade iii osmf in the institute of sri rajiv gandhi college of dental sciences, bengaluru for a span of 9 months. this study was carried out after obtaining the patient s consent and with the approval of the institution s research ethical committee. clinical diagnosis of osmf was based on symptoms of burning sensation in the mouth on consumption of spicy or hot food, dryness of mouth, presence of vesicles oral ulcers in the mouth, and restriction of mouth opening were observed. patients who were medically compromised and those who received previous treatments were not included in the study. of 28 patients, the number of male subjects were 18 and female subjects were 10. the patients were informed about the condition and its precancerous potential and were instructed to discontinue the use of are canut with tobacco. their history of personal habits with regard to frequency of chews, duration of use and symptoms like burning sensation and mouth opening were recorded. the burning sensation was assessed using visual analogue scale marked from 0 to 10 where 0 indicates no burning sensation and 10 indicates maximum burning sensation. extraorally, the patient s mouth opening was measured with reference to interincisal points between upper and lower incisor teeth, the maximum mouth opening was assessed with geometric divider and metallic scale. intraorally, the findings like blanching of oral mucosa, presence of vesicles and ulcers, palpable bands, limitation of tongue movement were observed. the patients were grouped based on their age : 21 - 30 (group i), 31 - 40 (group ii), 41 - 50 (group iii) and 51 - 60 (group iv). 12 patients belonged to age group 21 - 30 where 8 were males and 4 were females, 7 patients 31 - 40 where 4 were males and 3 were females, 6 patients 41 - 50 where 4 were males and 2 were females and 3 patients 51 - 60 group where 2 were males and 1 was female. the patients were administered hyaluronidase 1500 iu mixed in 1.5 ml of dexamethasone and 0.5 ml of lignocaine hcl injected intralesionally biweekly for 4 weeks. outcome assessment was done by measuring postoperative mouth opening each week and burning sensation using a visual analogue scale, (graph 1a and b). improvement in the patient s mouth opening with a net gain of 6 2 mm (92%), the range being 4 - 8 mm. definite reduction in burning sensation, painful ulceration and blanching of oral mucosa and patient followed up for an average of 9 months (table 1). it was observed that in group i prior to treatment, the mouth opening was limited to19%, following the treatment the mouth opening was 30.5% ; the improvement observed was by 11.5%. in group ii, prior to treatment, the mouth opening was limited to16.5, following the treatment the mouth opening was 27.2%, the improvement observed was by 10.7%. in group iii, prior to treatment, the mouth opening was 17.4%, following the treatment, the mouth opening was 29.3%, improvement was by 11.9%. in group iv, prior to treatment the mouth opening was 16.33 following the treatment the mouth opening was 28.7, the improvement observed was by 12.4% (table 2). percentage of relief of symptoms post - treatment. as proposed by kakkar and puri, for the purpose of treatment, the patients can be graded on the basis of the clinical condition.6 grade i : only blanching of oral mucosa without symptoms grade ii : burning sensation, dryness of mouth, vesicles or ulcer in the mouth without tongue involvement grade iii : in addition of grade ii, restriction of mouth opening grade iv : in addition to grade iii palpable bands all over the mouth without tongue involvement grade v : grade iv and also tongue involvement grade vi : osmf along with histopathlogically proven cancer. injection hyaluronidase 1500 iu, 0.5 ml injected intralesionally twice a week for 10 weeks. osmf is a precancerous condition and reports suggest that it is present since the time of sushruta8 reported by schwartz in 1962 and by joshi in 1953 ; who described its singleton among the indians. many trials have been conducted but as such no definitive treatment is currently available.9 however, improvement can be obtained passably by intralesional injection of cortisone and hyaluronidase.10 it was observed that patients receiving hyaluronidase alone showed a quicker improvement in the burning sensation and painful ulceration produced by the effects of local by - products, although combination of dexamethasone and hyaluronidase gave better long - term results than other regimens.11 however, the addition of dexamethasone has its own advantages and contraindications and a slight improvement in the overall result observed in the combination group justifies the addition of dexamethasone to hyaluronidase. with the exception of the individual s habit, the systemic conditions like chronic iron deficiency and vitamin b complex deficiency subsists.12 study by borle and borle postulated that treatment following intralesional injections of various drugs leads to aggravated fibrosis and pronounced trismus.13 the resultant worsening of this condition with submucosal injections are attributable to repeated needle stick injury14 to the soft tissues at multiple sites, clinical irritation from drugs being injected, and to the progressive nature of the disease. the same outcome has been observed with some surgical methods employed to treat osmf. conservative line of treatment like topical steroids, vitamins, antioxidants, physiotherapy would give expected symptomatic relief of pain and burning sensation.15 treatment modalities like intralesional injections of placental extracts that acts essentially by biogenic stimulation based on tissue therapy are also encouraged.16 clinical trial by haque. using gamma - interferon treatment has shown improvement in the patients mouth opening17 (inter incisal distance) with net gain of 8 4 mm (42%), the range being 4 - 15 mm. excision of fibrous bands is also managed by co2 and ktp laser,18 a potassium - titanyl - phosphate that doubles the frequency of pulsed neodymium : yttrium - aluminium garnet laser energy to 532 nanometer wavelength.19,20 injection of hyaluronidase with dexamethasone is an effective method of managing grade iii osmf and can possibly eliminate the morbidity associated with surgical management. this study is an added effort in providing evidence - based support to optimize patient care. the subjects falling prey to osmf can be reduced by educating the upcoming generation in schools and colleges. | background : oral submucous fibrosis (osmf) is a chronic debilitating and potentially malignant condition of the oral cavity. it is resistant and progressive affecting the entire oral cavity that sometimes causes a gradual reduction in mouth opening that may even extend up to the pharynx. although the medical treatment is not completely systematized, optimal doses of its treatment with local injection of corticosteroids with hyaluronidase or placental extract is effective to some extent. however, a combination of steroids and topical hyaluronidase shows better long - term results than either agents used individually. to evaluate the efficacy of dexamethasone and hyaluronidase in the treatment of grade iii osmf.materials and methods : a total of 28 patients diagnosed with osmf were treated in sri rajiv gandhi college of dental sciences for a time period of 9 months, by obtaining the patient s consent and with the approval of the institution s research ethical committee. they were treated by administering an intralesional injection of dexamethasone1.5 ml, hyaluronidase 1500 iu with 0.5 ml lignocaine hcl injected intralesionally biweekly for 4 weeks.results:improvement in the patient s mouth opening with a net gain of 6 2 mm (92%), the range being 4 - 8 mm. definite reduction in burning sensation, painful ulceration and blanching of oral mucosa and patient followed up for an average of 9 months.conclusion:injection of hyaluronidase with dexamethasone is an effective method of managing grade iii osmf and can possibly eliminate the morbidity associated with surgical management. |
cone beam computed tomography (cbct) is currently a valuable imaging modality with numerous applications in dental implant treatment, endodontic therapy, surgery, orthodontic assessment and evaluation of pathological lesions. the effects of factors such as system resolution and voxel size are undeniable on diagnostic accuracy of images, and there is the possibility of using a smaller voxel size to increase image resolution. however, use of a smaller voxel size to increase the resolution can create noise, and thus, significant improvement in the diagnostic value of images may not occur. also, the scanning time of objects becomes longer by increasing the resolution, the radiation dose increases and there would be higher risk of movement of patient during imaging. field of view (fov) refers to the scan volume of a particular cbct unit. a voxel describes the smallest distinguishable box - shaped part of a three - dimensional image. lowering the resolution may reduce the quality of images, increase noise and artifacts, and reduce the amount of anatomical information of the target areas. multiple factors such as fov, voxel size and the number of basis projections and image artifacts significantly affect the cbct image quality. in most cases, previous studies have reported inconsistencies in cbct measurements depending on the position of object within the fov. the accuracy of linear measurements is lower at the periphery compared to the center of the fov in cbct. because of the lack of any current protocol on selection of appropriate voxel size and also the proper location within the fov for cbct imaging in dentistry, the present study was conducted to assess the effects of voxel size (resolution) and the location of bone defects in different regions of the fov on diagnostic accuracy of cbct for their detection. four pieces of human mandible were selected after soft tissue removal and fixed in formalin. bone defects with 11 mm dimensions bone blocks were then fixed on a platform, which was parallel to the horizontal plane of the cbct unit ; cbct images of bone defects were obtained using alphard-3030 unit (asahi roentgen ind. kyoto, japan) operating at 4ma, 80kvp and 17s time in two different resolutions and five locations in the fov (fig. bone defects were not created in the mandible of the negative (healthy bone) cases (a total of eight cases) and thus cbct images were obtained only from healthy bone under the same experimental conditions (i.e. five locations in the fov at both high and low resolutions). locations in the fov were center, left, right, anterior and posterior, and resolutions used for cbct imaging included high resolution (mode i : 0.2 mm voxel size, 1010 cm fov) and low resolution (mode p : 0.3 mm voxel size, 1515 cm fov) (figs. 2 and 3). three observers evaluated the images using the same display monitor and romexis software (planmeca, helsinki, finland), and then information was recorded in the relevant forms. the bone blocks were scanned at five different areas within the field of view of the cbct system a : anterior ; c : center ; l : left ; r : right ; p : posterior cbct scan obtained with 0.2 mm voxel size of a mandibular bone defect ; (a) center of the field of view ; (b) posterior region of the field of view cbct scan obtained with 0.3 mm voxel size of a mandibular bone defect ; (a) center of the field of view, (b) posterior region of the field of view in the first session, the observers were trained to record the information in the relevant forms. then, each sample was coded and the images were randomly presented to the observers. the ability to detect bone defects was assessed using a dichotomous scale (seen and not seen). the images were coded again and randomly shown to the observers ; the related information was recorded. detection of bone defects (seen and not seen) was determined in positive cases (bone defects) and negative cases (healthy bone) based on the opinion of the observers ; the frequency of observations, high and low resolutions, and the location of object in the fov, and reported using number and percentage. intra- and inter - observer agreements for the detection of bone defects were calculated at different resolutions and locations in the fov using the kappa test. the sensitivity, specificity and 95% confidence interval values are presented in tables 1 to 3. the highest sensitivity was found in the central region in both high and low resolutions, which was significantly different from other fov regions at 95% confidence level using the wilson formula. also, the least sensitivity was recorded in the posterior region of the fov at low resolution and significantly increased by increasing the resolution (table 1). resolution of images (0.3 mm and 0.2 mm) and fov regions had insignificant effects on the specificity values ; because it was observed that the values were almost the same in all three observers in two resolutions and five different regions (table 2). the value of inter - observer agreement in different fov regions was 0.3 to 0.69 in high - resolution images and 0.46 to 0.69 in low - resolution images, and the lowest value was obtained in the posterior region in high resolution (0.3). the highest value was obtained at the center of the fov in both resolutions (0.69). the intra - observer agreement coefficients for all groups were adequately high (kappa0.75). sensitivity for detection of simulated bone defects at different object locations in the field of view (fov) and two different resolutions of cbct specificity for detection of simulated bone defects at different object locations in the field of view (fov) and two different resolutions of cbct system mean (95% confidence interval) of simulated bone defects measured at different object locations in the field of view (fov) and two different resolutions of the cbct system according to the results of this study, some variations were observed in the diagnostic accuracy of cbct for bone defect detection in positive and negative cases during scanning in different regions of the fov. although the sensitivity significantly decreased in cbct images with the object in the posterior region of the fov in low resolution, it increased in high resolution, especially in the posterior region. high sensitivity was observed by increasing the resolution of cbct images from 0.3 mm to 0.2 mm ; and the values were significantly higher at the center of the fov compared to other regions in both high and low resolutions at 95% confidence interval using the wilson formula. in a study by ibrahim, in 2014 on the effects of different locations of object in the fov in two cbct systems, a significant difference in trabecular bone microstructure measurements in the central region was found relative to the peripheral areas in the fov in newtom system, but this difference was not significant in the accuitomo system, which may be due to differences between the two systems. the inherent artefacts of the system and the object region in a limited fov could also result in deviations of measurements. these factors increase the linear measurement values at the periphery compared to the central region of the fov of the cbct system. in the current study, defects were observed best at the center of the fov and had statistically significant difference with object in other regions. the non - uniformity of the beam intensity causes inconsistency in grey values within the cbct fov, in which it is relatively higher at the center than at the peripheral regions. a higher quality image can be obtained with the object at the center. in the current study, the lowest sensitivity was observed in the posterior region of the fov in low resolution, which could be due to under - sampling from the posterior region of the fov compared to other regions because of of device limitations. variations in diagnostic parameters with the object in different regions of the fov have also been observed in high - resolution peripheral ct and multislice ct. voxel size, contrast - to - noise ratio and image artifacts are among the factors associated with the fov. a small voxel size with a small fov is generally recommended to improve diagnostic accuracy. variations in the quality of images of bone defects in different fovs and resolutions may be attributed to increased image artefacts particularly in smaller fovs. also, an image with a bigger voxel and a higher contrast - to - noise ratio can have a higher resolution than an image with a smaller voxel and lower contrast - to - noise ratio. therefore, while the selection of voxel size depends on the nature of the diagnostic task, the clinician should be aware of the variations of resolution and selected fov regions. although cbct has been reported as a precise technique for assessment and detection of bone defects, some limitations of the systems should be well acknowledged. based on the results, the observer s performance with regard to cbct images taken at high resolution was estimated to be better than that with images with low resolution ; thus, the sensitivity values improved by increasing the resolution of cbct images from 0.3 mm to 0.2 mm ; and the frequency of correct diagnoses increased in both positive and negative cases. by rotation of x - ray beam around however, as patients vary in size, the fov parameter would be different as well. in addition, the selected fov remains the most important scanning parameter in limiting the radiation dose and image quality. theoretically, more uniform images can be obtained in the maxillofacial region due to high - density structures. substantial amount of scattered beams is produced in cbct technique that negatively affects the image quality. selected smaller fovs, anatomical structures located outside the defined area are imaged by the detector, and since only a small imaging range evaluates the tissue, the resulting images will not be uniform. kwong, in 2008 used 6-inch fov and showed that the resultant images had the highest resolution and the lowest voxel size ; although high resolution alone does not necessarily mean higher image quality. different fovs contain various voxel sizes and cbct devices also vary in voxel size for each fov. hassan, in 2010 showed that voxels larger than 0.6, 0.9, or 1.2 mm in the x and y plane significantly reduced the visibility of occlusal surfaces of the teeth, interproximal spaces between the teeth and alveolar bone ; whereas, the choice of larger voxels reduces the image noise. factors such as voxel size in imaging, exposure factors, various applied fovs, slice thickness, imaging systems and detectors are effective in diagnostic results. these parameters vary between the cbct units and different imaging protocols in the same unit. although short scanning time and low patient radiation dose are important factors in diagnostic imaging, risk of misdiagnosis and consequent complications should also be taken into account. in general, voxel size should be determined based on the severity of patient s condition and treatment plan. image quality variations can be reduced or increased by altering the fov, and the image reconstruction parameters and post - processing methods are studied based on the type of cbct system used. the selected fov for cbct images is directly related to voxel size and affects the resolution and contrast. extended fov creates lower contrast and resolution compared to smaller fov and thus directly affects the detection of anatomical structures on cbct images. in accordance with the results of the current study, fov position seems to have little impact on diagnostic specificity because the values were more or less the same in all three observers at five different positions. appropriate diagnostic accuracy of cbct images for bone defects in the fov and different resolutions in this study may be related to adequate ability and skills of the observers, good quality of cbct scans and lack of motion artifacts in images. in this study, bone defects caused by drilling had distinctive and appropriate form, enhancing their detection by the observers. also, kamburoglu, in 2014 reported the same performance of observers in detection of simulated buccal peri - implant defects using different parameters of fov and various resolutions. in another study, no difference was observed in diagnostic accuracy of cbct images for detection of simulated buccal peri - implant defects with and without pattern artifact reduction in planmeca promax system. pattern artifact reduction was not used in this study or in the study by kamburoglu, in 2014. according to the results of the current study, the sensitivity for detecting bone defects by the observers and the frequency of detection was in the range of 0.251 in low resolution and 0.8751 in high resolution at different fov regions, showing that sensitivity was enhanced by increasing the resolution. on the other hand, the specificity value was between 0.75 and 1 for high - resolution images and 0.691 for low - resolution images. in the study by hedesiu, in 2012, sensitivity for diagnosis of apical defects in cbct scans was estimated to be 0.720.8 and specificity was between 0.60.77. however, higher levels (a rate of 1) of sensitivity and specificity for cbct system were recorded in examining the human mandible by patel, in 2009. the sensitivity parameter for cbct images in the study by patel, in 2014 was in the range of 79.2%91.7%. significant effects of resolution on sensitivity were not observed in the current study, which is different from the results of the above - mentioned studies. in total, the highest sensitivity was recorded in the central position in both high and low resolutions ; increased resolution generally led to improved sensitivity especially in the posterior region of the fov, but had no effect on specificity. the fov parameter in cbct should be adjusted to reduce patient radiation exposure for detection of bone defects. concerning some false - positive results in the negative cases, cbct should be used as an adjunct to clinical examination to make a correct diagnosis and for treatment planning. if the diagnostic purpose can be achieved by minimum radiation dose in the fov, there is no need to change this parameter or increase the absorbed dose of patient. improving the cbct image resolution from 0.3 mm to 0.2 mm increased the sensitivity values for detection of simulated bone defects. image resolution and position of the object in the fov seem to have little impact on specificity values. also, comparison of different fov regions revealed that the highest sensitivity rates were recorded in the central position in both high and low resolutions, and sensitivity significantly decreased in the posterior position at low resolution | objectives : this study aimed to assess the effect of voxel size and object location in the field of view (fov) on diagnostic accuracy of cone beam computed tomography (cbct) for detection of simulated bone defects.materials and methods : in this in vitro study, bone defects were drilled in four sections of a dry human mandible. bone blocks were fixed on a platform parallel to the horizontal plane and cbct images were acquired using 0.2 mm and 0.3 mm resolutions and five locations of fov (anterior, posterior, left, right and center). three reviewers viewed the images twice and the presence or absence of simulated bone defects was determined in positive and negative cases.results:sensitivity in different locations of fov ranged between 0.251.0 and 0.751.0 in low and high resolutions, respectively. these values were 0.6251.0 and 0.691.0, respectively for specificity. intra - observer agreements were in the range of 0.841.0 and 0.751.0 and inter - observer agreements were in the range of 0.30.61 and 0.460.69 in high and low resolutions, respectively. the highest sensitivity was seen at the center of the fov and with an increase in resolution from 0.3 mm to 0.2 mm, the sensitivity increased specially in the posterior region of the fov while image resolutions and fov locations did not affect specificity.conclusions:the highest sensitivity values were obtained at the center of the fov and lowest values were seen in images acquired in the posterior region in low resolution. diagnostic accuracy improved with increased resolution. |
although longevity in the uk is increasing, average increases mask important differences within the population [13 ]. furthermore as the uk population as a whole grows older, the demographics within it are changing (see figure 1). currently most ethnic minorities have younger populations than the majority white british population. however by 2051, the ethnic groups with the highest proportions of people, aged 50 and over will be other white, chinese, other asian, indian, other, and white irish alongside white british. in the non - white ethnic group alone, there will be 2.7 million people aged 65 and over and 1.9 million people aged 70 and over. whilst ethnic minorities already make up around half the local population in some parts of the country, by 2056 they will make up 43 percent of the total national population. together these changes highlight the need to focus attention to commissioning health services for an increasingly multiethnic older population. the purpose of this research was to review the evidence to guide a programme of applied research to address the key areas and processes for reducing inequalities in diabetes care for older people from ethnic minority groups. we focussed in this instance on south asians in the uk as this group has an established history in the uk (making up fifty percent or more of the population in some uk locations) and so would be likely to feature in the relevant research literature. we define ethnicity as a consciousness of belonging to a particular group based on commonality of family origin and culture of shared values and beliefs which is socially constructed and loosely related to country of birth, ancestral country of birth, language spoken at home, nature of geographical origin, racial group, and religion. the broad south asian ethnic group descriptor used in this review (unless otherwise stated) refers to the majority south asian populations in the uk : indian punjabi, indian gujarati, bengali, pakistani, and sri lankan. the term older people is used variously according to context and different age - related dimensions : chronological, biological, functional, psychological, and social. in western societies, it broadly aligns with age of retirement 60 or 65 + [7, 8 ] but this is a socially constructed time point which does not take into account other factors relevant to diabetes care and ethnicity such as the onset of complications ; and we account for this in our strategy for searching the literature. this study 's principal interest was diabetes care and prevention in relation to inequality, ethnicity, and the older population rather than the aetiology of diabetes per se, although this was necessarily touched on where it related to clinical practice and management of older people who are living with diabetes. diabetes care in the uk is a context specific and complex activity because it takes place across nhs settings through consultations in primary and secondary care and in people 's home through self - management and care support. the starting point was that our previous research in related areas had found inadequate care of older people with diabetes particularly those being cared for in residential settings ; that providing equitable care through the diabetes care pathway was a challenge for care providers ; and that people with a south asian background and diabetes can be doubly disadvantaged by having increased risk of developing diabetes compared to people with a white european background in the uk and additional access barriers. these separate but related findings suggested to us that it was important to look at the evidence as a whole and to understand the processes which could help inform action on inequalities. although familiar with some of the concepts associated with reducing inequalities in access to diabetes care, such as cultural competency and concordance, we had not considered these specifically in relation to older people and the increasingly diverse and ageing uk population before. this was the first review, as far as we were aware, to do so and it was by nature and design exploratory. we used a realist review methodology to help us search the literature and to start to build a theoretical base for our research programme. the review had two parts : part 1, a mapping phase where we thematically synthesised the relevant studies into the main areas of research evidence ; and part 2, a theory building phase where we hypothesised, by abstracting from the evidence, a theoretical framework for moving forward from this base. as the work was early stage the emergent theory raised further questions which will help test and refine the theory in the future. as it stands however the review highlights a number of issues for policy makers, providers, and researchers concerned with reducing and preventing inequalities in diabetes care and these are summarised at the end of this paper. we reviewed literature at the intersection of three areas : quality diabetes care, older people, and ethnicity (see figure 2). the review was conducted by a multidisciplinary team comprising researchers with interest and expertise in public health, diabetes, gerontology, and diversity research. following an initial exploratory phase we decided to apply a realist approach to review the literature. this methodology was considered the most appropriate because it accommodated the broad research question ; was compatible with the complex and context related nature of diabetes care ; was sympathetic to the usage of a multimethod, multidisciplinary evidence base ; and would facilitate the exposition of theory through emerging and generalisable mechanisms. this could inform our programme of research but also be useful to policy makers and practitioners working with other ethnic minority groups in the uk. we conducted an initial scoping phase in which we hand - searched for research publications and tested different search strategies with available electronic databases. we made a number of decisions about the search strategy which are listed as follows : the search would be limited to the previous 30-year timeframe and to studies from the uk. the period 1985 to 2015 spanned several changes of uk governing parties and associated health policies some of which addressed health inequalities in relation to diabetes care, the impact of which would be captured in literature published during this time.the search would focus specifically on the uk population. ethnic minorities and health systems are different in different countries and diabetes care is context related. literature on ethnicity, access, and cultural competency from other countries such as the us, canada, and australia was drawn on where appropriate in the analysis and discussion of mechanisms.the search would focus on the uks ' south asian population rather than other or all ethnic minorities living in the uk. previous research by members of the team provided insight into some of the inequalities that people with a south asian background experience. as these related to this particular population group 's migration and settlement in the uk they are likely we anticipated that some of the concepts and mechanisms emerging from this review would be applicable to other minority communities.the search would use common age descriptors for older people as well as specific age categories from 55 years upwards. in the context of diabetes and care for minority groups age as a descriptor could be relative and variable depending on the population and phase of care (i.e., prevention, treatment, and palliation).the search would use descriptors for diabetes that included the key complications : diabetic neuropathy, retinopathy, and nephropathy. terms for the latter would be expanded as studies of diabetic nephropathy and end stage kidney disease would be likely to include the older south asian population because of the links between ethnicity, diabetes, chronic kidney disease, and longevity [11, 15].the search would be inclusive of research using qualitative and quantitative methods as well as grey literature in line with the realist methodology to prioritise relevance and contribution to theory building. the quality standards applied in assessing potential publications were based on those appropriate for the type of publication, intervention, method, and design described [16, 17 ]. the search would be limited to the previous 30-year timeframe and to studies from the uk. the period 1985 to 2015 spanned several changes of uk governing parties and associated health policies some of which addressed health inequalities in relation to diabetes care, the impact of which would be captured in literature published during this time. ethnic minorities and health systems are different in different countries and diabetes care is context related. literature on ethnicity, access, and cultural competency from other countries such as the us, canada, and australia was drawn on where appropriate in the analysis and discussion of mechanisms. the search would focus on the uks ' south asian population rather than other or all ethnic minorities living in the uk. previous research by members of the team provided insight into some of the inequalities that people with a south asian background experience. as these related to this particular population group 's migration and settlement in the uk they are likely be reflected in uk evidence from this timeframe. despite a focus on one group we anticipated that some of the concepts and mechanisms emerging from this review would be applicable to other minority communities. the search would use common age descriptors for older people as well as specific age categories from 55 years upwards. in the context of diabetes and care for minority groups age as a descriptor could be relative and variable depending on the population and phase of care (i.e., prevention, treatment, and palliation). the search would use descriptors for diabetes that included the key complications : diabetic neuropathy, retinopathy, and nephropathy. terms for the latter would be expanded as studies of diabetic nephropathy and end stage kidney disease would be likely to include the older south asian population because of the links between ethnicity, diabetes, chronic kidney disease, and longevity [11, 15 ]. the search would be inclusive of research using qualitative and quantitative methods as well as grey literature in line with the realist methodology to prioritise relevance and contribution to theory building. the quality standards applied in assessing potential publications were based on those appropriate for the type of publication, intervention, method, and design described [16, 17 ]. the following databases were searched : academic search elite, cinahl plus with full text, medline, medline with full text, psycarticles, psycinfo, socindex with full text, and global health. publication abstracts were searched using keyword criteria as follows : diabetes or diabetes mellitus or type 1 diabetes or t1 dm or type 2 diabetes or t2 dm or hyperglycem or hypoglycem or non insulin dependent diabetes mellitus or niddm or insulin or insulin resistance or glucose level or glucose regulation or haemoglobin a1c or hba1c or metabol or foot problems or amputation or lower extremity or lower limb or complications or nephropathy or retinopathy or kidney disease or chronic kidney disease or renal or renal impairment or kidney damage or albuminuria or proteinuria or microalbuminura or renal replacement therapy or ckd or esrd or esrf or eskd or eskf or rrt or end stage renal disease or end stage kidney disease or end stage kidney failure or end stage renal failure or dialysis or primary care and older people or older persons or elder or old age or ageing or aging or late life or frail or non frail or end of life or geriatrics or gerontology or post menopausal or over 55 years or over 60 years or over 65 years or over 70 years or over 75 years or over 80 years or over 85 years or over 90 years or over 95 years or over 100 years or end of life or functional disability or functional decline or mortality and ethn or race or culture or bame or bme or minorit or ethnic minority or asia or indoasia or south asia or indian or pakistani or bangladeshi or sri lankan or racial or black or culturally and linguistically diverse group or cald diabetes or diabetes mellitus or type 1 diabetes or t1 dm or type 2 diabetes or t2 dm or hyperglycem or hypoglycem or non insulin dependent diabetes mellitus or niddm or insulin or insulin resistance or glucose level or glucose regulation or haemoglobin a1c or hba1c or metabol or foot problems or amputation or lower extremity or lower limb or complications or nephropathy or retinopathy or kidney disease or chronic kidney disease or renal or renal impairment or kidney damage or albuminuria or proteinuria or microalbuminura or renal replacement therapy or ckd or esrd or esrf or eskd or eskf or rrt or end stage renal disease or end stage kidney disease or end stage kidney failure or end stage renal failure or dialysis or primary care and older people or older persons or elder or old age or ageing or aging or late life or frail or non frail or end of life or geriatrics or gerontology or post menopausal or over 55 years or over 60 years or over 65 years or over 70 years or over 75 years or over 80 years or over 85 years or over 90 years or over 95 years or over 100 years or end of life or functional disability or functional decline or mortality and ethn or race or culture or bame or bme or minorit or ethnic minority or asia or indoasia or south asia or indian or pakistani or bangladeshi or sri lankan or racial or black or culturally and linguistically diverse group or cald electronic search results were screened for duplication and relevance to the review area and question. copies of the full publication were obtained for included abstracts which were screened and those considered relevant included in the analysis. this process was conducted by ew and mw jointly, with assistance from an information specialist and with input and oversight from the other members of the author team. the research question what are the key mechanisms for reducing inequalities in diabetes care in the uk for older people with a south asian background ? was the basis of capturing learning from the published literature. in realist terms it was conceptualised as a complex intervention comprising government policy, applied research, and evidence based practice from the uk which addressed inequalities in diabetes outcomes and care for older people with diabetes from ethnic minorities and spanned the diabetes care system as a whole. we drew on the rameses guidance for reporting realist review to help make our review and its findings as clear as possible. the realist programme theory developed iteratively through the scoping, mapping, and theory building stages of the literature review and the findings are reported in two parts in section 3 : part 1 : the overview and mapping of literature relevant to the research question and search criteria. part 2 : the building of a theoretical framework for research in response to the question. for each mapped area we considered the context, mechanisms, and outcomes and considered how these related to common concepts also emerging from the literature that could be explanatory in terms of observed inequalities in diabetes care and interventions to reduce them (see table 1). part 1 : the overview and mapping of literature relevant to the research question and search criteria. part 2 : the building of a theoretical framework for research in response to the question. for each mapped area we considered the context, mechanisms, and outcomes and considered how these related to common concepts also emerging from the literature that could be explanatory in terms of observed inequalities in diabetes care and interventions to reduce them (see table 1). there were very few studies which specifically investigated diabetes, older people, and ethnicity, and even fewer (none) which specifically addressed diabetes care for older south asian people in the uk. although studies which included south asian people with diabetes often stated in their background that diabetes was a leading cause of mortality and morbidity and south asians were the largest ethnic minority in the uk, the majority of studies identified by our literature search concerned prevalence and incidence of diabetes, diabetes related complications, and associated conditions particularly cardiovascular disease. exceptions to this were the uk asian diabetes study (ukads) and the prevention of diabetes and obesity in south asians (podosa) which were intervention studies of enhanced diabetes care and prevention respectively, within the uk south asian population. we found however that the data and findings concerning age within included studies tended to be embedded within the results section of the publication, not detailed in the aims of the research nor discussed in more than a cursory way in relation to the timing of interventions in the population being studied. in south asians, the prevalence of type 2 diabetes is 4 times greater than that of white europeans. most of the research papers which focussed on ethnicity included it as a demographic descriptor and independent variable of the outcome or outcomes being examined. policy documents for diabetes, kidney care, and care of older people in contrast highlighted ethnicity as a key variable associated with inequality in access to quality care and in terms of interventions being culturally acceptable [2224 ]. recent guidelines for diabetes care for older people [25, 26 ] suggested that care should be individualised within an overarching theme of person centred diabetes care and that it should be tailored to individuals taking into consideration relevant factors. one such factor could be the person 's ethnicity, but this was not explicitly stated within the guidelines. where ethnicity was discussed in relation to inequalities in the research literature it was mainly to explain variations in outcomes or patterns of distribution within a given population and there was a dearth of studies which analysed inequalities as it related to diabetes care specifically for older people with a south asian ethnic background. there were very few studies about diabetes and diabetes care which explicitly included older south asian people as participants and a similar number of papers which discussed the lack of participation of older people and ethnic minorities in studies as a research issue [27, 28 ]. studies of diabetes which include an analysis by ethnicity invariably noted the earlier onset of diabetes in south asians compared to white europeans as an important factor in understanding both aetiology and disease progression as well as indicating a timeframe for intervention and prevention which is different to the majority population. south asians experience diabetes approximately 10 years before white europeans and show signs of more rapid progression of complications [29, 30 ]. research studies of diabetes complications in ethnicity minorities did not explicitly identify older people for inclusion, but because complications are related to time since diabetes diagnosis and age, they included a large proportion of older people within their study populations by default [31, 32 ]. together, key uk government guidelines, the quality outcomes framework and the national service frameworks for diabetes and kidney disease, have encouraged gps to consider ethnicity as a factor for earlier diagnosis and targeted care. these quality initiatives have gone some way to redress inequalities in diabetes care but there are concerns that, as they stand, they may perpetuate the existing status quo and not reduce inequalities further. several of the studies which detail south asian ethnicity describe the heterogeneity within the broad south asian descriptor for the uk 's diverse south asian population and some, depending on the data source, were able to break down their results across the main south asian groups (indian, pakistani, and bangladeshi) in the uk. ethnicity was linked to socioeconomic status in some studies including use of income level as a proxy for age as an alternative explanatory variable to capture some of the social and cultural associations with age. the complicating associations between diabetes and cardiovascular disease (cvd) were the subject of over half the studies identified through our electronic search. these were seeking to understand the aetiology of morbidity and mortality of cvd and included diabetes and ethnicity as established risk factors in the analysis. similarly, in relation to high blood pressure and atherogenic lipid profile, key risk factors for circulatory diseases, these have been found to have an association with south asian ethnicity both in comparison with other ethnic groups and amongst the main uk south asian groups. differences in diabetes related mortality and morbidity between different ethnic groups outlined in a small number of publications point to different mechanisms through which ethnicity exerts influence. for example, south asian and black groups both have increased risk of diabetes, cvd, and stroke compared to white europeans but show differences in level of risk and type of stroke. this in turn suggests particular genetic differences in addition to social and behavioural factors all or some of which may be linked. furthermore these studies have shown that when diabetes and age are controlled for, ethnicity exerts an independent effect on cardiovascular outcomes [40, 41 ]. studies which focus on diabetic nephropathy show that south asians also experience complications at an earlier age and their progression is faster than in white europeans. south asians ' risk of diabetic nephropathy is 13 times that of the white european population. as a group they are disproportionately represented in the population for renal replacement therapy, and because of this and the additional and independent risk of mortality from cvd that chronic kidney disease confers, together with a lack of ethnically compatible kidneys for transplantation, they are disproportionately represented in the group of people in need of end of life care. other diabetes complications, retinopathy and neuropathy, have a similar association with ethnicity ; that is, they have been found to be associated with increased risk factors and are indicators of microvascular damage. furthermore south asian populations are at increased risk of developing vascular dementia because of the increased incidence of diabetes, hypertension, and chronic kidney disease [4446 ]. there is a higher rate of cognitive impairment in older people with ckd ; it is largely unidentified and associated with severity of ckd [4749 ]. as the south asian population is ageing and as longevity is main risk factor for comorbidities in older people, the incidence of end stage renal failure and dementia are set to increase in south asian ethnic group. both these complications are ultimately life limiting but have a disease trajectory which can last many years, and as retinopathy and neuropathy affect sight and pain symptoms, care provision of older people with diabetes and complications incorporates preventive activity, treatment of symptoms, and comorbidities and end of life care [2224 ], which in the case of diabetic nephropathy may include renal replacement therapy. depression as a comorbid condition for people with diabetes is associated with both increased risk of developing cardiovascular problems as well as being secondary to cardiovascular complications and increasing risk of mortality. it is also a prevalent and costly burden to end stage renal patients and south asian patients are disadvantaged if it is not identified or they are unable to access services. individualised assessment of need and cultural sensitivity are included within the national service frameworks for diabetes, kidney disease, and care of older people [2224 ] as means of delivering person centred care. the equality impact assessment for the national dementia strategy however acknowledged that although south asians together with black caribbeans represent the largest ethnic minority in the uk, evidence about dementia care in these communities is lacking. research into the extent of how well healthcare services are able to meet the needs of south asian people who have diabetes has found that whilst services have implemented the organisational element of quality improvement policy such as the quality outcomes framework and shifts of diabetes care from secondary to primary care they may not have resulted in quality of care from the patient perspective [56, 57 ] nor in reduction of inequalities. this is attributed to lack of awareness about diabetes complications and services and communication barriers in healthcare encounters and research, although studies have not specifically addressed these in connection to age and ageing. the small number of trials testing culturally appropriate self - management programmes and structured education has found some short term effects on diabetes control and increased knowledge ; however they conclude that more research is needed to test different types and intensities of intervention and with different south asian groups. the patient experience research referred to, however, did not specifically include older people in their inclusion criteria. pilots of integrated diabetes care such as the north west london integrated care pilot for people over 75 years of age considered ethnicity in their design and analysis [61, 62 ]. the attendance by south asian people aged 4075 in the first year of the health checks programme was higher than previous studies of screening programmes in diverse groups highlighting the role of primary care in access for south asian patients compared to other parts of the nhs particularly in areas with high south asian populations with gps who have the same ethnicity. however whether the programme as a whole will achieve its target 75% uptake has been queried and the need for a combined population and high risk approach to prevention and targeting of care which considers age as the most powerful predictor of cardiovascular and diabetes risk and takes into account the earlier onset of diabetes in people with south asian ethnicity is a possible pragmatic solution. patient experience research with south asian people with diabetes in primary care identified barriers one of which was a need for information and health education to be delivered in a culturally appropriate way that matches an individual 's understanding of health and disease, as well as taking into account the broader social context for ethnic minority groups and common psychological responses [6668 ]. findings related to some dimensions of ageing and south asian ethnicity, for example, age - related expectations of health and health related behaviours, but ageing was not a specific focus of these studies although they called for multidimensional approach to understanding the preventable diabetes related mortality and morbidity. a care pathway approach to exploring patient experience of diabetes care across primary care and specialist renal care found that south asian patients referred to renal care lacked awareness of kidney complications of diabetes despite familiarity with diabetes over more than 10 years. furthermore reflecting back on diabetes care patients felt there had been missed opportunities for information and self - management support. the small number of studies of south asian patients ' experiences of care in secondary care kidney services also tells us more about the care of older south asian people with diabetes as nearly half the south asian patients requiring renal replacement therapy also have t2 dm. communication difficulties are a challenge in the day - to - day provision of renal care as well as for end of life care services to south asian patients who are often older and do not speak english as their first language [70, 71 ]. the exploratory mapping of the literature in this review created a context for the second part of our analysis. explanatory concepts which emerged from the literature alongside the observational data were cultural stratification and comorbidities, cultural competency, and access. the relationship between these concepts and the cmo analyses in each mapped area is shown in table 1. together the mechanisms and explanatory concepts formed a theoretical framework (see figure 4) for responding to the review question and identifying key areas for future enquiry which we articulated as broad research questions below. as diabetes complications are associated with longevity and length of time with diabetes as well as south asian ethnicity, it is common that older south asian people with diabetes will have multiple comorbidities requiring some sort of prioritisation and integration of treatment and care according to which conditions are of most concern or life limiting. stratification of patients by risk, comorbidities, patient experience, and diagnosis is therefore a key part of informing effective care [72, 73 ] and determines the context for care. the fact that south asian people develop diabetes earlier and experience the complications younger means that in the context of diabetes care the descriptor the changing demographics of the uk mean that there will be more older south asian people in the future and half will have developed diabetes by age 80. studies which identified the cardiovascular risk and outcomes associated with diabetes and south asian ethnicity and the small number breaking it down further into the predominant south asian groups in the uk provide evidence for the high risk that south asians with diabetes have for cardiovascular disease mortality and morbidity and persisting inequalities. this finding is not new, but it points towards the importance of understanding the heterogeneity within ethnic categories as well as the specific genetic and social influences on health outcomes. in the future it will be possible to draw more on the findings of biomarker and bariatric metabolic surgery research but at present accurate monitoring of ethnicity within the health system, the use of available data, targeting of screening programmes, and adaptability in day - to - day practice are ways of tailoring care towards individualised risk. detection of prediabetes, incident diabetes, and diabetes complications is important for prevention of the onset and progression of complications through the provision of appropriate and timely care which may need to be more aggressive for south asians because of the greater risk for cardiovascular (including cognitive and renal) complications. measures to detect complications which are culturally mediated, that is, dependent on language or ideas of dependency and quality of life, such as depression and cognitive impairment, need to be sensitive enough to identify complications in heterogeneous populations [53, 76 ]. the range of complications which are associated with older age and diabetes may contribute to frailty which results in vulnerability to sudden changes in health states and increased risk of falls, disability, long term care, and death. a recognised frailty descriptor for the clustering of comorbidities and associated indicators has been suggested to be more meaningful in a clinical context [78, 79 ] than chronological age and particularly within a model for integrated care. if frailty is to be useful indicator for stratifying and tailoring diabetes care greater understanding of what it means for clinical care and prevention is required both across different ethnic groups and in relation to individual culture. how can knowledge about diabetes comorbidities and associated impacts for older people with a south asian background improve care that maximises quality of life and nhs resources ? whereas stratification on the basis of disease, comorbidities, and symptoms dictates the context for clinical care, the way that information is conveyed and discussed to people with diabetes is important for supporting self - management and decision making in patient care. the opportunities for prevention of diabetes and complications are an important part improving outcomes for older south asian people with diabetes because of the earlier and extended timeframe that they are living with diabetes. the focus on primary care and integrated care as a means of delivering patient centred outcomes, if supported by systemic knowledge and awareness of culture within the nhs, aligns with the concept of culturally competent care : understanding the importance of social and cultural influences on patients ' health beliefs, and behaviours ; considering how these factors interact at multiple levels of the health care delivery system (e.g. at the level of structural processes of care or clinical decision making) ; and finally, devising interventions that take these issues into account to assure quality health care delivery to diverse patient populations. research which investigated ethnicity and quality of diabetes care in south asian patients in primary and secondary care identified the importance of individualising care within a culturally competent approach to support concordance in the care process. for individualised care to be supported practitioners therefore not only need culturally valid tools for assessing and diagnosing comorbid conditions, but also require a culturally adaptable approach which encourages concordance, that is, mutual agreement and involvement in their care. understanding the importance of social and cultural influences on patients ' health beliefs, and behaviours ; considering how these factors interact at multiple levels of the health care delivery system (e.g. at the level of structural processes of care or clinical decision making) ; and finally, devising interventions that take these issues into account to assure quality health care delivery to diverse patient populations. to achieve this one on one with patients requires the resources within the system to be in place and a full understanding of the challenge. the evidence as it stands suggests that although it is possible to target diabetes interventions and make cultural adaptations these have not been shown to be cost effective or to have fully addressed motivation as a key issue which requires a better understanding of culture and healthcare interactions at an individual and family as well as organisational level. peer support interventions have been identified as a potentially effective way of achieving culturally competent care but evidence is lacking from the uk of its usefulness with particular population groups [83, 84 ]. the concept of cultural intelligence takes the theory of cultural competency further [85, 86 ] by suggesting that care providers and the healthcare system as a whole are able to work effectively with all people of any culture. on an organisational level this concerns availability of sound data to inform decisions and at the level of the clinical encounter it involves open and adaptable communication skills. what are the most effective communication methods for promoting concordance in diabetes care with older people with a south asian background ? a person has access to quality care when the care they experience is meaningful and effective [87, 88 ]. as older people with diabetes and complications receive care in various settings : in general practice, acute departments of nhs hospitals, renal units, at home and in residential, and nursing care homes, commissioners require evidence of what constitutes quality care in these different contexts and in relation to inequalities within their local population. whilst the national quality improvement frameworks for diabetes and kidney services have improved diabetes care in terms of the infrastructure for monitoring in primary care with incentives for practices to do this, the evidence suggests that these do not support access to all aspects of diabetes care and that it can be fragmented and variable for all patients particularly for south asian groups for whom there can be more barriers [8991 ]. it has been estimated that a quarter of care home residents are likely to have diabetes and whilst data on care home residency by ethnicity is sparse, it is reasonable to anticipate that numbers of south asian older people requiring residential and social care services will grow in line with demographic changes. we also know there are growing numbers of south asian people requiring palliative and end of life care so that policy makers and commissioners must work with the range of care providers to ensure equitable access to care. our review of the literature highlights there is a dearth of research studies which have considered access as a collective function of providers within local systems and which include older patient and carer participation in diabetes care at local and individual levels. this is despite the growing awareness of the diabetes epidemic and observations that older age is when cultural differences and sensitivities can be most acutely experienced ; healthcare utilisation is at its greatest [8, 96 ] ; and when the costs are directly felt by individuals and their carers through morbidities, disability, and reduced quality of life. the prevention imperative to reduce levels of diabetes and complications requires intervention to raise public awareness of the issues of diabetes care for older people from ethnic minority groups and to shift attitudes of patients and clinicians towards a more empowered approach to care planning. to enable access to holistic diabetes care for older people requires primary care commissioners to lead and facilitate an integrated approach with care providers, people with diabetes, and their carers. whilst evaluation of on - going programmes such as integrated care initiative, national diabetes audit with patient experience of diabetes services, and house of care will contribute to this process, primary research with patients, care providers, and formal and informal carers is necessary to understand the clinical and cultural contexts of ageing with diabetes better and to maximise ways to improve access and quality of care for older people and people with or at risk of diabetes and cardiovascular complications. what are patients and their informal and formal carers experience of involvement in care planning and how can these inform service improvement for older people living with diabetes who have a south asian background ? current policy and interventions to reduce inequalities in diabetes care in older people with south asian ethnicity have not resulted in a knowledge base of what works to reduce complications and the poorer outcomes for this population. this exploratory synthesis of the literature is the first to put forward a theory based framework for doing so. the lack of a body of research evidence which addresses inequality and quality of diabetes care for older south asian people with diabetes reflects many and complex relationships between diabetes and macro- and microvascular complications ; the different settings where care is provided ; the lack of specific inclusion of older south asian people in research ; and the heterogeneity within ethnic and age descriptors. studies which, by default, have included this group highlighted that the ethnic specific and ageing effects of diabetes require further enquiry. limitations of this review relate to complexity ; diffuse literature ; a broad research question ; and the multidimensional influence of ethnicity and culture on health. we mitigated any shortfalls in capturing relevant literature via electronic databases by hand searching and including grey literature and including broad age descriptors which was in line with the exploratory nature of this study. the realist approach taken helped to expand the knowledge base by identifying common mechanisms across different contexts which together contributed to a theoretical framework for policy, research, and practice. it is both a strength and a limitation that our review was conducted by a team with familiarity with particular areas of the literature, that is, diabetic nephropathy and end stage renal failure in south asians, and frailty in relation to diabetes and older people. whilst it helped inform the search strategy and theory building it could constitute bias as published research of inequalities in diabetic kidney disease and kidney care made an important contribution and the subsequent analysis applied some of the concepts from diabetic nephropathy research previously published by two of the authors. to mitigate this risk the team rereviewed the analysis and synthesis at key stages during development and invited critical analysis of the review prior to finalising the work. team composition comprised public health researchers and senior academics who have been involved in guideline development, some of whom are practicing clinicians, strengthened our analysis and interpretation in policy and practice terms. theory building from such a broad question and diverse literature base identified mechanisms which were mid - level, conceptual, and compatible with a systems viewpoint, and interpretation into practical questions for policy makers, clinicians, and researchers was a useful element of this review (see table 2). although this piece of work was limited to a uk perspective and a focus on one (albeit broad and heterogeneous) ethnic grouping, the rationale, realist methodology used, and the resulting theoretical framework could equally well be applied to other groups and other diverse populations in other countries. the focus of the review was on understanding the mechanisms which could be useful for reducing inequalities in diabetes care and because the work was exploratory the theoretical ideas are at an early stage and conceptual so also relevant to other health systems. figure 4 illustrates the review areas and emerging concepts and mechanisms described in the results. at the centre of this model, a theme which underpins uk diabetes policy is individualised, culturally intelligent, and ethical care for older people living and dying with diabetes this review suggests that better understanding of how risk, disease trajectories, and comorbidities affect people differently (stratification) ; of how culture, and not just ethnicity, influences care (cultural competency) ; and of how services can be delivered so they are meaningful and effective for individuals in different settings (access) is all key mechanisms to achieve these objectives. our theory building went further to identify submechanisms : concordance ; the use of culturally meaningful measures for comorbidities affecting older people such as depression and cognitive impairment ; and care planning, in particular understanding ways that older people with diabetes can be involved to ensure that it is as person centred as possible. these submechanisms, articulated as future research questions, were at the next level of abstraction from the evidence reviewed. addressing these will enable us to revisit and refine this early theoretical framework to further improve understanding of how to ensure equitable care at the intersection represented with a ? underpinning individualised care and pertinent to understanding these mechanisms is the ability of the healthcare system to work with the intersectionalities of individuals and groups within a population. the heterogeneity within broad ethnicity and age descriptors is lost in much of the research literature and a more nuanced approach to understanding individual identity and influences on health will be needed to take forward the different research elements we have identified. research with diverse groups of older people and their care providers in different clinical and community settings requires a culturally intelligent approach by researchers [28, 102 ]. conducting research with older people with diabetes also presents particular practical and ethical challenges particularly if the person has comorbidities such as cognitive impairment or is at end of life. however a focus on the lived experience and meaning of diabetes for older people with different comorbidities and cultural backgrounds is important to fill some of the evidence gaps in this area. in practice terms too the awareness of multiple identities and individual experiences affecting diabetes care including, but not exclusive to ethnic group and age, requires closer involvement between patients and practitioners in negotiating care in order for it to be truly person centred. although this review focussed on south asian ethnicity the mechanisms and recommendations made are transferrable and relevant to care delivery with other population groups. in a similar way the relevance of this review in policy terms should be seen in the context of other influences on health inequalities, that is, the psychological, sociological, economic, and life course factors [104, 105 ]. although we investigated inequalities and access to diabetes care in relation to ethnicity, the mechanisms identified are ways through which the diabetes care system can work with the individuals and the intersectionalities that influence diabetes risk, prevention, and management. this review has found that there are very few studies which address care of older people with diabetes who have a south asian background. as policy makers need evidence to help them respond to the changing demographic profile of the uk to commission effective services to prevent avoidable mortality and morbidity and maximise resources, this is an important limitation in the existing evidence base. there is commissioning guidance for diabetes services and integrated care which by default covers care for chronic conditions and older people and points to earlier onset, need for services to consider ethnicity, but it seems that there has been limited organisational engagement, it has been low priority, and there are limited skills. south asian people experience diabetes earlier than white europeans and have a greater risk of complications and faster progression so that care providers and patients would benefit from a better informed and targeted approach to intervention. for policy, practice and research to make an impact on reducing inequalities in diabetes care for older people with diverse backgrounds we suggest attention is given to all three of the mid - level mechanisms : access, comorbidities, and stratification and cultural competency. research that specifically includes older people with a south asian background would go some way to providing knowledge about the best way to do this. the definition of older people needs to be redefined in the context of diabetes care and south asian ethnicity and the influence of intersectionalities require more attention to understand and apply these mechanisms for reducing inequalities in diabetes care. | the impact of type 2 diabetes on ageing societies is great and populations across the globe are becoming more diverse. complications of diabetes unequally affect particular groups in the uk older people, and people with a south asian background are two population groups with increased risk whose numbers will grow in the future. we explored the evidence about diabetes care for older people with south asian ethnicity to understand the contexts and mechanisms behind interventions to reduce inequalities. we used a realist approach to review the literature, mapped the main areas where relevant evidence exists, and explored the concepts and mechanisms which underpinned interventions. from this we constructed a theoretical framework for a programme of research and put forward suggestions for what our analysis might mean to providers, researchers, and policy makers. broad themes of cultural competency ; comorbidities and stratification ; and access emerged as mid - level mechanisms which have individualised, culturally intelligent, and ethical care at their heart and through which inequalities can be addressed. these provide a theoretical framework for future research to advance knowledge about concordance ; culturally meaningful measures of depression and cognitive impairment ; and care planning in different contexts which support effective diabetes care for aging and diverse populations. |
a 60-year - old man was diagnosed with type 3 chronic aortic dissection and scheduled for the replacement of the descending thoracic aorta. the patient complained of no symptoms, and the extent of aortic dissection was between the left subclavian artery and the bifurcation of the common iliac artery. he had a history of hypertension and underwent thrombectomy of the right iliac artery 3 years before this presentation. without premedication, invasive monitoring of blood pressure was executed through the right radial artery, and central venous catheters were inserted into the right internal carotid artery and left subclavian vein. bispectral index (bis), cerebral oximeter, electroencephalogram (eeg), somatosensory evoked potential and motor evoked potential were monitored during anesthesia. after induction of anesthesia, dural puncture was conducted at l4 - 5 in the right decubitus position using a tuohy needle through the median approach. after identifying csf drainage, a drain catheter with an outer diameter of 1.5 mm (hermetic lumbar catheter) the drain was connected to a 3-way stopcock ; a pressure transducer was connected to one end for continuous monitoring of csf pressure, and a closed drain bottle was connected to the other end for continuous drainage of csf. during the procedure, the initial csf pressure was 10 mmhg, and there were no specific events such as bleeding. ten minutes after cross - clamping, csf pressure was increased from 10 to 17 mmhg. csf pressure was periodically measured at a 10-minute interval as 10 ml of csf were drained through natural drainage by gravity. after 60 ml of csf were drained, the csf turned out to be red. after the completion of the operation, he was transferred to the intensive care unit. total aortic clamping time was 22 minutes, the lowest bladder temperature was 23.5, cardiopulmonary bypass time was 97 minutes, and total operation time was 305 minutes. since csf was bloody at the intensive care unit, csf drainage was stopped and the csf pressure was maintained at 11 to 16 mmhg. thirteen hours after the operation, his consciousness returned to an alert state, and neurologic examination revealed normal findings. although he had no postoperative complications, he required a mechanical ventilator due to pulmonary edema, which was weaned and extubated 18 days after the operation. paralysis of the lower extremities due to spinal ischemia is a fatal complication of the replacement of the descending thoracic aorta. aorta clamping leads to an increase in csf pressure and a decrease in systolic pressure of the terminal aorta with a significant decrease in spinal perfusion pressure, which subsequently induces ischemic injury to the spinal cord. methods for preventing spinal cord injury associated with operation include distal aortic perfusion, sufficient anastomoses of the intercostal and lumbar arteries, hypothermia and csf drainage. among them, intraoperative and postoperative csf drainage decreases csf pressure, increase spinal blood flow, decrease the frequency of spinal cord injury due to the replacement of the aorta and improves neurologic symptoms and signs such as paralysis of the lower extremities. complications associated with csf drainage include csf leakage, headache, meningitis, intracranial hemorrhage, bleeding in the spinal canal and breakdown of the drain catheter. as for bleeding, weaver. reported cases of neurologic complications due to bleeding in the spinal canal, murakami. reported cases of cauda equina syndrome due to hematoma around the cauda equina and those of subdural hematoma, and settepani. is suspected when bloody csf is found. in our patient, although bloody csf was found, his clinical course was closely observed because there were neither neurologic abnormalities nor coagulopathy. consciousness, extremity movement and sensory function were closely observed without mri. in patients with bloody csf, surgeons usually stop csf drainage and correct coagulopathy, and thereafter continue the csf drainage after identifying clear csf. however, in patients with persistent bloody csf, surgeons remove the drain catheter and insert a new one into a different site on the basis of neurologic symptoms. in patients with delayed neurologic deficits or paralysis of the lower extremities, ct of the brain and spine was performed to establish a diagnosis of intracranial hemorrhage or spinal hematoma. however, in cases of aortic surgery, the drain catheter can not be immediately removed because activated clotting time should be maintained 400 seconds by using anticoagulants for correcting coagulopathy and also because csf should be drained after operation if needed. therefore, in such cases it is desirable to perform postoperative neurologic examination, close observation of clinical symptoms, identification of restoration of coagulation function and radiologic examination if needed. wynn. reported that in a total of 24 patients with bloody csf during or after thoracic aortic surgery, 17 had intracranial hemorrhage on brain ct, only 3 of whom exhibited neurologic symptoms. this result implies that all patients with bloody csf do not have neurologic symptoms. in this respect, we might have performed brain ct on our patient who had no neurologic symptoms. in our patient, it is thought that bloody csf was not to the extent that neurologic abnormalities were induced by coagulopathy during the operation. bloody csf is produced by drainage of a large amount of csf, a high velocity of csf drainage, venous congestion, rupture of the vein, the pre - existing intracranial lesion and subdural or intracranial hemorrhage due to coagulopathy or hypertension. reported that the amount of csf drainage was 690 ml in 230 patients with subdural hemorrhage, whereas it was 359 ml in those without, suggesting that there is no significant correlation between the occurrence of bloody csf and the amount of csf.. demonstrated that in patients with a csf pressure of 10 mmhg, csf drainage at a velocity of 10 ml / hr did not cause intracranial hemorrhage. taken together, although csf drainage can be performed during aortic surgery in order to protect the spinal cord, neurologic complications may be induced. for this reason, surgeons should be careful not to cause damage to the spinal cord while inserting a drain catheter and should closely monitor the amount and velocity of csf drainage. in addition, early protection of the brain, neurologic examination and radiologic imaging studies are necessary when there are abnormal findings in drainage, bis and eeg during the operation. | cerebrospinal fluid (csf) drainage is a routinely used adjunct to thoracoabdominal aortic surgery which may reduce the incidence of preoperative paraplegia by improving spinal cord perfusion. however, this procedure infrequently causes complications. bloody or bloody - tinged csf may be associated with intracranial or spinal hematoma. we present herein a case of bloody csf during the replacement of the descending thoracic aorta. |
acute fatty liver of pregnancy (aflp) is a rare and potentially life - threatening complication which tends to manifest in the third trimester of pregnancy / early postpartum period. the condition occurs more commonly in primigravida, twin pregnancy, and pregnancies carrying a male fetus. maternal mortality is now estimated to be 12.5%18%, with a neonatal mortality rate of 7%66%. the incidence of aflp is 1 in 7,000 to 1 in 16,000 pregnancies. a prospective uk - based research study, involving 229 centers identified 57 confirmed cases in a total of 1,132,964 pregnancies, giving an incidence of 5 in 100,000 pregnancies. 74% of cases were identified at a median gestation age of 36 weeks, with 60% of cases delivered within 24 h of diagnosis. though, it is important to further lower the mortality rate by early diagnosis and prompt treatment of this disease. the aim of the present study is to explore the clinical outcome of patients with aflp, evaluate the effect of early diagnosis and treatment, and analyze the influence of the mode of delivery on 14 mol / l), leukocytosis (> 11 10/l), elevated uric acid (> 340 mol / l), hypoglycemia (42 mol / l), elevated ammonia (> 47 mol / l), coagulopathy (prothrombin time > 14 sec or activated partial thromboplastin time > 34 sec), and microvesicular steatosis on liver biopsy. the most striking feature of this syndrome is a high level of bilirubin associated with moderate increases of transaminases. the platelet count may be decreased with or without other signs of disseminated intravascular coagulation (dic). classical features of ascites or bright liver were only seen in a quarter of these. this observation is reflected in other studies which report that hepatic ultrasound is not sufficiently sensitive or specific to make a definite diagnosis. the highly suspicious or confirmed cases of aflp must be treated as obstetrics emergency, with the prompt termination of pregnancy. the management of aflp requires maternal stabilization following delivery and supportive care. in our data, all pregnancies were terminated within 24 hours of highly suspected or diagnosed aflp, and the outcomes were good. if vaginal delivery can not be achieved quickly, cesarean section is the preferred method, as it is beneficial to stop the progress of the patient 's condition and to shorten labor as much as possible. in conclusion mentioned data, of the 126 patients, 109 (86.5%) pregnancies were terminated by cesarean section and of those cases 14 patients died. the mortality rate of the mothers who underwent cesarean section 12.8% was lower than those who delivered vaginally 35.2%. there were 12 (8.9%) perinatal deaths delivered by cesarean section and 7 (28%) perinatal deaths from vaginal delivery. so, analyzing these data prompt delivery after confirmed diagnosis is essential for better pregnancy outcome and lesser maternal and fetal mortality. all patients should be hospitalized as soon as a diagnosis of aflp is suspected. moderately or severely affected patients (those presenting with encephalopathy, deep jaundice, or prothrombin times less than 40% of control) or patients with any extrahepatic complication should be attended to in the intensive care unit. glucose infusions should be maintained until a full metabolic recovery is achieved due to the risk of sudden hypoglycemia, which can occur at any time, even during clinical recovery. patients with severe hepatic dysfunction are best treated in an icu setting before and after delivery. blood sugar levels should be monitored and severe coagulation disorders treated with platelets and fresh frozen plasma transfusions. initial treatment involves supportive management with intravenous infusion, intravenous glucose, fresh frozen plasma, and packed red blood cells for patients with coagulation dysfunction to reduce blood loss and cryoprecipitate to correct dic. once the patient is stabilized, further course of management includes the appropriate mode for delivery. this may occurs by vaginal delivery, while in cases of severe bleeding and compromise status of mother, caesarian section may be done. when coagulation dysfunction exists, patients have an additional degree of risk with cesarean section ; in these cases, a longitudinal incision is beneficial to reduce bleeding. therefore, we suggest that cesarean section is the method of choice to terminate pregnancy, if vaginal delivery can not be performed promptly. because the probability of postpartum hemorrhage is high, hysterectomy and uterine artery embolization should be considered at the time of pregnancy termination. the clinical conditions deteriorate rapidly and progress to multiorgan dysfunctions, if there is delay in the diagnosis and the prompt termination of pregnancy. patients with severe hepatic injury remain at risk for respiratory failure, renal failure, gi bleeding, and nephrogenic diabetes insipidus and should be closely monitored during the immediate postpartum period. the rare patient who progresses to fulminant hepatic failure can be treated by liver transplantation.. systemic complications of aflp are due to fulminant hepatic failure and include encephalopathy, and renal insufficiency, coagulopathy, sepsis, hypoglycemia, and gastrointestinal hemorrhage. other systemic complicating effects include acute respiratory distress syndrome sometimes requiring assisted ventilation, ascites, and upper gastrointestinal bleeding from gastric ulceration, and mallory - weiss syndrome. maternal deaths occurs due to hemorrhage, gastrointestinal bleeding, sepsis, aspiration, pancreatitis, renal failure. aflp is an uncommon, life - threatening complication of third trimester with variable presentation. it is recommended that patients with nausea, vomiting, or epigastric pain, and persistent jaundice in the third trimester, with alter in liver function, should be suspected for the diagnosis of aflp. the patients, who are critically ill at the time of clinical presentation, develop complications, or continue to deteriorate despite emergency delivery, and require collaborative management in the intensive care unit (icu). early diagnosis, prompt delivery, adequate supportive care, and a multidisciplinary approach are the key to a good outcome. | objectives. our aim is to explore the clinical outcome of patients with acute fatty liver of pregnancy (aflp), and evaluate the effect of early diagnosis and treatment. methods. seven patients who were diagnosed with aflp were retrospectively analyzed from february 2005 to january 2013. the clinical records of the patients with aflp were reviewed for clinical features, laboratory examinations, and maternal and perinatal prognosis. routine laboratory evaluation revealed hyperbilirubinemia, moderately elevated liver transaminase, but negative serum hepatitis virus in each patient. for additional evidence, 126 cases of aflp were reviewed retrospectively from original articles researched in a medline - based english and chinese knowledge infrastructure between the same periods. results. the initial symptoms of all the 7 cases with aflp were gastrointestinal symptoms ; anorexia, nausea, vomiting, and progressive jaundice. complications revealed with renal insufficiency in all 7 patients. hepatic failure, mods, hypoglycemia and dic were seen in 4 patients (57.1%). hemorrhagic shock, ards, and hepatic encephalopathy were seen in 3 patients (42.8%). there was only one case of maternal death (14.2%), three cases of perinatal death (30%) and one postnatal death (10%). conclusion. aflp occurs in late pregnancy is a rare clinical syndrome occurs at about 36 weeks of gestation. early diagnosis and prompt termination of pregnancy is the key of management with multidisciplinary collaboration, comprehensive treatment and effective prevention are helpful to improve prognosis of the cases with aflp and perinatal death. |
with current techniques for mapping receptive fields, it is impossible to resolve the contribution of single cone photoreceptors to the response of central visual neurons. using adaptive optics to correct for ocular aberrations, we delivered micron - scale spots of light to the receptive field centers of neurons in the macaque lateral geniculate nucleus. parvocellular lgn neurons mapped this way responded with high reliability to stimulation of single cones. |
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rare germline variants are difficult to identify using traditional sequencing due to relatively high cost and low throughput. using second - generation sequencing, we report a targeted, cost - effective method to quantify rare snps from pooled genomic dna. we pooled dna from 1,111 individuals and targeted four genes. our novel base - calling algorithm, snpseeker, derived from large deviation theory, can detect snps present at frequencies below the raw error rate of the sequencing platform |
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obstetric anal sphincter injuries (oasis) are the leading cause for anal incontinence (ai) in women.13 indian and other women of south asian origin have been shown to have higher rates of oasis compared with caucasian women.46 a short perineal body has been suggested as the cause for the higher incidence of oasis in women of asian origin. it has been suggested that a prophylactic episiotomy might be used in women of indian and oriental origin. the numbers needed to treat (nnt) to prevent oasis in indian women resident in the uk undergoing forceps delivery is 1.88, and vacuum delivery is 10.5 another study described a fivefold - increased risk of oasis in asian women.6 the episiotomy angle has been described as a significant factor in the causation of oasis. studies have shown an increased incidence of oasis in association with postdelivery episiotomy angles that are very acute (suture angle less than 30), as these can directly injure anal sphincters. episiotomies that are too lateral (suture angle greater than 60) do not relieve the pressure on the perineum.7 andrews found a mean angle of 37 in episiotomies without oasis, while eogan9 found a mean angle of 38. based on this, the concept of a safe zone of episiotomies with a suture angle between 40 and 60 has been proposed.7,10 recently, a new type of episiotomy scissors (episcissors-60 ; medinvent, llc, romsey, uk) were introduced that direct the episiotomy at 60 to the perineal midline at the time of cutting. these were a modification of the mayo scissors with a guide limb that points toward the anus. a median angle of 43 was achieved in a case series of caucasian women undergoing instrumental deliveries.10 more recently, another version of the episcissors-60 was commercially introduced with blades angled at 60 to the scissors shaft. to test the efficacy of these scissors in spontaneous vaginal deliveries, consecutive patients delivering in two private maternity hospitals in thane, india undergoing clinically indicated episiotomies were included. the scissors were introduced vaginally at crowning, and aligned to orient the guide limb vertically from the posterior fourchette to the anus (figures 1 and 2). while a single cut was preferred, a stagger cut was needed for some women. postdelivery angles were measured by the obstetrician by placing a protractor transparency on the perineum after delivery, and the angle was marked with an indelible ink pen (figure 3). per rectal examination was performed prior to suturing to detect oasis. a total of 25 women underwent clinically indicated episiotomies for conditions like fetal distress, prolonged second stage of labor, and maternal exhaustion. of these, 16 women were nulliparous, eight women were para 1, and one woman was a para 2. one woman had a vaginal breech delivery (para 2), and the rest were cephalic deliveries. the median birth weight was 2,800 g (standard deviation 312 g, interquartile range 2,5003,000 g). the median postdelivery suture angle of the episiotomy was 50 (standard deviation 3.5, interquartile range 4854, range 4555). the angled version of the episcissors-60 achieved a postdelivery suture angle of 50 in indian women having spontaneous vaginal deliveries. this was 7 greater than the angles achieved with the scissors in instrumental births by freeman.10 this could be explained by the greater degree of perineal distension in instrumental births at the time of the episiotomy being performed. the mean birth weight in their study was 3,410 g, which differed significantly from that of the present study (2,800 g). it is possible that a higher - birth - weight fetus would distend the perineum more at crowning, leading to a lower postdelivery angle. it could also be related to differences in the position of the women while measuring the angles (in poles for instrumental births versus free - standing for spontaneous births). nevertheless, we did not have a single patient with a postdelivery angle of less than 45, which is reassuring. clearly, the incision angle of the episiotomy needs to be greater than the postdelivery suture angle to allow for perineal distension. the suture angle will vary between normal and instrumental deliveries, the individual accoucheur s timing of the episiotomy, and the indication for the episiotomy, ie, fetal distress versus prolonged second stage. a 40 episiotomy (premarked with gentian violet staining) achieved an angle of 22 postdelivery, and a 60 premarked episiotomy achieved a postdelivery angle of 45. studies using stereophotogrammetry13 have described a perineal distension of 2.77-fold in the transverse plane and 1.43-fold in the vertical plane. this is similar to the perineal distension of 3.26-fold noted in magnetic resonance imaging studies.14 a limitation of this study was that since it became our normal practice to use the episcissors-60, we could not measure the angles in women undergoing episiotomies with the ordinary scissors as a comparator group. however, the literature suggests that postdelivery recorded episiotomy angles are quite close to the midline.8 the authors believe that their episiotomies were angled more acutely prior to the change in practice to use the episcissors-60. however, andrews showed that truly occult anal sphincter injuries are very minimal. a randomized controlled trial would be needed to definitively compare the current practice with using the episcissors-60. however, given the fact that only 13% of clinicians are able to achieve an angle of 40 by visual estimation alone, and that the scissors are a fixed - angle device, there would be legitimate concerns as to whether randomization was ethical. the angled - version episcissors-60 demonstrated a postdelivery suture angle of 50 in a cohort of indian women undergoing spontaneous vaginal deliveries. | backgroundobstetric anal sphincter injuries (oasis) are the leading cause of anal incontinence in women. episiotomies with a postdelivery suture angle of less than 30 to the midline are more likely to injure the anal sphincter directly, while those with a suture angle of more than 60 are associated with increased incidence of oasis, as they do not relieve the pressure on the perineum. a safe zone of 4060 has been proposed. recently, two new types of episiotomy scissors (episcissors-60 straight version and angled version) were introduced to ensure a standardized cutting angle of 60 to the midline. we audited our results with the angled episcissors-60 in spontaneous vaginal deliveries.materials and methodsconsecutive patients delivering in two private maternity hospitals in thane, india undergoing clinically indicated episiotomies were included. only patients delivering spontaneously were included. the scissors were introduced vaginally at crowning, and aligned to orient the guide limb vertically from the posterior fourchette to the anus. while a single cut was preferred, a stagger cut was needed for some women. postdelivery angles were measured by placing a protractor transparency on the perineum after delivery and marking the angle with an indelible ink pen. per rectal examination was performed prior to suturing to detect oasis.resultsa total of 25 women underwent clinically indicated episiotomies. of these, 16 women were nulliparous, eight women were para 1, and one woman was a para 2. one woman had a vaginal breech delivery (para 2), and the rest were cephalic vertex deliveries. the average age was 27 (range 2035) years. the median birth weight was 2,800 g (standard deviation 312 g, interquartile range 2,5003,000 g). the median postdelivery suture angle of the episiotomy was 50 (standard deviation 3.5, interquartile range 4854, range 4555). no cases of oasi were detected in this series.conclusionthe episcissors-60 angled version demonstrated a postdelivery suture angle of 50 in a cohort of indian women undergoing spontaneous vaginal deliveries. |
a large variety of environmental carcinogens are metabolically activated to electrophilic metabolites that can bind to nucleic acids and protein, forming covalent adducts. the formation of dna - carcinogen adducts is thought to be a necessary step in the action of most carcinogens. recently, a variety of new fluorescence, immunochemical, and radioactive - postlabeling procedures have been developed that allow the sensitive measurement of dna - carcinogen adducts in organisms exposed to environmental carcinogens. in some cases, similar procedures have been developed for protein - carcinogen adducts. in an organism with active metabolic systems for a given carcinogen, adducts are generally much longer lived than the carcinogens that formed them. thus, the detection of dna- or protein - carcinogen adducts in aquatic foodstuffs can act as an indicator of prior carcinogen exposure. the presence of dna adducts would, in addition, suggest a mutagenic / carcinogenic risk to the aquatic organism itself. vertebrate fish are characterized by high levels of carcinogen metabolism, low body burdens of carcinogen, the formation of carcinogen - macromolecule adducts, and the occurrence of pollution - related tumors. shellfish, on the other hand, have low levels of carcinogen metabolism, high body burdens of carcinogen, and have little or no evidence of carcinogen - macromolecule adducts or tumors. the consumption of carcinogen adducts in aquatic foodstuffs is unlikely to represent a human health hazard. there are no metabolic pathways by which protein - carcinogen or dna - carcinogen adducts could reform carcinogens. incorporation via salvage pathways of preformed nucleoside - carcinogen adducts from foodstuffs into newly synthesized human dna is theoretically possible.(abstract truncated at 250 words)imagesfigure 1.figure 1.figure 2. |
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patients with inflammatory bowel disease (ibd) have underlying immune dysregulation felt to be due to several factors, including intestinal dysbiosis, increased intestinal epithelial permeability, and medications. evidence suggests that the balance between microbes, particularly commensal flora, plays an important role in the pathogenesis of ibd. tnf - alpha inhibitors have been associated with increased risk of reactivation of latent tuberculosis, in large part due to their role in granuloma formation. thiopurine agents such as azathioprine (aza) or 6-mercaptopurine (6-mp) have been shown to predispose patients to bacterial and viral infections. corticosteroids used as monotherapy and dual therapy with either anti - tnf or thiopurines have been shown to increase risk of infections. although minimizing exposure to corticosteroids and monitoring for leukopenia in patients on thiopurine analogs are important in infection control, vaccination remain one of the hallmarks of preventative care in these patients. guidelines for vaccination in ibd patients were published in 2004. despite this, poor vaccination rates persist due to several factors, including lack of awareness on the part of clinicians, fear of potential adverse effects, or ambiguity as to which provider should take responsibility for immunizations. additionally multiple studies have shown increased risk of nonmelanoma skin cancer, cervical cancer, osteoporosis, and eye manifestations among patients with ibd. we sought to expand on the concept of quality improvement within the va and evaluate for other possible areas that could be addressed to improve the healthcare of ibd patients. therefore, we assessed rates of recommended vaccinations and preventative screening among patients with ibd using the va for care, and analyzed the association of these factors with 5-year mortality. data were extracted from the administrative data repository maintained by the va and accessible for research. included patients were veterans diagnosed with ibd per icd-9 diagnosis codes (555.0, 555.1, 555.2, 555.9, 556.0, 556.2, 556.3, 556.5, 556.6, 556.9) during fiscal year 2005 to 2014 (fy05fy14 ; october 2005september 2014) with prior - year use including comorbid diagnosis data. veteran status was determined by a valid va priority code in the range 1 to 8 indicating why the patient was eligible for va care based on military experiences, disability, and poverty. veterans with va priority 26 have lesser levels of disability or special military circumstances and pay copays for pharmacy benefits only. priority 7 to 8 are veterans who agreed to copayments for both pharmacy and care. the source files are extracts of va 's all - electronic medical records system and capture all care episodes. we used inpatient and outpatient data on encounters, lab tests, procedures, and pharmacy fills as contained in the stopp research data repository. comorbidity was assessed by the selim chronic disease score counting 30 physical conditions and 6 mental illnesses. selim conditions are anemia, cancer, skin cancer, cataract, hepatitis, chronic obstructive pulmonary disease (copd), chronic heart failure (chf), diabetes, diverticula of the colon, colitis or enteritis, enlarged prostate, inflammatory disease of the prostate, gall bladder disease, gout, osteoarthritis, hip problems, other arthropathies, rheumatoid arthritis, hypertension, heart attack, angina, arrhythmia, peripheral vascular disease, transient ischemic attack, stroke, low back pain, ulcers, seizures, thyroid disorders, and urinary tract infection ; anxiety, depression, bipolar disorder, schizophrenia, post - traumatic stress disorder, and alcohol use disorder. additional conditions were osteoporosis and osteopenia (icd9 codes 733), nonmelanoma skin cancers (basal cell carcinoma, squamous cell carcinoma : 173), and eye disorders (360.11, 365.0005, 365.2024, 366.0004, 366.1018, 370, 371.23, 371.43, 371.49, 379, 710.2x). vaccines included pneumococcal (cpt codes 90732, 90669, 90670, g0009), varicella (cpt 90716), and tdap (cpt 90715, 90700) as well as hepatitis b vaccination (cpt codes 90739, 90740, 90746, 90747) and ppd (cpt code 90585). specialty care was captured by clinic type (ophthalmology ; dermatology ; women 's health) and screenings by cpt codes or icd9 visit / diagnosis codes (eye : cpt 92225, 92226 ; papanicolau test : cpt 87621, 88164, 88141, 88142, 88143, 88174, 88175 ; hpv : icd9 v73.1, 795.0x, 795.1x, 622.1x ; gynecological exam : icd9 v72.31 ; dexa scan : cpt 77080), whereas abnormal dermatology results were indicated by icd9 codes v76.2, v76.47, v76.49. the study was approved prior to initiation by the institutional review board of the central texas veterans health care system using expedited procedures ; consent was waived. analyses included descriptive means and frequencies, gender comparisons, and a multiple logistic regression of 5-year mortality as a function of demographics, comorbidity, corticosteroid use, pre-/post - ibd primary care and pneumococcal vaccination with an indicator for truncated follow - up for those with 65 ; see table 1). most (62%) patients used va care each of the 10 years of the observation period (mean 8.4 ; sd 2.5). reflecting patients with ibd diagnosed in prior years, 36% entered the study cohort in the first fiscal year, then 7% to 8.5% each year thereafter, and only 6% in fy2014. veterans treated for inflammatory bowel disease (ibd) during fy2005 to fy2014 (n = 62,002). patients used primary care before their ibd diagnosis (91%) ; after diagnosis, this increased to 96% (chi - square = 31.6 ; df = 1 ; p 2000 women veterans from 2011 and 2012, 91% had documentation of cervical cancer screening or refusal. in our study, only 66% of women had undergone a pap smear in the previous 10 years. the latest 2013 guidelines from the american college of obstetricians and gynecologists recommended cervical cancer screening with pap tests every 3 years or 5 years with human papilloma virus (hpv) testing for women with no risk factors. notably, however, guidelines for hiv - infected patients recommend annual screening without hpv testing and have been applied to all immunocompromised patients regardless of the cause of their immunocompromised state. the increased risk of cervical dysplasia among immunocompromised patients is felt to be due to prolonged persistence of hpv infection compared to the general population. in a 2015 meta - analysis by allegretti, there was an increased risk of cervical high - grade dysplasia / cancer among patients with ibd on any immunosuppressive medication, with an overall odds ratio of 1.34. patients on thiopurine analogs, particularly azathioprine, have consistently shown the highest risk of nonmelanoma skin cancer. in 2010, long showed over a 4-fold increased risk in patients who had been on azathioprine for over 1 year. in our study, only 11% of the patients had utilized dermatology clinic. lastly, the side effects resulting from prolonged exposure to corticosteroids have been well documented, including osteoporosis, glaucoma, and cataracts. studies on glucocorticoid induced osteoporosis have shown that a prednisone dose of 5 mg per day for a total of 3 months increases fracture risk. the american college of rheumatology has suggested osteoporosis screening with dual x - ray absorptiometry (dexa) scan based on the world health organization fracture risk assessment tool (frax), taking into account corticosteroid use. screening for higher risk individuals is recommended as early as < 50 years of age. although we did not have data on dose or duration of prescription of glucocorticoids, our findings do accord with these previously noted risks of corticosteroid exposure. in addition to cataracts and glaucoma induced by corticosteroids, rare complications including uveitis and episcleritis are also more prevalent among the ibd patient population. given these risks, patients with ibd should be closely monitored for osteoporosis and considered for dexa scan and should be closely monitored by an ophthalmologist. patients with private insurance, medicare, or medicaid may seek care in the community. as such they may have gotten screening and preventative care in the community with va pcp documenting them as compliant ; we were unable to ascertain this. our study did not review clinical notes, but our patients used the va an average of 8 out of the 10 years studied. it should also be noted that not all patients with ibd require all the screening tests examined in this study. guidelines recommend screening for osteoporosis in ibd patients with ongoing steroid use, cumulative prior steroid use greater than 3 months, history of low trauma fractures, age over 60, or in postmenopausal females. our data should be interpreted in this context, realizing that not all patients in our study required a dexa scan or the other screening tests. we were also unable to obtain dose and frequency data on the immune mediators, and had no information on sources and tissue types of infections. this also contributes to uncertainty about whether the observed rate of dexa scans was low, as we have no data on long - term steroid use. another inherent limitation in studies conducted through the va healthcare system is that the patient population is predominantly male ; thus, conclusions may not generalize to the general population. various explanations for the low rates of preventative care measures in ibd patients have been proposed. one possibility is that there is uncertainty about which physician is responsible for preventative care in ibd patients. primary care physicians may be hesitant to order routine vaccinations and screening tests due to lack of awareness of ibd - specific guidelines. gastroenterologists, on the other hand, may assume that the primary care physicians are ordering vaccinations and routine screening. another possibility is that in patients with complex medical problems such as ibd, primary care physicians, as well as subspecialists invest more energy in planning out management of the disease and therefore are more prone to neglect routine preventative care. in conclusion, our study suggests that preventative care rates for ibd patients in the va healthcare system lag behind national standards. future research should be aimed at identifying interventions that may improve these rates, such as standardized patient intake processes, template notes, and patient surveys. | abstractpatients with inflammatory bowel disease (ibd) have underlying immune dysregulation. immunosuppressive medications put them at risk of infection. this study assessed rates of recommended vaccinations and preventative screening in patients with ibd.nationwide data on patients diagnosed with ibd in the veterans health administration (vha) october 2004 to september 2014 were extracted. variation in vaccination, screenings, and risk of death by demographic factors (age group, gender) were estimated in bivariate and multivariable analyses.during the 10-year study period, 62,002 patients were treated for ibd. nonmelanoma skin cancer was found in 2.6%, and these patients more commonly accessed dermatology clinic (22.5% vs 15.2% ; chi - square = 66.6 ; df = 1 ; p < 0.0001). in total, 15% received dexa scans, especially women (34.7% vs 13.2% men ; chi - square = 1415.5 ; df = 1 ; p < 0.0001). eye manifestations were noted in 38.3% yet only 31% were referred to ophthalmology. abnormal pap smears were found for 15% of women < 65 (compared to 5% among normal patient populations) ; 34% had no record of pap smear in vha data. vaccination rates were modest : pneumococcal 39% ; tdap 23% ; hepatitis b 3% ; varicella and ppd < 0.5%. in an adjusted logistic regression model, 5-year mortality was lower among those using primary care prior to ibd diagnosis (odds ratio [or ] = 0.61 ; 95% ci 0.550.68).despite the current ibd guidelines, vaccination and preventative screening rates were unacceptably low among patients diagnosed with ibd. interventions such as education and increased awareness may be needed to improve these rates. |
in this issue of critical care, camporota and colleagues report interesting observations on the relationship between the initial gas exchange response to high - frequency oscillatory ventilation (hfov) and clinical outcomes. hfov seems ideally suited to protecting the lung during mechanical ventilation in patients with acute respiratory distress syndrome (ards). by delivering very small tidal volumes and allowing higher mean airway pressures however, the recently published oscillate (oscillation in ards treated early) and oscar (high - frequency oscillation in ards) trials - both of which postdate the article by camporota and colleagues - found that hfov failed to reduce mortality. in particular, oscillate raised questions of potential harm from hfov. in the face of these disappointing results, trialists and clinicians alike are left wondering how to reconcile theory to practice. a number of potential mechanisms for harm have been postulated, including (a) the deleterious effects of increased sedation, (b) hemodynamic embarrassment due to reduced right ventricular preload or increased right ventricular afterload, or (c) an increase in mechanical alveolar stress and strain within the baby lung. a critical factor influencing the importance of these various mechanisms may be the response of the individual patient to the application of high mean airway pressures. imaging studies have demonstrated profound heterogeneity in the extent of alveolar recruitment in response to increases in airway pressure. in patients with ' recruitable lung ', higher airway pressures increase the size of the functional ' baby lung ', reduce alveolar stress and strain, and may even reduce right ventricular afterload without significantly compromising right ventricular filling. in patients lacking ' recruitable lung ', higher mean airway pressures achieve the opposite : worsened alveolar stress and strain and increased right ventricular afterload. consequently, the potential for benefit or harm may depend critically on the degree of lung recruitment in response to increasing mean airway pressure. enter the interesting observations of camporota and colleagues, who found that in patients with moderate or severe ards who were placed on hfov by their attending physicians, the change in arterial partial pressure of oxygen / fraction of inspired oxygen (pao2/fio2) ratio 6 hours after hfov initiation was associated with 30-day survival. improvements in oxygenation were also associated with reductions in arterial partial pressure of carbon dioxide (paco2), particularly in patients with more severe respiratory failure, contradicting the widely held belief that hfov inevitably worsens respiratory acidosis. the oxygenation response to increased positive end - expiratory pressure (peep) is the product of a complex interplay between alveolar recruitment and cardiac output. classic physiological studies by dantzker and colleagues and lynch and colleagues demonstrated that increased peep reduces intrapulmonary shunt both by re - opening collapsed lung units and by reducing cardiac output. in the latter case, non - ventilated lung units are preferentially affected by reduced blood flow, possibly because of the effects of hypoxic pulmonary vasoconstriction. in the present study, camporoto and colleagues reported that cardiac output was unchanged ; thus, the short - term oxygenation response likely signifies lung recruitment. the authors speculate that the gas exchange response may be a useful predictor of the utility of hfov in the difficult - to - oxygenate patient and further propose that failure to demonstrate improved oxygenation after a 6-hour trial of hfov should prompt a consideration of alternative rescue modalities such as extracorporeal life support. it is difficult, however, to do anything more than speculate on the basis of these data. first, it is difficult to be certain whether the association between early improvements in oxygenation and reduced mortality arises from the effects of hfov - induced lung recruitment or simply represents the natural history of survivors (whose oxygenation will tend to improve with time) in contrast to non - survivors (whose oxygenation will tend to worsen with time). second, even if the improvements in oxygenation were directly related to lung recruitment, it is impossible to discern whether oxygenation ' responders ' have lower mortality than ' non - responders ' because ' responders ' have a less fatal form of ards or because they accrue greater benefit from hfov. finally, from these data in which all patients received hfov, it is impossible to know whether increases in mean airway pressure on conventional ventilation would have been equally effective. such issues can be resolved only by a carefully designed randomized trial of an open - lung ventilation strategy that stratifies patients by oxygenation response prior to random assignment. in the meantime, in light of the available evidence, hfov should usually be reserved for patients with refractory hypoxemia. when applying hfov, clinicians should consider monitoring lung recruitment (by oxygenation response or other means available) and right ventricular function (by echocardiography or other hemodynamic monitoring) to ensure that this unique mode of ventilation is achieving appropriate physiological goals in the individual patient. hfov : high - frequency oscillatory ventilation ; peep : positive end - expiratory pressure. | high - frequency oscillatory ventilation (hfov) seems ideal for lung protection in acute respiratory distress syndrome, but randomized trials have not shown a mortality reduction. the initial oxygenation response to hfov appears to be associated with survival. here, we discuss the mechanisms of oxygenation response to increases in airway pressure and the interpretation of the oxygenation response observed in the present study. |
the best - corrected visual acuity (bcva) was 20 / 200, od and 20 / 63, os. iops were 45 mmhg, od and 14 mmhg, os, with maximally tolerated medical treatment (topical brimonidine, topical dorzolamide / timolol fixed combination [dtfc ], and oral acetazolamide [250 mg, three times a day ]) a total of 2.5 mg/0.1 ml of bevacizumab was injected at two locations (7 and 9 o'clock, approximately 1.25 mg/0.05 ml each) using a 26-gauge needle. two days after the injection, the iop in the right eye was decreased to 21 mmhg. two months after the bevacizumab injections, new vessels were not observed in either the iris or the angle (fig. however, the iop was 28 mmhg in the right eye with medication, and ahmed glaucoma implant surgery was subsequently performed. two months after the surgery, the iop was controlled at 12 mmhg without any iop - lowering medication. a 63-year - old male who had been diagnosed with central retinal vein occlusion in the right eye visited the emergency room with ocular pain and decreased visual acuity. he had been using topical dtfc and brimonidine twice a day for three months because of the iop elevation after intravitreal injection of triamcinolone acetonid to control the macular edema. with medication, the iop, od had been controlled below 20 mmhg for three months. at the time of the emergency room visit, the patient 's vision was hand movement, and the iop was 48 mmhg in the right eye. slit lamp examination of the right eye disclosed corneal edema and gross hyphema. iris neovascularization was suspected in the inferior iris although it could not be confirmed due to corneal edema (fig. subconjunctival injections, 3.75 mg/0.15 ml in total, were completed at the 5, 8, and 11 o'clock areas. one day later, iop, od decreased to 32 mmhg, and the cornea was much clearer. two days after the injections, iris neovascularization was observed only on the margin of the pupil (fig. the iop, od was 29 mmhg. with the conditions much more amicable for surgery, ahmed glaucoma implant surgery was performed on the right eye on the fourth day after the subconjunctival injections. preoperative iop, od was consistently 29 mmhg. although the iop was decreased to 15 mmhg at the next surgery, vigorous iris neovascularization was noted in the inferior iris (fig. repeat subconjunctival injections of bevacizumab (1.25 mg/0.05 ml) were performed on this day. three weeks after surgery, the iop, od was 17 mmhg, and no neovascularization was visible on the iris (fig.. a 39-year - old male with type i diabetes, who had been previously diagnosed with bilateral proliferative diabetic retinopathy associated with nvg in his left eye, presented with visual disturbance in the left eye. ophthalmic examination showed a bcva of 20 / 25, od, and 20 / 32, os. the iop was 11 mmhg, od and 38 mmhg, os despite treatment with both topical dtfc and brimonidine twice a day in the left eye. slit lamp examination revealed neovascularization of the iris at the pupillary margin and neovascularization of the inferior, nasal, and temporal anterior chamber angle in the left eye (fig. a subconjunctival injection of bevacizumab (2.5 mg/0.1 ml) was performed in the left eye. in the following days after the injection, a significant regression of the neovascularization was observed in the angle (fig. 3b and 3c), and the iop, os was decreased to 19 mmhg. three months later, the iop, os was maintained at 14 mmhg with no neovascularizations visible in either the iris or angle. the best - corrected visual acuity (bcva) was 20 / 200, od and 20 / 63, os. iops were 45 mmhg, od and 14 mmhg, os, with maximally tolerated medical treatment (topical brimonidine, topical dorzolamide / timolol fixed combination [dtfc ], and oral acetazolamide [250 mg, three times a day ]) a total of 2.5 mg/0.1 ml of bevacizumab was injected at two locations (7 and 9 o'clock, approximately 1.25 mg/0.05 ml each) using a 26-gauge needle. two days after the injection, the iop in the right eye was decreased to 21 mmhg. two months after the bevacizumab injections, new vessels were not observed in either the iris or the angle (fig. however, the iop was 28 mmhg in the right eye with medication, and ahmed glaucoma implant surgery was subsequently performed. two months after the surgery, the iop was controlled at 12 mmhg without any iop - lowering medication. a 63-year - old male who had been diagnosed with central retinal vein occlusion in the right eye visited the emergency room with ocular pain and decreased visual acuity. he had been using topical dtfc and brimonidine twice a day for three months because of the iop elevation after intravitreal injection of triamcinolone acetonid to control the macular edema. with medication, the iop, od had been controlled below 20 mmhg for three months. at the time of the emergency room visit, the patient 's vision was hand movement, and the iop was 48 mmhg in the right eye iris neovascularization was suspected in the inferior iris although it could not be confirmed due to corneal edema (fig. subconjunctival injections, 3.75 mg/0.15 ml in total, were completed at the 5, 8, and 11 o'clock areas. one day later, iop, od decreased to 32 mmhg, and the cornea was much clearer. two days after the injections, iris neovascularization was observed only on the margin of the pupil (fig. the iop, od was 29 mmhg. with the conditions much more amicable for surgery, ahmed glaucoma implant surgery was performed on the right eye on the fourth day after the subconjunctival injections. preoperative iop, od was consistently 29 mmhg. although the iop was decreased to 15 mmhg at the next surgery, vigorous iris neovascularization was noted in the inferior iris (fig.. repeat subconjunctival injections of bevacizumab (1.25 mg/0.05 ml) were performed on this day. three weeks after surgery, the iop, od was 17 mmhg, and no neovascularization was visible on the iris (fig. a 39-year - old male with type i diabetes, who had been previously diagnosed with bilateral proliferative diabetic retinopathy associated with nvg in his left eye, presented with visual disturbance in the left eye. ophthalmic examination showed a bcva of 20 / 25, od, and 20 / 32, os. the iop was 11 mmhg, od and 38 mmhg, os despite treatment with both topical dtfc and brimonidine twice a day in the left eye. slit lamp examination revealed neovascularization of the iris at the pupillary margin and neovascularization of the inferior, nasal, and temporal anterior chamber angle in the left eye (fig. 3a). a subconjunctival injection of bevacizumab (2.5 mg/0.1 ml) was performed in the left eye. in the following days after the injection, a significant regression of the neovascularization was observed in the angle (fig. 3b and 3c), and the iop, os was decreased to 19 mmhg. three months later, the iop, os was maintained at 14 mmhg with no neovascularizations visible in either the iris or angle. we reported on three eyes with nvg where iris or angle neovascularization regressed rapidly after subconjunctival injections of bevacizumab with accompanying iop reduction. regression of the iris or angle neovascularization allowed for successful ahmed glaucoma implant surgery, which might have otherwise been complicated with intraoperative and postoperative intracameral bleeding. recently, intravitreal [1 - 6 ] or intracameral injections of bevacizumab have emerged as a new option in the treatment of nvg. they resulted in a rapid regression of the iris and angle neovascularization, which impeded the progression of peripheral anterior synechia in the period before the effect of prp occurs. however, intravitreal and intracameral injections could be associated with the risk of infectious endophthalmitis, vitreous hemorrhage, retinal detachment, and further iop elevation [9 - 11 ]. this method is now often used for the treatment of corneal neovascularization or to reduce corneal graft rejection. since subconjucntival bevacizumab injections for nvg are yet to be a widely practiced procedure, there is no established standardized amount of bevacizumab that is recommended for each injection. a number of reports have demonstrated a dramatic therapeutic effect of this agent with this particular concentration [16 - 19 ]. since we focused on the difference in the route of entry (intravitreal vs. subconjunctival), and not the minimal effective volume of the drug, the same amount known to be effective for intravitreal injections was used in this study. when 0.05 ml was injected, an injection bleb was created near the limbus across the 2 o'clock hour width. an amount greater than 0.05 ml expanded the bleb to the posterior direction, which was speculated not to have an additional effect in dispersing the bevacizumab into the anterior chamber or the iris. we injected the bevacizumab at the near - limbal location which was close to the neovascularization of the iris (nvi). it has been suggested that macromolecules may diffuse through the sclera and directly into the iris after subconjunctival injections. therefore, in eyes with massive nvi distributed in wide regions of the iris, multiple injections of bevacizumab were performed. in eyes with localized, small degrees of nvi nonetheless, our cases show that it is still useful in preventing the aggravation of the disease during the time window before applying further treatment such as prp and filtering surgery. moreover, associated iop - lowering and decrease of hyphema, or corneal edema allows for prompt prp. the regression of iris or angle neovascularization also results in conditions that are much more amicable for surgery. in conclusion, our cases demonstrate that subconjunctival bevacizumab injections can be potentially useful for treatment of nvg as an initial treatment before prp and/or filtering surgery. this maneuver may be particularly effective in patients presenting with high iop or vigorous iris vascularization where intravitreal or intracameral injections of bevacizumab may be complicated by further iop elevation. | to describe three cases of neovascular glaucoma (nvg) where iris or angle neovascularization regressed remarkably after subconjunctival bevacizumab injections used as the initial treatment before pan retinal photocoagulation (prp) and/or filtering surgery. three consecutive nvg patients whose intraocular pressure (iop) was not controlled with maximal medication were offered an off - label subconjunctival injection of bevacizumab (2.5 - 3.75 mg/0.1 - 0.15 ml, avastin). bevacizumab was injected into the subconjunctival space close to the corneal limbus in two or three quadrants using a 26-gauge needle. serial anterior segment photographs were taken before and after the injection. following subconjunctival injection of bevacizumab, iris or angle neovascularization regressed rapidly within several days. such regression was accompanied by lowering of iop in all three cases. the patients underwent subsequent prp and/or filtering surgery, and the iop was further stabilized. our cases demonstrate that subconjunctival bevacizumab injection can be potentially useful as an initial treatment in nvg patients before laser or surgical treatment. |
diverticula of the jejunum and ileum (excluding meckel 's diverticulum) are rare clinicopathological entities and most patients are asymptomatic. due to the rarity of the condition, the diagnosis is difficult and often delayed until complications arise. kayexalate (sodium polystyrene sulfonate) in sorbitol is used frequently for treatment of hyperkalemia and has been associated with colonic necrosis and upper gastrointestinal injuries in a subset of uremic patients. necrosis of the gastrointestinal (gi) tract due to kayexalate in sorbitol is underrecognized by pathologists as well as clinicians. small intestinal damage related to kayexalate has been described only infrequently in the terminal ileum. we report a case of jejunal diverticulosis with mucosal injury and diverticulitis in a uremic patient treated with kayexalate. to our knowledge the two conditions and the potential role of kayexalate in the pathogenesis of diverticulitis are discussed. a 87-year - old male patient under dialysis for a terminal chronic renal insufficiency was admitted to the surgery department for correction of an occlusion located at the vascular access of his left upper limb fistulae. there was a history appendectomy, cholecystectomy, radical prostatectomy and of ischemic cardiopathy with instable angor that had been relieved by stenting of the left circumflex coronary artery placed 2 months earlier. the patient received sorbisterit - calcium (calcium - polystyrol) with sorbitol among other medications. during the current admission, he developed abdominal pain that was more severe in the right hemi - abdomen. an abdominal ct scan was performed and revealed a small intestinal diverticulosis with 5 giant diverticula (fig. there was an infiltration of mesenteric fat around 2 diverticula measuring 5.5 and 3 cm, which was suggestive of diverticulitis., an exploratory laparotomy was performed with resection of a 25-cm - long jejunal segment (fig. the serosal surface was partially covered with a fibrinous exudate but there was no evidence of perforation. the smallest measured 4 3.5 1.2 cm and the largest 6.5 4.5 4.4 cm (fig. histological examination showed that the diverticula were in fact pseudo - diverticula, which consisted of mucosa and muscularis mucosae invaginating through the muscularis propria (fig. however, some pseudo - diverticula also contained some thin and discontinuous smooth muscle bundles in addition to the muscularis mucosae (fig. multiple ulcerations of various depth and areas of transmural necrosis were seen in most diverticula (fig. numerous irregular, sharply angulated (broken - glass like) crystals were seen adherent on the mucosal side and on the ulcerations (fig. they were basophilic on routine stain, with a mosaic or striped pattern, and stained bright red on periodic acid schiff (pas) stain. they were also found in the necrotic wall, and rarely in the exudate on the peritoneal surface consistent with microscopic perforations. the serosal surface was partially covered with a fibrinous exudate but there was no evidence of perforation. the smallest measured 4 3.5 1.2 cm and the largest 6.5 4.5 4.4 cm (fig. histological examination showed that the diverticula were in fact pseudo - diverticula, which consisted of mucosa and muscularis mucosae invaginating through the muscularis propria (fig. however, some pseudo - diverticula also contained some thin and discontinuous smooth muscle bundles in addition to the muscularis mucosae (fig. multiple ulcerations of various depth and areas of transmural necrosis were seen in most diverticula (fig. numerous irregular, sharply angulated (broken - glass like) crystals were seen adherent on the mucosal side and on the ulcerations (fig. they were basophilic on routine stain, with a mosaic or striped pattern, and stained bright red on periodic acid schiff (pas) stain. they were also found in the necrotic wall, and rarely in the exudate on the peritoneal surface consistent with microscopic perforations. to our knowledge, this is the first description of an association between jejunal ulcerated diverticulitis and kayexalate intake in a uremic patient. in this unusual case, kayexalate might be just an innocent bystander but could also be on the other hand implicated to some extent in the pathogenesis of the mucosal injury and diverticulitis. before discussing the possible role of kayexalate in the pathogenesis of the diverticulitis, we will first review the two conditions (ileo - jejunal diverticulosis and kayexalate related gi tract injury) separately. diverticula of the jejunum and ileum are rare clinical entities in contrast to meckel 's diverticulum, which is fairly common [1, 4, 5 ]. reported incidence of ileo - jejunal diverticula ranges from 0.3 to 4.5% in autopsy series and from 0.5 to 2.3% in radiological studies and depends largely on examination technique. highest incidence was found either when filling the small bowel with water under moderate pressure in autopsy studies or when using enteroclysis in radiological studies. they are more frequently encountered in the elderly, with a peak incidence in the sixth and seventh decades, and have a slight male predominance. they are preferentially localized to the jejunum (80%), to a lesser degree to the ileum (15%) and sometimes they involve both parts (5%). they can measure from a few millimeters up to more than 10 cm and tend to be more numerous and larger in the proximal jejunum [7, 8 ], such as in our case. strictly speaking, in most instances they are not true diverticula such as meckel 's diverticula, which comprise all parts of the bowel wall, but they are pseudo - diverticula formed by herniation of mucosa and submucosa through the muscular layer of the bowel wall on the mesenteric border of the bowel where the arteries enter the intestine. histologically they are lined by mucosa, muscularis mucosae and submucosa and lack a muscularis propria. in contrast, meckel 's diverticula are located on the side opposed to the mesentery, are delineated by mucosa, submucosa and muscularis propria and may contain heterotopic mucosa. although they can be congenital the vast majority is acquired [8, 9 ]. the cause of small bowel diverticulosis seems to be motor dysfunction of the smooth muscle or the myenteric plexus. the resulting intestinal dyskinesia might increase intraluminal pressure that will push the mucosa and submucosa through the weakest site of the bowel wall, which are the entry points of the blood vessels from the mesentery [8, 9, 10 ]. the predominance of diverticula in the jejunum is attributed to the greater diameter of the penetrating jejunal arteries. the diverticula usually remain asymptomatic (over 80%) and become evident as an incidental finding at the time of surgery, during a radiographic examination or at autopsy. their clinical importance is based on the fact that like colonic diverticulosis they may cause serious complications, such as infection, hemorrhage, intestinal obstruction and ileovesical fistula. complications related to the diverticula occur in about 15 to 20% of patients and complications or symptoms requiring surgery have been reported to occur in up to 10%. the most frequent acute complication of the jejunoileal diverticula is probably diverticulitis (with or without perforation), occurring in 2.3 to 6.4% of cases. the occurrence of gi necrosis due to kayexalate in sorbitol is well documented [2, 3, 11, 12, 13, 14, 15 ] and supported by experimental evidence. however, the true incidence of colonic necrosis after kayexalate intake is unknown. gerstmann. noted a 0.27% overall incidence, with a higher (1.8%) incidence in the postoperative period. factors that contribute to this entity include uremia, hypovolemia, peripheral vascular disease, and immunosuppressive therapy. one of the first studies from lillemoe. reported five uremic patients who developed catastrophic colonic necrosis that was associated temporally with the use of kayexalate in sorbitol and contributed to death in four of the five patients. that study also provided experimental evidence implicating sorbitol rather than kayexalate as the agent responsible for colonic necrosis in a rat model. no pathologic changes developed in rats receiving enemas of kayexalate in water, but transmural colonic necrosis developed in six of 10 rats receiving sorbitol enemas. production of prostaglandins may be stimulated by sorbitol and could play a part in causing mucosal injury. previous reports of gi injury associated with administration of kayexalate in sorbitol have focused almost exclusively on colonic or ileocolonic necrosis in patients with hyperkalemia [3, 11, 12, 13, 14, 15 ]. more recently, abraham. have shown that kayexalate can also induce upper gi tract injury. in the differential diagnosis, kayexalate crystals also need to be distinguished histologically from cholestyramine crystals that look very similar. the distinction is important since, in contrast to kayexalate, cholestyramine has not been associated with gi tract injury to date. both consist of irregular, sharply angulated (broken - glass like) fragments of an amorphous substance, but the former are slightly basophilic on routine stain with a mosaic or striped pattern. the latter appear bright red on routine stain and are more homogeneous, without a mosaic or striped pattern (fig. it can be difficult to define the role of kayexalate in sorbitol in the development of mucosal injury and diverticulitis in elderly patients who presents with multiple medical problems and who are also at risk for ischemic complications. in our case, while it is possible that kayexalate might be just an innocent bystander in the pathogenesis of the diverticulitis, we believe that kayexalate likely played a role in the diverticulitis and mucosal injury either as a causative agent or as a cofactor. the pattern of ulceration and diverticulitis present in our case is not the one that is usually seen in diverticulitis in the colon or in a meckel 's diverticulum. the ulcerations were often associated with wall necrosis and there was mild acute inflammation which might point to a local ischemia. in contrast, the mucosae next to the diverticulitis did not show any sign of ischemia, which suggests that bowel segment ischemia is not the cause of the diverticulitis. rashid and hamilton reported similar findings to ours in their study about colonic specimens from nine patients with kayexalate - sorbitol induced colonic necrosis. they described in the involved segments mucosal ulceration, dusky mucosal coloration, mural oedema, serosal fibrinous exudate, mucosal or transmural necrosis and perforation with luminal kayexalate crystals [3, 11 ]. in the gi tract, diverticula are likely to be sites vulnerable to kayexalate injury since the content in the lumen including kayexalate crystals and sorbitol is trapped inside the diverticula and is more likely to stay in contact with the mucosa for prolonged time. the same is also true for any mechanical factors such as tumors or other obstructing lesions that delay gi transit and prolong contact between the mucosa and sorbitol, hence predisposing to injury. furthermore, impairment of blood - flow by the pseudodiverticula, which compress the branches of the mesenteric blood vessels at the site of passage through the muscularis propria, may add additional stress to the mucosa. since the pseudodiverticula wall is also significantly thinner than the normal bowel wall, the risk of perforation and peritonitis is also increased. in conclusion, in addition to the more commonly reported risk of colonic necrosis, kayexalate in sorbitol could induce injury to other parts of the gi tract, particularly in sites where a prolonged contact with the mucosa might be expected. although the histologically visible kayexalate crystals are not directly responsible for mucosal damage, their recognition as a marker for the patient 's ingestion of kayexalate in sorbitol is a clue in providing the correct diagnosis in patients with mucosal injury, which is essential for appropriate treatment. diverticula of the jejunum and ileum are rare clinical entities in contrast to meckel 's diverticulum, which is fairly common [1, 4, 5 ]. reported incidence of ileo - jejunal diverticula ranges from 0.3 to 4.5% in autopsy series and from 0.5 to 2.3% in radiological studies and depends largely on examination technique. highest incidence was found either when filling the small bowel with water under moderate pressure in autopsy studies or when using enteroclysis in radiological studies. they are more frequently encountered in the elderly, with a peak incidence in the sixth and seventh decades, and have a slight male predominance. they are preferentially localized to the jejunum (80%), to a lesser degree to the ileum (15%) and sometimes they involve both parts (5%). they can measure from a few millimeters up to more than 10 cm and tend to be more numerous and larger in the proximal jejunum [7, 8 ], such as in our case. strictly speaking, in most instances they are not true diverticula such as meckel 's diverticula, which comprise all parts of the bowel wall, but they are pseudo - diverticula formed by herniation of mucosa and submucosa through the muscular layer of the bowel wall on the mesenteric border of the bowel where the arteries enter the intestine. histologically they are lined by mucosa, muscularis mucosae and submucosa and lack a muscularis propria. in contrast, meckel 's diverticula are located on the side opposed to the mesentery, are delineated by mucosa, submucosa and muscularis propria and may contain heterotopic mucosa. although they can be congenital the vast majority is acquired [8, 9 ]. the cause of small bowel diverticulosis seems to be motor dysfunction of the smooth muscle or the myenteric plexus. the resulting intestinal dyskinesia might increase intraluminal pressure that will push the mucosa and submucosa through the weakest site of the bowel wall, which are the entry points of the blood vessels from the mesentery [8, 9, 10 ]. the predominance of diverticula in the jejunum is attributed to the greater diameter of the penetrating jejunal arteries. the diverticula usually remain asymptomatic (over 80%) and become evident as an incidental finding at the time of surgery, during a radiographic examination or at autopsy. their clinical importance is based on the fact that like colonic diverticulosis they may cause serious complications, such as infection, hemorrhage, intestinal obstruction and ileovesical fistula. complications related to the diverticula occur in about 15 to 20% of patients and complications or symptoms requiring surgery have been reported to occur in up to 10%. the most frequent acute complication of the jejunoileal diverticula is probably diverticulitis (with or without perforation), occurring in 2.3 to 6.4% of cases. the occurrence of gi necrosis due to kayexalate in sorbitol is well documented [2, 3, 11, 12, 13, 14, 15 ] and supported by experimental evidence. however, the true incidence of colonic necrosis after kayexalate intake is unknown. noted a 0.27% overall incidence, with a higher (1.8%) incidence in the postoperative period. factors that contribute to this entity include uremia, hypovolemia, peripheral vascular disease, and immunosuppressive therapy. one of the first studies from lillemoe. reported five uremic patients who developed catastrophic colonic necrosis that was associated temporally with the use of kayexalate in sorbitol and contributed to death in four of the five patients. that study also provided experimental evidence implicating sorbitol rather than kayexalate as the agent responsible for colonic necrosis in a rat model. no pathologic changes developed in rats receiving enemas of kayexalate in water, but transmural colonic necrosis developed in six of 10 rats receiving sorbitol enemas. production of prostaglandins may be stimulated by sorbitol and could play a part in causing mucosal injury. previous reports of gi injury associated with administration of kayexalate in sorbitol have focused almost exclusively on colonic or ileocolonic necrosis in patients with hyperkalemia [3, 11, 12, 13, 14, 15 ]. more recently, abraham. have shown that kayexalate can also induce upper gi tract injury. in the differential diagnosis, kayexalate crystals also need to be distinguished histologically from cholestyramine crystals that look very similar. the distinction is important since, in contrast to kayexalate, cholestyramine has not been associated with gi tract injury to date. both consist of irregular, sharply angulated (broken - glass like) fragments of an amorphous substance, but the former are slightly basophilic on routine stain with a mosaic or striped pattern. the latter appear bright red on routine stain and are more homogeneous, without a mosaic or striped pattern (fig. it can be difficult to define the role of kayexalate in sorbitol in the development of mucosal injury and diverticulitis in elderly patients who presents with multiple medical problems and who are also at risk for ischemic complications. in our case, while it is possible that kayexalate might be just an innocent bystander in the pathogenesis of the diverticulitis, we believe that kayexalate likely played a role in the diverticulitis and mucosal injury either as a causative agent or as a cofactor. the pattern of ulceration and diverticulitis present in our case is not the one that is usually seen in diverticulitis in the colon or in a meckel 's diverticulum. the ulcerations were often associated with wall necrosis and there was mild acute inflammation which might point to a local ischemia. in contrast, the mucosae next to the diverticulitis did not show any sign of ischemia, which suggests that bowel segment ischemia is not the cause of the diverticulitis. rashid and hamilton reported similar findings to ours in their study about colonic specimens from nine patients with kayexalate - sorbitol induced colonic necrosis. they described in the involved segments mucosal ulceration, dusky mucosal coloration, mural oedema, serosal fibrinous exudate, mucosal or transmural necrosis and perforation with luminal kayexalate crystals [3, 11 ]. in the gi tract, diverticula are likely to be sites vulnerable to kayexalate injury since the content in the lumen including kayexalate crystals and sorbitol is trapped inside the diverticula and is more likely to stay in contact with the mucosa for prolonged time. the same is also true for any mechanical factors such as tumors or other obstructing lesions that delay gi transit and prolong contact between the mucosa and sorbitol, hence predisposing to injury. furthermore, impairment of blood - flow by the pseudodiverticula, which compress the branches of the mesenteric blood vessels at the site of passage through the muscularis propria, may add additional stress to the mucosa. since the pseudodiverticula wall is also significantly thinner than the normal bowel wall, the risk of perforation and peritonitis is also increased. in conclusion, in addition to the more commonly reported risk of colonic necrosis, kayexalate in sorbitol could induce injury to other parts of the gi tract, particularly in sites where a prolonged contact with the mucosa might be expected. although the histologically visible kayexalate crystals are not directly responsible for mucosal damage, their recognition as a marker for the patient 's ingestion of kayexalate in sorbitol is a clue in providing the correct diagnosis in patients with mucosal injury, which is essential for appropriate treatment. | small intestine diverticulosis is a rare entity that is asymptomatic in the majority of cases. however, it may cause serious complications, such as infection, hemorrhage, intestinal obstruction and diverticulitis. kayexalate (sodium polystyrene sulfonate) in sorbitol has been associated with colonic necrosis and less frequently with upper gastrointestinal injuries in a subset of uremic patients treated for hyperkalemia. we report a case of jejunal diverticulosis with mucosal injury and diverticulitis in a uremic patient treated with kayexalate and discuss the potential role of kayexalate in the pathogenesis of diverticulitis. |
the study of the biological foundations of speech and language processing is a lively and controversial area of research. broadly speaking, there are three areas of biology that are widely discussed : evolutionary biology (under which one can include evo devo as well as comparative ethology), genetics, and neurobiology. all these areas of investigation are stimulating vigorous debate (e.g., rice 1996, 1997 ; pinker 1999 ; jackendoff 2003 ; chomsky 2007 ; larson. 2010 ; fitch 2010 ; disciullo and boeckx 2011 ; maggie and gibson 2012). notwithstanding the enthusiasm for the questions and controversies, the empirical basis of much of the research is relatively thin at least if one is looking at evolutionary biology and genetics. the evolutionary biologist richard lewontin has argued that because we do not have access to earlier forms at least with respect to overt, measurable, and quantifiable behavior or output, since the individuals are dead and their central nervous systems inaccessible basically all accounts remain post hoc narrative interpretations of potential evolutionary scenarios, often called just so stories borrowing from the author rudyard kipling, totally unsatisfying from a mechanistic, explanatory point of view (lewontin 1998). the spectrum of evolutionary scenarios that are entertained in this literature is wide (e.g., we first imitated animal sounds, or first made noises of cooperation, or first felt a need to gossip about conspecifics, or social motivation drives changes in the system, etc.), and the stories range from the plausible to the charming to the ridiculous (in part reviewed in pinker and bloom 1992 ; for current perspectives, see larson. recent considerations from evolutionary developmental biology provide a new angle (chomsky 2010) ; nevertheless, these are largely data about structural properties of the biological system and structural similarities across species and we remain largely in the dark about what putatively early functional forms of linguistic representation or communication did in fact look like. this area of research holds significant promise, but there are as yet no major results that one can point to as foundational for an understanding of the foundations of representation and computation in language. a different angle on evolution, comparative ethology, has a relatively rich body of data, namely the extensive research on animal communication and cognition, but the results remain puzzling (and often equally speculative) in light of the system that is ultimately under investigation, human speech and language processing. this work is, to be sure, a potentially highly informative source of data on the biological infrastructure underpinning the human system ; in particular, as researchers converge on what are the kinds of questions about speech / language that one can plausibly investigate in other species, this work will become increasingly influential. for example, it stands to reason that attributes of the input and output systems (i.e., sensory and motor processing) can be profitably tested in nonhuman preparations, on the view that much of the machinery is highly conserved across evolution (e.g., hauser 1998). on the other hand, there are aspects of language processing that appear highly idiosyncratic, and it is not at all obvious how study of nonhuman species will inform us in this regard (for example, the operations underlying inflectional and derivational morphology, the representation of structurally conditioned long - distance relationships between reflexives and their antecedents, the nature of the words / concepts that are the atoms of language, and so on). one strategy that has yielded results in comparative ethological research is focused on identifying the inventory of elementary operations underlying language processing. assuming (reasonably) that the broader domains we use to talk about language processing (e.g., phonology, syntax, semantics) can be decomposed into more elementary operations (say, concatenation, labeling, construction of a constituent ; see poeppel & embick 2005), one can begin to assess whether this inventory of elementary operations is available, usable, and used in different animal species. (the strategy to decompose language processing into elementary subroutines is further discussed below.) that is to say, perhaps we are not prepared (or interested) to attribute syntax to some species, but we would not be offended to characterize that animal as using, say, a concrete example of research in this vein concerns recursion as a hypothesized primitive computational operation (hauser. research on the sensorimotor foundations of speech has tested parallels between human and animal systems. in summary, the evolution of speech and language is widely discussed and hotly debated, but the empirical basis of the arguments remains problematic. one issue that has been an obstacle to more rapid progress is that there is no agreement on how to evaluate evolutionary hypotheses in this context because there is serious disagreement about the nature of the linguistic system itself. it is fair to say that the vast majority of scholars assume that the system (the faculty of language) is quintessentially designed for communication. on this view, whatever parts make up the language system (phonology, morphology, syntax, lexical and compositional semantics), their major role is in the service of communication. therefore, precursors and evolutionary scenarios are constructed with communication (a concept that itself is rather underspecified) as the critical causal centerpiece. in contrast, some research arising from a more linguistic theoretical tradition (biolinguistics ; for a recent perspective, see chomsky 2007) does not presuppose that communication lies at the core ; rather, the computational and representational mental inventory is considered to be the key question. what is the what are putatively language - specific (domain specific) aspects (say morphology) and what aspects are arguably generic and likely to be shared with other species (say a long - term memory system) ? on this kind of view, some of the subroutines may be used for communication whereas others may be serving other purposes (e.g., externalization of thought). language is used for communication but not necessarily optimally designed for this purpose. indeed, some parts may make it more ill - suited for communication, e.g., the rampant ambiguity that exists in virtually every aspect of language processing. its properties and quirks may derive from other aspects of psychology, say being able to combine constituent thoughts, generating new internal representations, etc. it goes without saying that the construction of alternative evolutionary scenarios is closely linked to the underlying theories about the system itself, and as a consequence, the hypotheses that have been articulated differ sharply between scholars adopting such different foundational perspectives, i.e., language as an internal computational system that can be used for communication versus language as a system evolved to optimize communication. to adjudicate between these options, one will want to consider the arguments for the two theoretical positions as well as hope for a considerable enrichment of the available evidence. a different area of biological research in which the empirical basis is steadily growing is genetics. a number of papers in the special issue elegantly deal with the advances in this domain. just a few general remarks are in order, to situate the work and motivate the perspective that is outlined below. there is a small, albeit growing, amount of psycholinguistically motivated research focused on twin and adoption studies (stromswold 2001). the bulk of the data, however, comes, predictably, from the study of speech and language disorders, which offer a useful entry point into genetic analyses. with regard to the input and output systems, there is interesting research on dyslexia and stuttering (e.g., drayna, this volume, on stuttering) ; with regard to more central language systems, there is increasing interest in characterizing the genetics of specific language impairment (rice, this volume). the question that is briefly raised here concerns the relationship between potential results in genetic research and the online representation and computation of speech and language. specifically, let us assume that the genetic studies succeed perfectly and that we have available a detailed map of the genetic foundations of the linguistic phenotype. something very foundational is missing for causal models : an understanding of the actual circuits executing the processing. in particular, it is the brain part of people that does the speaking (and hearing, comprehending, and interpreting). therefore, to begin to have an explanatory understanding of how our genetic makeup underlies the structural and functional properties at the basis of speech and language to have satisfying mechanistic linking hypotheses between linguistic behavior and its genetic foundations the correlative data mediating between genetics and linguistics (and psychology / cognitive science, more generally) can not do without an understanding of how the genome / epigenome relates to the neuronal circuitry that is the implementational infrastructure for cognition. i take it to be the goal of this research direction to provide a mapping from genetics to neural circuitry to computational neuroscience to language processing. presumably, the yearning is to identify the genetic basis of the specific neural circuits (on whatever microscopic, mesoscopic, or macroscopic scale turns out to be relevant) that in turn constitute the basis for the operations that underpin speech and language, i.e., the representations and computations that lie at the foundation of the faculty of language. the third area of biological inquiry, neurobiology, is obviously closely related to evolutionary biology and genetics and inconceivable without these but has a more immediate available basis for testing hypotheses about the relevant biological infrastructure. perspective on some cognitive neuroscience foundations of speech perception and language comprehension, with the goal of providing examples of the kinds of circuits and operations that both evolutionary theory and genetic characterization ultimately must aim to capture. here i briefly sketch three ideas that illustrate at what level of analysis both evolutionary and genetic considerations might be able to make some substantial progress. first, a more up - to - date functional anatomy of language is briefly presented. second, some recent physiological data on timing are summarized, highlighting their potential relation to genetic work. third, we discuss the problem of aligning the analytic constituents of language research and neurobiology : the granularity mismatch problem.the pathways of speech and language : functional neuroanatomyuntil at least the late 1990s, consultation of a textbook on neurology, neuropsychology, neuroscience, linguistics, or any associated discipline showed the classical model of the brain basis of language : on a schematic of the left hemisphere were depicted broca 's area in the left inferior frontal lobe and wernicke 's area in the posterior superior temporal lobe. typically, the frontal area was associated with aspects of production (and later, syntactic aspects of language processing), and the temporal area was associated with perception (and later, aspects of meaning). the classical intuition rather behaviorist in nature in terms of its philosophical underpinning, i.e., language processing is reflexive was that the input (speech) was taken in and processed in the posterior areas and then shuttled forwards to frontal areas for generating an output (production). there was no notion of internal representation or computation in any mentalistic context ; rather, the most sophisticated models, such as wernicke 's, described the phenomena in terms of acoustic and motor images of words that were related to each other. it is now uncontroversial that this classical model, while immensely useful historically especially in a clinical context, is hopelessly underspecified biologically as well as linguistically (poeppel and hickok 2004). experimental research deriving from noninvasive recording (fmri, pet, eeg, meg) as well as from patient research has enriched our understanding of the anatomic basis of speech perception and language comprehension and production. there are many more cortical and subcortical regions that we now know to play integral roles beyond the two classically defined regions ; both left and right hemispheres are implicated across various tasks (e.g., voice recognition ; lexical semantics), and the analyses of what these various regions accomplish are now linked to linguistics, psychology, computational neuroscience, and other relevant approaches. below i very briefly and superficially discuss a few examples that illustrate some of the areas of progress.one new model attempting to capture these recent developments in neuroscience (in particular, data from imaging) as well as in linguistics and psycholinguistics postulates that there is a dual stream of information processing (hickok and poeppel 2004, 2007). (a related model that focuses on the analysis of meaning also emphasizes concurrent pathways ; lau. 2008.) focusing on speech recognition, an incoming signal 's acoustic attributes are initially analyzed in the dorsal and posterior superior temporal gyrus and superior temporal sulcus. importantly, these initial stages of perceptual analysis are computed bilaterally in the superior temporal cortex (binder. 2000), although the left and right cortical regions have important computational specializations (especially with regard to timing properties, as discussed in the next section) that contribute differentially to perceptual analysis (hickok and poeppel 2007 ; poeppel. a ventral pathway includes the superior temporal sulcus, the anterior temporal lobe, the middle temporal gyrus, inferior temporal sulcus, and perhaps the inferior temporal gyrus. the ventral stream underlies the mapping from sensory / phonological representations to lexical or conceptual representations (i.e., from sound to meaning). a dorsal pathway, including the sylvian parietotemporal area as well as the inferior frontal gyrus, anterior insula, and premotor cortex, provides the substrate for mapping from sensory / phonological representations to articulatory - motor representations. while early analysis is indisputably bilateral and much of the processing in the ventral stream is more bilateral than previously assumed (binder. 2000 ; hickok and poeppel 2007), the dorsal pathway appears to be more left - lateralized. in addition to positing multiple streams as well as multiple areas within each stream it must now also be acknowledged that each cortical region has fine - grained subdivisions. as we know from the study of visual areas, the laminar and columnar structure in the cortex offers intricate micro - circuitry underlying neural coding.adopting such a model has a variety of consequences for discussing language processing. for example, such a distributed functional anatomy of speech processing proper suggests there must be a representational format for speech sounds that facilitates the rapid transformation between articulatory and auditory coordinate systems. in other words, furthermore, it is obviously a new kind of research challenge to assign elementary functions or computational subroutines to these independently defined subregions. rather, it is taken for granted that successful language processing is a consequence of the coordinated action of these distributed elements ; each contributes some particular operation, and jointly they form the basis for recognizing words, combining words to form phrases, and so on. finally, this kind of architecture lends itself naturally to investigate both feedforward and feedback processing, the interaction of which lies at the center of most current theories of language processing. and, with respect to the larger issue at stake, one can now formulate hypotheses about the evolutionary trajectory of an anatomic subregion and its putative functional role. one can dissect the neural circuitry within a region and ask what aspect of the genetic toolbox might yield the observed anatomic cell and circuit structure. a particular region and its function may be implicated only in language processing or, alternatively, may show up in the processing of other cognitive domains. critically, the functional anatomy reflects that the problem is spatially decomposed into a number of subroutines. the quip that anatomy is destiny should at least be taken seriously as a research strategy in this domain of research. in the next section, we turn to a similar strategy in the time domain, breaking the processing problem down at different time scales.multi-time resolution processingone of the fascinating challenges of perceptual analysis is how to break the input stream which comes at a listener continuously (one might think of the arrow of time)into chunks of the appropriate size for further processing. in the last 10 years, both behavioral and neurophysiological research have revealed that processing of this type happens on multiple timescales concurrently (poeppel 2001, 2003 ; boemio. by analogy to breaking down the complex problem of language representation and processing in space (by creating a distributed functional anatomy with localized expertise in different regions), we now have good reason to conjecture that the perceptual system breaks the problem down along multiple timescales that have natural relations to the units of processing.one model that has received some attention and generated controversy, asymmetric sampling in time, ast ; poeppel (2003) posits that the input stream is sampled, simultaneously, at a relatively rapid rate (approximately 25 to 50 hz) and a significantly slower rate (below 8 hz). such sampling over quite distinct time scales would allow the processor to concurrently analyze syllabic / lexical information at one scale and much more rapid segmental / phonemic information at the other. both the rationale and the empirical support for this model have been reviewed extensively (poeppel. 2008 ; giraud and poeppel 2012), but recent research has begun thinking about this multi - time resolution notion in the context of genetic work. in particular, one of the more provocative hypotheses suggests that a selective dysfunction of the circuits for slow versus fast sampling could underlie deficits such as dyslexia. on one hypothesis, a selective dysfunction of the slow sampling scale leads to phonological representations that are poorly mapped to orthography, therefore leading to poor reading performance (rise time hypothesis ; goswami 2010). an alternative hypothesis argues that it is the compromised rapid processing system that underlies dyslexic performance. there is a long and complex history trying to map elementary processing routines to language performance. it is not yet clear how these theories will relate to both linguistic theory and neurobiology. however, it is encouraging that there exist new models that lend themselves to extensive empirical testing, whether in biological or genetic studies. the central message, again, is that a system that breaks processing down in the time domain offers a further, more granular view of what constitutes the substrate for evolutionary change in what might be a profitable target for genetic investigation. one reason this area of research holds promise is that there are clear possibilities for linking hypotheses across levels of analysis, as was espoused at the outset. 2009) has identified in great detail how local cell types and circuits of a certain type regulate gamma band activity, including for sensory processing ; this gamma band activity has been hypothesized to be important for certain analyses of speech (rapid sampling rate discussed above), and these rates in turn have been suggested to provide the basis for processing linguistic representations of a particular grain size (segmental and featural information). in short, the work in this domain stands a chance of being able to relate across levels genetics, the circuitry enabling specific computations, and the relevant cognitive mechanisms. a research program that outlines how oscillations mediate between spike trains at one end and speech signals at the other is provided in giraud and poeppel (2011).the granularity mismatch problemwe finally turn to the problem that lies at the very center of cognitive neuroscience, and we ask how a potential solution can inform both genetic research and evolutionary theorizing. suppose we ask a large number of neuroscientists to define what are the primitive elements (the alphabet) of neurobiology. a long list will be generated of putatively elementary units of anatomy and units of function. presumably, the list will include concepts such as neuron, synapse, oscillation, long - term potentiation, etc. there, the primitive elements will include, arguably, concepts such as distinctive feature, syllable, noun phrase, question formation, etc. now, it is the pretense of these fields of inquiry that there is a mapping between the elementary constituents of neurobiology and the elementary constituents of cognitive science. but if we are honest, it is quite clear that we do not have the vaguest idea of how the (empirically well - supported) elementary representations or processes from language (say distinctive feature) map to the (well - supported) elementary representations or processes from neuroscience (say dendritic spine) ! the fact of the matter is that the mapping from the biological substrates to cognitive representation and processing is simply not understood at a satisfying level. in some sense, this is simply a restatement of the philosophical mind body problem.in part, this problem comes from the fact that these domains are studied at very different levels of granularity. in linguistics, the concepts used to study the knowledge of language one has as an adult speaker / listener, its acquisition, and its processing are exceedingly fine grained. in contrast, in cognitive neuroscience research, the typical study seeks to understand phenomena at the level of where is syntax. this mismatch is not just practical the granularity mismatch problem but also speaks to the ontological commitments that different areas of research make to what they take to be the set of primitive elements (poeppel and embick 2005).is there a productive way forward ? clearly, we need suitable linking hypotheses. these are, incidentally, precisely the linking hypotheses that can potentially provide quite different targets for investigation for evolutionary theorizing and genetic experimentation. what will such linking hypotheses look like, and how can we begin to address the complicated relations between the primitives of biology and the primitives of cognitive science ? in earlier work on this problem, it is argued that linking hypotheses between speech / language and brain are most likely to bear fruit if they make use of computational analyses that appeal to generic computational subroutines (poeppel and embick 2005 ; smolensky and legendre 2006). the recommendation is to take a given domain of language processing, say word recognition, and decompose it into elements that could be plausibly mapped onto circuits that can be instantiated in nervous tissue. note that this is not an exercise in reductionism as it is typically understood. while the goal is indeed to determine the basic bits and pieces, the goal is not simply to map what we take to be linguistic primitives on to what we know to be biologically available constituents (say something like a mapping from word to neuron). in part, the problem lies in the fact that we do not know what the mapping should even be. maps on to dendrite, or neuron, or ensemble, or cortical column, or some heretofore unknown assembly of parts. reduction is a sensible game plan if the ontological structure of the underlying domain is known, but i contend that neurobiological too, is not yet understood at a level that licenses reduction in the standard sense. so, broadly speaking, the aim is to find generic representations / computations of the sort that can at the same time be constitutive of a linguistic representation or process and be a plausible element that can be instantiated in a neuronal circuit. the kinds of generic operations one might have in mind here include concepts such as concatenation (take an x and a y and generate a chain x y) or linearization. a different example might derived from the temporal analyses discussed above, i.e., how does the circuitry underlying gamma band responses related to the hypotheses about gamma mediate analysis in speech. there are interesting research programs going in these directions, so future empirical work will show whether this kind of strategy has legs. the pathways of speech and language : functional neuroanatomy until at least the late 1990s, consultation of a textbook on neurology, neuropsychology, neuroscience, linguistics, or any associated discipline showed the classical model of the brain basis of language : on a schematic of the left hemisphere were depicted broca 's area in the left inferior frontal lobe and wernicke 's area in the posterior superior temporal lobe. typically, the frontal area was associated with aspects of production (and later, syntactic aspects of language processing), and the temporal area was associated with perception (and later, aspects of meaning). the classical intuition rather behaviorist in nature in terms of its philosophical underpinning, i.e., language processing is reflexive was that the input (speech) was taken in and processed in the posterior areas and then shuttled forwards to frontal areas for generating an output (production). there was no notion of internal representation or computation in any mentalistic context ; rather, the most sophisticated models, such as wernicke 's, described the phenomena in terms of acoustic and motor images of words that were related to each other. it is now uncontroversial that this classical model, while immensely useful historically especially in a clinical context, is hopelessly underspecified biologically as well as linguistically (poeppel and hickok 2004). experimental research deriving from noninvasive recording (fmri, pet, eeg, meg) as well as from patient research has enriched our understanding of the anatomic basis of speech perception and language comprehension and production. there are many more cortical and subcortical regions that we now know to play integral roles beyond the two classically defined regions ; both left and right hemispheres are implicated across various tasks (e.g., voice recognition ; lexical semantics), and the analyses of what these various regions accomplish are now linked to linguistics, psychology, computational neuroscience, and other relevant approaches. below i very briefly and superficially discuss a few examples that illustrate some of the areas of progress. one new model attempting to capture these recent developments in neuroscience (in particular, data from imaging) as well as in linguistics and psycholinguistics postulates that there is a dual stream of information processing (hickok and poeppel 2004, 2007). (a related model that focuses on the analysis of meaning also emphasizes concurrent pathways ; lau. 2008.) focusing on speech recognition, an incoming signal 's acoustic attributes are initially analyzed in the dorsal and posterior superior temporal gyrus and superior temporal sulcus. importantly, these initial stages of perceptual analysis are computed bilaterally in the superior temporal cortex (binder. 2000), although the left and right cortical regions have important computational specializations (especially with regard to timing properties, as discussed in the next section) that contribute differentially to perceptual analysis (hickok and poeppel 2007 ; poeppel. a ventral pathway includes the superior temporal sulcus, the anterior temporal lobe, the middle temporal gyrus, inferior temporal sulcus, and perhaps the inferior temporal gyrus. the ventral stream underlies the mapping from sensory / phonological representations to lexical or conceptual representations (i.e., from sound to meaning). a dorsal pathway, including the sylvian parietotemporal area as well as the inferior frontal gyrus, anterior insula, and premotor cortex, provides the substrate for mapping from sensory / phonological representations to articulatory - motor representations. while early analysis is indisputably bilateral and much of the processing in the ventral stream is more bilateral than previously assumed (binder. 2000 ; hickok and poeppel 2007), the dorsal pathway appears to be more left - lateralized. in addition to positing multiple streams as well as multiple areas within each stream it must now also be acknowledged that each cortical region has fine - grained subdivisions. as we know from the study of visual areas, the laminar and columnar structure in the cortex offers intricate micro - circuitry underlying neural coding. for example, such a distributed functional anatomy of speech processing proper suggests there must be a representational format for speech sounds that facilitates the rapid transformation between articulatory and auditory coordinate systems. in other words, furthermore, it is obviously a new kind of research challenge to assign elementary functions or computational subroutines to these independently defined subregions. rather, it is taken for granted that successful language processing is a consequence of the coordinated action of these distributed elements ; each contributes some particular operation, and jointly they form the basis for recognizing words, combining words to form phrases, and so on. finally, this kind of architecture lends itself naturally to investigate both feedforward and feedback processing, the interaction of which lies at the center of most current theories of language processing. and, with respect to the larger issue at stake, one can now formulate hypotheses about the evolutionary trajectory of an anatomic subregion and its putative functional role. one can dissect the neural circuitry within a region and ask what aspect of the genetic toolbox might yield the observed anatomic cell and circuit structure. a particular region and its function may be implicated only in language processing or, alternatively, may show up in the processing of other cognitive domains. critically, the functional anatomy reflects that the problem is spatially decomposed into a number of subroutines. the quip that anatomy is destiny should at least be taken seriously as a research strategy in this domain of research. in the next section, we turn to a similar strategy in the time domain, breaking the processing problem down at different time scales. multi - time resolution processing one of the fascinating challenges of perceptual analysis is how to break the input stream which comes at a listener continuously (one might think of the arrow of time)into chunks of the appropriate size for further processing. in the last 10 years, both behavioral and neurophysiological research have revealed that processing of this type happens on multiple timescales concurrently (poeppel 2001, 2003 ; boemio. by analogy to breaking down the complex problem of language representation and processing in space (by creating a distributed functional anatomy with localized expertise in different regions), we now have good reason to conjecture that the perceptual system breaks the problem down along multiple timescales that have natural relations to the units of processing. one model that has received some attention and generated controversy, asymmetric sampling in time, ast ; poeppel (2003) posits that the input stream is sampled, simultaneously, at a relatively rapid rate (approximately 25 to 50 hz) and a significantly slower rate (below 8 hz). such sampling over quite distinct time scales would allow the processor to concurrently analyze syllabic / lexical information at one scale and much more rapid segmental / phonemic information at the other. both the rationale and the empirical support for this model have been reviewed extensively (poeppel. 2008 ; giraud and poeppel 2012), but recent research has begun thinking about this multi - time resolution notion in the context of genetic work. in particular, one of the more provocative hypotheses suggests that a selective dysfunction of the circuits for slow versus fast sampling could underlie deficits such as dyslexia. on one hypothesis, a selective dysfunction of the slow sampling scale leads to phonological representations that are poorly mapped to orthography, therefore leading to poor reading performance (rise time hypothesis ; goswami 2010). an alternative hypothesis argues that it is the compromised rapid processing system that underlies dyslexic performance. there is a long and complex history trying to map elementary processing routines to language performance. it is not yet clear how these theories will relate to both linguistic theory and neurobiology. however, it is encouraging that there exist new models that lend themselves to extensive empirical testing, whether in biological or genetic studies. the central message, again, is that a system that breaks processing down in the time domain offers a further, more granular view of what constitutes the substrate for evolutionary change in what might be a profitable target for genetic investigation. one reason this area of research holds promise is that there are clear possibilities for linking hypotheses across levels of analysis, as was espoused at the outset. 2009) has identified in great detail how local cell types and circuits of a certain type regulate gamma band activity, including for sensory processing ; this gamma band activity has been hypothesized to be important for certain analyses of speech (rapid sampling rate discussed above), and these rates in turn have been suggested to provide the basis for processing linguistic representations of a particular grain size (segmental and featural information). in short, the work in this domain stands a chance of being able to relate across levels genetics, the circuitry enabling specific computations, and the relevant cognitive mechanisms. a research program that outlines how oscillations mediate between spike trains at one end and speech signals at the other the granularity mismatch problem we finally turn to the problem that lies at the very center of cognitive neuroscience, and we ask how a potential solution can inform both genetic research and evolutionary theorizing. suppose we ask a large number of neuroscientists to define what are the primitive elements (the alphabet) of neurobiology. a long list will be generated of putatively elementary units of anatomy and units of function. presumably, the list will include concepts such as neuron, synapse, oscillation, long - term potentiation, etc. there, the primitive elements will include, arguably, concepts such as distinctive feature, syllable, noun phrase, question formation, etc. now, it is the pretense of these fields of inquiry that there is a mapping between the elementary constituents of neurobiology and the elementary constituents of cognitive science. but if we are honest, it is quite clear that we do not have the vaguest idea of how the (empirically well - supported) elementary representations or processes from language (say distinctive feature) map to the (well - supported) elementary representations or processes from neuroscience (say dendritic spine) ! the fact of the matter is that the mapping from the biological substrates to cognitive representation and processing is simply not understood at a satisfying level. in some sense, this is simply a restatement of the philosophical mind body problem. in part, this problem comes from the fact that these domains are studied at very different levels of granularity. in linguistics, the concepts used to study the knowledge of language one has as an adult speaker / listener, its acquisition, and its processing are exceedingly fine grained. in contrast, in cognitive neuroscience research, the typical study seeks to understand phenomena at the level of where is syntax. this mismatch is not just practical the granularity mismatch problem but also speaks to the ontological commitments that different areas of research make to what they take to be the set of primitive elements (poeppel and embick 2005). these are, incidentally, precisely the linking hypotheses that can potentially provide quite different targets for investigation for evolutionary theorizing and genetic experimentation. what will such linking hypotheses look like, and how can we begin to address the complicated relations between the primitives of biology and the primitives of cognitive science ? in earlier work on this problem, it is argued that linking hypotheses between speech / language and brain are most likely to bear fruit if they make use of computational analyses that appeal to generic computational subroutines (poeppel and embick 2005 ; smolensky and legendre 2006). the recommendation is to take a given domain of language processing, say word recognition, and decompose it into elements that could be plausibly mapped onto circuits that can be instantiated in nervous tissue. while the goal is indeed to determine the basic bits and pieces, the goal is not simply to map what we take to be linguistic primitives on to what we know to be biologically available constituents (say something like a mapping from word to neuron). in part, the problem lies in the fact that we do not know what the mapping should even be. maps on to dendrite, or neuron, or ensemble, or cortical column, or some heretofore unknown assembly of parts. reduction is a sensible game plan if the ontological structure of the underlying domain is known, but i contend that neurobiological too, is not yet understood at a level that licenses reduction in the standard sense. so, broadly speaking, the aim is to find generic representations / computations of the sort that can at the same time be constitutive of a linguistic representation or process and be a plausible element that can be instantiated in a neuronal circuit. the kinds of generic operations one might have in mind here include concepts such as concatenation (take an x and a y and generate a chain x y) or linearization. a different example might derived from the temporal analyses discussed above, i.e., how does the circuitry underlying gamma band responses related to the hypotheses about gamma mediate analysis in speech. there are interesting research programs going in these directions, so future empirical work will show whether this kind of strategy has legs. there are few more fascinating scientific questions than understanding the structure and function of the brain and precisely how it forms the basis for human cognition. the goal must be to develop an explanatory account, such that the circuits that are identified and studied provide a causal explanation for the representations and computations deployed in language comprehension and production. at the moment, it is fair to say that the research is by and large correlative, not explanatory. in imaging studies, we identify local brain activation and quantify the extent to which it correlates with some language task. in genetic studies, we correlate relatively broad patterns of the linguistic phenotype with genetic markers. in evolutionary studies, we construct scenarios over broad categories of experience, such as syntaxor even communication. the (admittedly brief and caricature - ish) argument presented here suggests that a more aggressive decomposition of these concepts specifically the putative primitives of neurobiology and of language research might provide a new and unconventional view to study both evolution and genetics. if the target of investigation is not a broad category such as syntax, but a narrow category that is plausibly instantiated in neural circuits such as, say, concatenation or rapid sampling, then one can imagine constructing a relatively narrow empirical test. moreover, one could begin to investigate in which way specific computational subroutines go wrong and lead to pathologies. there are some notable efforts in this direction especially in the research on specific language impairment (see, e.g., rice, this volume). overall, what is advocated is no more than to be a splitter, not a lumper. we now have excellent toolboxes to be theoretically well motivated, computationally explicit, and biologically plausible splitters (using the tools of linguistics, computational neuroscience, neurobiology) ; if we end up splitting into the appropriate granularity, it will provide fresh new ground for evolutionary theories and genetic and epigenetic characterization. | the paper argues that both evolutionary and genetic approaches to studying the biological foundations of speech and language could benefit from fractionating the problem at a finer grain, aiming not to map genetics to languageor even subdomains of language such as phonology or syntaxbut rather to link genetic results to component formal operations that underlie processing the comprehension and production of linguistic representations. neuroanatomic and neurophysiological research suggests that language processing is broken down in space (distributed functional anatomy along concurrent pathways) and time (concurrent processing on multiple time scales). these parallel neuronal pathways and their local circuits form the infrastructure of speech and language and are the actual targets of evolution / genetics. therefore, investigating the mapping from gene to brain circuit to linguistic phenotype at the level of generic computational operations (subroutines actually executable in these circuits) stands to provide a new perspective on the biological foundations in the healthy and challenged brain. |
bovine tuberculosis (btb) is not only serious animal and zoonotic disease that causes significant financial loss but also a public health hazard. while the main host of mycobacterium bovis, the causative agent of btb, is cattle, other animals, including humans, infection in humans occurs when unpasteurized milk (or derivatives) is consumed or when people are in contact with infected cattle. btb is a factor that undermines the development of the dairy and meat industry and international commerce. therefore, it is essential to control and eradicate this disease and an efficient vaccination strategy would help in the control of bovine tuberculosis. vaccination of cattle to control btb is particularly demanding in high - prevalence zones where it is economically unfeasible to slaughter animals. rationally attenuated, the advantage of using attenuated m. bovis strains is that they produce a large number of protective antigens, including those absent from bcg. thus, vaccination with live attenuated m. bovis can induce a stronger and persistent immune stimulation, conferring higher levels of protection against tuberculosis than bcg. these operons, which encode membrane and exported proteins, are highly conserved in pathogenic and nonpathogenic mycobacteria. in a previous study, we have demonstrated that the mce2 operon is essential for the survival of mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. in that study, we found that m. tuberculosis knockout in the mce2 operon replicated less than its parental strain in mouse organs after intratracheal inoculation of animals. our findings were consistent with previous observations supporting the involvement of mce operons in the host - pathogen interaction [2, 49 ]. in particular, in a recent study marjanovic. demonstrated that mice infected with an m. tuberculosis mutant in the mce2 operon survive longer than those infected with its parental and virulent strain, although the replication of both strains in organs was equivalent. the use of the m. tuberculosis mce2 strain as a vaccine confers better protection than bcg in both mice and guinea pigs challenged with a hypervirulent m. tuberculosis strain [11, 12 ]. m. bovis is closely related to m. tuberculosis, and both species are included in the m. tuberculosis complex. in addition, as both organisms can cause identical clinical disease in humans and are genetically extremely similar, it is likely that many of the virulence factors of m. bovis are the same as those of m. tuberculosis. based on these facts, we propose an m. bovis knockout strain in the mce2 operon as a candidate vaccine against btb. in order to determine the immunogenic properties of this candidate, in this study we characterized the immunological profile elicited in cattle by the m. bovis mce2 mutant strain towards m. bovis antigens and compared it with that elicited by its parental and virulent strain nctc 10772. to this end, we measured the cytokine mrna expression in peripheral blood mononuclear cells (pbmcs) and determined the lymphocyte subsets involved in recalling activation of pbmcs from cattle inoculated with the candidate vaccine in response to m. bovis antigens. we found that inoculation of cattle with the candidate vaccine stimulates both cd4 + and cd8 + t - cell responses to produce th1-associated cytokines. by using the gene knock - out system described by parish and stoker, we created a mutant strain of m. bovis carrying a unmarked - chromosomal deletion of the region spanning the mce2a - mce2b genes (figure 1(a)). the deletion was confirmed by pcr using primers that hybridize outside the deleted region. as shown in figure 1(b), an amplicon of 1,831 bp corresponding to the mce2a and mce2b genes and adjacent regions was obtained in the wild type strain, while in the mutant, the amplified dna fragment was of 177 bp and corresponded to the mce2a - mce2b locus. primers that hybridize in siga amplified the expected fragment in both strains, indicating the integrity of the dna samples used in the pcr reactions. the mutant strain was designated mbmce2. to evaluate the recall response to purified protein derivative (ppdb) of lymphocyte subsets in animals inoculated with either the candidate mbmce2 or its wild type parental strain (nctc 10772), we used a flow cytometry - based proliferation assay. in pbmcs isolated 15 and 90 days after infection (dpi), activation of cd4 + and cd8 + significantly increased upon stimulation with ppdb (figures 2(a) and 2(b)). after specific stimulation, the expression of il-2r in cd4 + cells, which indicates lymphocyte activation, increased along the time of infection (p < 0.01) with similar rates in both groups (figure 3(a)). the expression of il-2r in cd8 + cells also showed the same trend, but it was only significant in animals inoculated with the wild type strain (p < 0.05) (figure 3(b)). importantly, in pbmcs from animals before the infection, the percentages of cd4 + and cd8 + cells were not significantly altered upon ppdb stimulation, indicating that the animals used in this study were neither previously sensitized nor infected with m. bovis. in order to evaluate th1 and th2 immune responses in cattle inoculated with the candidate vaccine strain mbmce2, we assessed the cytokine expression profile in pbmcs by measuring cytokine mrnas after stimulating the cells with ppdb (figure 4). pbmcs obtained from cattle inoculated with mbmce2 at 15 dpi responded to ppdb stimulation by expressing 16.2-fold more ifn- mrna than cells from animals before inoculation. in contrast, a lower level of ifn- was detected in animals inoculated with the wild type strain (p < 0.05). however, at 90 dpi, the expression of ifn- increased in cattle inoculated with the wild type, while it showed only a slight reduction in those inoculated with the mutant. the expression of interleukin-2 (il-2) in pbmcs from both animal groups was significantly different from that in pbmcs from animals before the inoculation. however, the expression level of this cytokine was equivalent in both groups and maintained during the time points assayed. only the group inoculated with the mutant responded to ppdb stimulation with production of il-12 and tnf- (p < 0.0001). il-10 and stimulation with ppdb induced greater il-10 gene expression in animals inoculated with the mutant than in those inoculated with the wild type strain at both time points assayed (p < 0.005). at 15 dpi, the expression of il-4 was similar in both groups, while at 90 dpi, the level of il-4 was higher in the animals inoculated with the wild type strain (p < 0.005). the efficacy of bcg to protect against tuberculosis in humans has shown to be highly variable and depending on uncertain factors of the populations tested. field evaluation of the protective efficacy of bcg against bovine tuberculosis has indicated that bcg is not capable of protecting 100% of the animals [16, 17 ]. one possible explanation of this failure is that exposure to environmental mycobacteria compromises the protection efficacy of bcg in cattle, as it happens with the use of bcg in humans. to overcome the incomplete protection afforded by bcg, numerous vaccine strategies have been developed and tested in experimental vaccine assays. among them, prime - boost strategies combining single antigens and bcg have been shown to improve the protective efficacy of bcg significantly. inspections on the genomic sequences of m. bovis isolates and bcg strains have revealed that at least 16 regions of differences (rds) are absent from the bcg chromosomes [19, 20 ]. many of these rds encode for powerful and protective antigens [1, 21 ]. thus, an attractive alternative to the use of bcg as a veterinary vaccine is the development of attenuated m. bovis strains deleted in proteins that are nonantigenic but relevant for virulence. here, we describe the construction of a mutant strain of m. bovis and its immunological evaluation as a candidate attenuated live vaccine against btb. based on previous findings indicating that inactivation or deletion of the mce2 operon attenuates m. tuberculosis, we obtained an attenuated nonreverting m. bovis strain by deletion of two mce2 genes. importantly, since the experimental approach used to generate this mutant strain avoids the insertion of any selective and nonselective gene marker into the bacterial chromosome, we created this mutant in compliance with the criteria of tuberculosis vaccine development. another important aspect of this candidate vaccine strategy is that the absence of mce2a and mce2b proteins in the mutant strain could be used as marker to differentiate infected animals from vaccinated ones. given that there is no doubt about the essential protective role of cd4 + t cells to control tuberculosis infections, it is a condition that any candidate vaccine is capable of stimulating this cell population. therefore, the most effective vaccination strategies will be those that stimulate cd4 + t - cell responses to produce th1-associated cytokines. in accordance with these requirements, the candidate vaccine mce2 elicited strong t - cell responses in cattle, with activation of cd4 + following stimulation with the m. bovis - specific antigen ppdb. it has been demonstrated that cd8 + t cells are also required for immunity against tuberculosis in a variety of animal models as well as in humans [9, 2327 ]. in this regard, in the group of animals inoculated with the mutant strain, the activation of cd8 + t cells was sustained along the time of infection and increased at the last point assayed, but this increase was not statistically significant, likely due to the reduced number of animals included in the experiment. similarly, subcutaneous inoculation of cattle with mce2 induced activation of cd4 + and cd8 + following specific stimulation, but only the cd4 + t cells responded at significant levels (unpublished results). moreover, inoculation of the mutant strain in cattle induced the production of th1 expression of il-12 responses with production of il-2, il-12, tnf-, and ifn-. surprisingly, the level of and tnf- was higher in stimulated pbmcs from animals inoculated with the mutant than in those from animals inoculated with the wild type strain. these data indicate that, in cattle, mbmce2 induces a strong th1 response that is maintained for at least 90 days., who have shown that an m. tuberculosis mutant in the mce2 operon induces smaller amounts of proinflammatory cytokines (tnf-, il-6, and mcp-1) in raw cells than the wild type h37rv strain. however, considerable differences between the systems used in both studies could explain these disparities. in particular, these authors used an in vitro murine model to determine the cytokines produced after cell infection, while here we measured cytokines produced after antigen stimulation of total bovine pbmcs. although it is still unclear why the mutant strain elicited stronger proinflammatory response than the wild type strain, we speculate that the lack of mce2 lipid transporter could alter the m. bovis cell wall composition, which, in turn, could affect the host 's immune response elicited by the bacteria. production of the anti - inflammatory cytokine il-10 was early detected in animals inoculated with the mutant strain. in contrast, animals inoculated with the wild type strain showed low il-10 expression at 15 and 90 dpi. at 15 dpi, il-4 expression levels were similar in both groups, whereas at 90 dpi, the expression of this cytokine decreased in the wild type group. together, these results suggest that anti - inflammatory responses in animals inoculated with the mutant strain were generated to attenuate the adverse effect of an exacerbated inflammation in the host 's tissues driven by the highly expressed ifn-. the importance of the induction of multifunctional cd4 + t cells producing different cytokines simultaneously for protection against tuberculosis has been previously addressed in a mouse model. therefore, it will be of interest to carry out further studies to establish whether multifunctional cd4 + t cells are induced in mbmce2-vaccinated cattle. given that the immune responses between bovines and humans have been considered a determinant issue in the development of diagnostic tools and vaccines for both human and bovine tuberculosis, the results presented here make this mutant an attractive candidate vaccine to be tested against both bovine and human tuberculosis. here, we demonstrated that the lack of mce2 gene expression maintains the immunogenicity properties of m. bovis, favoring its ability to protect against bovine tuberculosis. although the results presented here make mce2 an attractive candidate vaccine, further studies addressing safety and innocuity concerns are necessary before testing this candidate in challenge assays. all cloning steps were performed in escherichia coli top10 (f - mcra(mrr - hsdrms - mcrbc) 80laczm15 lacx74 nupg reca1 arad139 (ara - leu)7697 gale15 galk16 rpsl(strr) enda1 -). e. coli were grown either in luria - bertani (lb) broth or on lb agar. when necessary, 50 g / ml kanamycin was added to the lb media. m. bovis strains were grown in middlebrook 7h9 medium supplemented with 0.05% tween 80 or middlebrook 7h10. middlebrook media were supplemented with albumin 0.5%, dextrose 0.4%, and 0.4% pyruvate (adp). to grow the knock - out m. bovis strain, middlebrook 7h10-adp was supplemented with either 20 g / ml kanamycin plus 50 g / ml x - gal or 2% sucrose. purification of plasmids and dna fragments was performed using the gfx micro plasmid prep kit (ge healthcare) and dna and gel band purification kit (ge healthcare), respectively, according to the manufacturer 's instructions. genomic regions of about 2 kb either upstream of mce2a or downstream of mce2b were obtained by pcr from m. bovis total dna by using the following pair of primers : 3mce2 and 5mce2 (table 1). the final delivery vector was generated by incorporation of the paci cassette from pgoal 17 into this last p2nil recombinant vector. the vector generated was pretreated with uv light (100 mj cm) to induce depurination and promote recombination. this uv - treated plasmid was used to electroporate the collection strain m. bovis nctc 10772. the unmarked mutant was obtained using a two - step strategy described previously, and mutant clones were identified by colony pcr, using the primers described in table 1. holstein - fresian calves (six months old) were inoculated intratracheally as described previously with 10 - 10 colony forming units (cfu) of the mutant mbmce2 (four animals) or its parental virulent strain m. bovis nctc 10772 (three animals). all experiments conformed to local and national guidelines on the use of experimental animals and category iii infectious organisms. all the animals used in this study were negative for ifn- by elisa assay (bovigam) and tuberculin skin test at the beginning of the experiments but positive by both assays at the end of the experiments (data not shown). blood samples were taken both at the beginning of the experiment for evaluation of preimmune status and at 15 and 90 dpi. heparinized blood (10 ml) from each animal was used for pbmc isolation by gradient centrifugation over histopaque 1077 (sigma aldrich) following the manufacturer 's protocol. pbmcs were incubated at 37c in rpmi complete medium supplemented with 10% of bovine fetal serum (internegocios) and 20 g / ml final concentration of ppdb (biocor) on 12-well tissue culture plates for 16 h for rna extraction and 48 h for flow cytometry determinations. after three months of infection, the calves were euthanized and then thin slices of lungs and lymph nodes of the head and pulmonary region were analyzed looking for granuloma formations. only one of the animals inoculated with the wild type nctc 10772 strain developed macroscopic lesions compatible with tuberculosis (data not shown). for flow cytometry determinations, 2 10 cells were incubated either with or without ppdb. to evaluate the expression of cd4 (mca 1653a647, igg2a), cd8 (mca837pe, igg2a), and cd25 (mca2430f and mca2430pe) surface markers, cells were stained with fluorescent - conjugated monoclonal antibodies (addserotec, oxford, uk). stained cells were analyzed in a facscalibur cytometer (bd, franklin lakes, nj, usa) using cell quest software. percentages of il-2r - expressing cells were calculated as the ratio of cd4 + or cd8 + cells expressing cd25 and total cd4 + or cd8 + cells. the data were organized in two sets, one for cd4 + cells and the other for cd8 + cells. each data set recorded cdc25 expression in cells infected with two different m. bovis strains over time. the dependent variable was the expression of cdc25 in ppdb stimulated cells, the bacterial strain was considered a fixed factor with two levels (mbmce2 and nctc 10772), the level of cdc25 in nonstimulated cells was included in the models as a covariate, and time of sampling was treated as a random factor. the analyses were performed using the nlme library of r [33, 34 ]. the quality and quantity of rna and the synthesis of cdna were assayed as described previously. the mrnaof cytokines (il-2, il-4, il-10, il-12p35, tnf-, and ifn-) was quantified by qpcr by using specific primers (table 1). for each animal, the preimmune condition was used as the calibrator, and pol ii and gadph were used as reference genes. | the generation of efficient candidate vaccines against bovine tuberculosis will contribute to the control of this zoonotic disease. rationally attenuated mycobacterium bovis strains generated by knockout of virulence genes are promising candidate vaccines. however, to be effective, these candidate vaccines should at least maintain the immunological properties of their virulent parental m. bovis strains. therefore, the aim of this study was to obtain an m. bovis strain deleted in the mce2 genes and evaluate the effect of the mutation on the immunological profile elicited by the bacteria in cattle. we showed that the activation of cd4 + t cells in cattle inoculated with the mutant strain was equivalent to that in animals inoculated with the parental strain. moreover, after in vitro stimulation, peripheral blood mononuclear cells from animals inoculated with the mutant produced higher levels of mrna th-1 cytokines than the parental strain. therefore, these results indicate that the mce2 mutant is a promising candidate vaccine against bovine tuberculosis. |
the human tropomyosin genes should be known as tpm1 through tpm4 (tpm1 through tpm4 for mouse and rat tropomyosin) to be consistent with other gene nomenclatures.the protein short name tm is historically well established but tpm is consistent with standard protein nomenclature and is therefore preferred in the formal name.tpm1 or tpm2 specifies the protein is from gene tpm1 or tpm2 etc.alternate isoforms are numbered systematically from one (e.g., tpm1.1 etc.), keeping as close as possible to any precedent. thus tpm2.3 is the tropomyosin isoform from gene 2 isoform 3.in the formal name a subscript designates the tissue most closely associated, historically, with the protein isoform. many such isoforms have been since found in multiple tissues and such designations are not always useful.st striated muscle, cardiac / skeletal tissuesm smooth muscle, br braincy other cytoplasmicthe splicing of the four exons that vary in vertebrates can be designated by a four letter code ; a.a.b.d indicates exon 1 is splice form a, exon 2 is splice form a, exon 6 is splice form b and exon 9 is splice form d.a dash as in b.-.b.d indicates that exon 2 is missing and therefore this is a short form of tpm.previous publications of tpm isoforms have indicated that exon 9 can be a combination of two splice forms at the mrna level (e.g., vindin and gunning 2013) but these reflect splice variants in non - coding regions at the 3 end of the mrna transcripts. we have therefore omitted such isoforms from our list of expressed proteins.the formal names of the two tropomyosins from the tpm1 gene, found in the contractile filaments of smooth muscle, are thentpm1.3sm(a.a.b.d) and tpm1.4sm(a.a.a.d) the human tropomyosin genes should be known as tpm1 through tpm4 (tpm1 through tpm4 for mouse and rat tropomyosin) to be consistent with other gene nomenclatures. the protein short name tm is historically well established but tpm is consistent with standard protein nomenclature and is therefore preferred in the formal name. alternate isoforms are numbered systematically from one (e.g., tpm1.1 etc.), keeping as close as possible to any precedent. thus tpm2.3 is the tropomyosin isoform from gene 2 isoform 3. in the formal name a subscript designates the tissue most many such isoforms have been since found in multiple tissues and such designations are not always useful.st striated muscle, cardiac / skeletal tissuesm smooth muscle, br braincy other cytoplasmic st striated muscle, cardiac / skeletal tissue the splicing of the four exons that vary in vertebrates can be designated by a four letter code ; a.a.b.d indicates exon 1 is splice form a, exon 2 is splice form a, exon 6 is splice form b and exon 9 is splice form d.a dash as in b.-.b.d indicates that exon 2 is missing and therefore this is a short form of tpm.previous publications of tpm isoforms have indicated that exon 9 can be a combination of two splice forms at the mrna level (e.g., vindin and gunning 2013) but these reflect splice variants in non - coding regions at the 3 end of the mrna transcripts. a dash as in b.-.b.d indicates that exon 2 is missing and therefore this is a short form of tpm. previous publications of tpm isoforms have indicated that exon 9 can be a combination of two splice forms at the mrna level (e.g., vindin and gunning 2013) but these reflect splice variants in non - coding regions at the 3 end of the mrna transcripts. the formal names of the two tropomyosins from the tpm1 gene, found in the contractile filaments of smooth muscle, are thentpm1.3sm(a.a.b.d) and tpm1.4sm(a.a.a.d) tpm1.3sm(a.a.b.d) and tpm1.4sm(a.a.a.d) the short form of the name would be tpm1.3 and tpm1.4 tables 1, 2, 3 and 4 list the current, known tpm protein isoforms and the proposed formal names. as far as possible the alternate names for the same protein that have been used in the past are included in columns two and three. we also include, in table 5, the full set of human tpm amino acid sequences in the order that the exons appear in the gene. this will allow any ambiguity in the literature to be checked against the names and exon sequences used here. table 5human tropomyosin amino acid sequences in order of the exons in the gene for additional information about tropomyosin sequences and exon organization in animals, refer to barua. 2011 human tropomyosin amino acid sequences in order of the exons in the gene for additional information about tropomyosin sequences and exon organization in animals, refer to barua. 2011 it is almost certain that additional isoforms of tropomyosin will be identified in these species. consideration of all possible splicing combinations already established in at least one isoform leads to a calculation of 48 possible distinct isoforms in humans without consideration of currently unknown splicing alternatives. for example, cooley and bergtrom (2001) identified an increased number of potential isoforms from the tpm1 gene using rt - pcr. our ability to detect and confirm additional isoforms will depend on both the extent of expression across, and the levels of expression in, different cell types. the ncbi accession numbers for each isoform are included in tables 1, 2, 3 and 4 and the human, mouse and rat genes on the ncbi website have been amended to use the nomenclature as listed here. | tropomyosin, a ubiquitous protein in animals and fungi, is associated with the actin cytoskeleton and is involved with stabilising actin filaments and regulating the interaction of the filament with other actin binding proteins. the protein is best known for its role in regulating the interaction between actin and myosin in muscle contraction but in recent years its role as a major player in the organisation and dynamics of the cytoskeleton has been increasingly recognised. in mammals tpm is expressed from four distinct genes and alternate splicing of each gene can produce a total of up to 40 different mrna variants most of which are expressed as proteins. we are expecting a renaissance in the study of tropomyosins as the roles of these different isoforms are beginning to be deciphered. however, it is our belief that such a renaissance is being limited by confusion over the naming systems for the tropomyosin isoforms. these result in even experienced workers struggling to reconcile work done in different laboratories and at different times. we propose here a systematic nomenclature for tropomyosin based on the best current practice. we recommend the adoption of these names and a cross - reference to the table of alternate names and accession numbers for protein sequences is included here. the national center for biotechnology information (ncbi) website has been amended to include the nomenclature for the human, mouse and rat genes. |
paracetamol is one of the most used antipyretic- analgesic preparation, which can be found in different pharmaceutical forms and in different doses. due to its wide utilization in the clinical practice, determination of paracetamol in pharmaceutical formulation is of a great importance since that over dosage with paracetamol may cause the hepatic fulminant necroses and other toxic effects. material and methods : study has included two formulations of paracetamol suppositories with doses of 125 mg widely used in the paediatric practice. suppositories prepared according to these two formulations by the melting method and spilling into forms was subject to the quality control by implementing a series of trials and analyses for that aim, such are : reactions of identification, average mass, disintegration time, and homogeneity whilst quantitative determination was performed by applying two methods of instrumental analyze : spectrophotometry in uv zone and cromatography in liquid phase with high pressure. results of these analyses, performed immediately following the preparation and 3 months after the preparation, showed that content of paracetamol in both of two formulations is within the norms of pharmacopoeia. suppositories of paracetamol in doses of 125 mg prepared as per formulation 1 are to be considered as more appropriate because it contains semi synthetic glycerides as excipient which has better features than other suppository excipients. paracetamol is one of the most used antipyretic - analgesic preparation, which can be found in different pharmaceutical forms and in different doses. it has an antipyretic and analgesic effects but it does not manifest any antiinflammatory effect (1). currently, in clinical practice, paracetamol is a safe alternative for substitution of the acetoacetic acid and of phenacetin. due to its wide utilization in the clinical practice, determination of paracetamol in pharmaceutical formulation is of a great importance since that over dosage with paracetamol may cause the hepatic fulminant necroses and other toxic effects (2). different methods for quality control of pharmaceutical products of paracetamol are used as described in literature. according to the monography of the paracetamol in british pharmacopoeia, 2 basic methods of analyzing the paracetamol are described ; spectrophotometric method for determination of paracetamol and hplc method for determination of 4-aminophenol. meanwhile, in the monography as per usp 30, the hplc method is described for paracetamol, but with no described methods for testing of the impurity of preparation (3). nevertheless, many methods of defining the paracetamol in pharmaceutical preparation are published in the professional scientific literature and some of them determine also the percentage of 4-aminophenol in simultaneous manner. (rp - hplc, liquid microemulsion cromatography, capillary electrophoresis, spectrophotometric electrophoresis uv) (4). process of producing the tablets requires a strict control of each individual phase of this process (processing of material, grinding, granulation, admixture, sieving, and tableting). granulation increases the size of granules in order to supply, in a uniform manner, the proper equipment for preparation of tablet matrix. this results in uniform pressure of product granules and it enables tablets to be uniform one as far as heft and consistency of physical - chemical properties of the pharmaceutical product are concerned (hardness, incoherence). this is why pharmaceutical industry usually utilizes granulates of the substance with narrow distribution (small difference in the size of particles comparing to average size of granulate) (5, 6). in our pharmaceutical market, formulations of paracetamol of different manufacturers are also present, and therefore, analyses of these formulations are important regarding medical practice and scientific pharmaceutical community in our country. aim of this research was to analyze the formulation, preparation, quality control, and follow - up of the stability of two formulations of paracetamol suppositories through hplc and spectrophotometry methods in the uv zone, presentation and analyses of these results commensurate to regulative of international pharmacopoeias. paracetamol suppositories of 125 mg of two formulations were analysed regarding the content of acting substance with methods of instrumental analyze. results of determining of the content of paracetamol in suppositories by spectrophotometry in uv zone. results of the paracetamol content in two formulations of these suppositories and statistic processing of results are provided in tables 1, 2 and graph 1., it can be seen that average mass of paracetamol suppositories of 125 mg complies with the theoretical mass of both formulations (mass which derives from tabulation of paracetamol s dose for 1 suppository (125 mg) with the mass of excipients used to obtain suppository). from the table 2, it can be seen that content of paracetamol in two formulations of suppositories of 125 mg complies with requirements of official pharmaceutical literature (95.0 105.0%). from the determination of paracetamol s content in two formulations of suppositories 125 mg, as per method of spectrophotometry in the zone, 3 months after the preparation, it is obvious that content of paracetamol in suppository has not changed, which speaks about the well known mentioned fact regarding tablets and syrup also in terms that this substance is quite stable. (table 3) results of determining of the content of paracetamol in suppositories by cromatography in the liquid phase with high pressure (hplc). these results indicate that the content of paracetamol in suppositories of 125 mg is within norms (95.0 105.0%). content of paracetamol in suppositories of 125 mg has not changed, which is also proved with statistical indexes represented in the above table. obtained results from the statistical data processing, emphasized also in cases of paracetamol syrup and tablets, speaks also for the precision of analytical methods applied by us. from both experimented formulations, we think that formulation 1 are to be considered as more appropriate because it contains semi synthetic glycerides used as excipient described in most of recent years pharmacopoeias. results of our research were compared with conditions, respectively criteria, set by international pharmacopeia, respectively british pharmacopeia (bp) and american pharmacopeia (usp) (7, 8, 9). permitted limit of mass deviation, as per bp, lies within a range of 5% of the declared mass. in our research, average mass of the analyzed formulations has not exceeded this limit set as per bp (10). time of absorption was also analyzed in our research as per conditions set by bp. time of disintegration for all of the formulations was within defined limits by bp, 1998, and it was less than 15 min, respectively it was comparable to results of other authors (11, 12). has defined the paracetamol absorption time in different percentages in temperature of 37 1c in accordance to the method described in british pharmacopoeia, 1998, whilst the process of defining the absorption was realized in the same conditions in terms of temperature according to the method also described in british pharmacopoeia and it was realized in the erweka - dt equipment (13). regarding stability of paracetamol formulations in our research, in a period following 3 months, no evident influential changes were seen in the content of these formulations. other authors also has ascertained that there were seen no significant changes in physical - chemical properties and dissolution velocity of paracetamol, provided that suppositories were stored in defined conditions within summarized requirements of british pharmacopoeia. in the market there are many boxes for dispensing of medicines to patients that enables protection of suppositories against air, humidity, and light by increasing the overall medicine compliance. results of a research conducted by haywood and associates showed that paracetamol suppositories can be repacked and stored in a dispensing box for medicines at patients for a period of 6 weeks and to provide adequate protection against air, humidity, and light by preserving physical - chemical properties of the paracetamol suppositories (14). therefore, generally paracetamol suppositories indicate a high scale of stability. results of our research enabled us an detailed reflection of qualitative and quantitative content of two formulations of paracetamol found in our country pharmaceutical market and indicated an high scale of compliance in between 2 methods of instrumental analyse : spectrophotometry in uv zone and cromatography in liquid phase with high pressure (hplc). two different formulations of paracetamol suppositories in doses of 125 mg, which are used in the paediatric practice, were experimented. suppositories prepared according to both formulations by the melting method and spilling into forms was subject to the quality control by implementing a series of trials and analyses, such are : reactions of identification, average mass, disintegration time, and homogeneity. quantitative determination of paracetamol in suppositories, as in the case with tablets, was performed by applying two methods of instrumental analyze : spectrophotometry in uv zone and cromatography in liquid phase with high pressure (hplc). as in the case with paracetamol tablets, results of defining the content of paracetamol by both analytical methods, immediately following the preparation and 3 months after preparation, have indicated that content of the acting matter in both two formulations is within the norms of specialty literature. even in the case of suppositories, changes in the results of analyses between two analytic methods were inconsiderable. paracetamol suppositories in doses of 125 mg as per formulation 1 are to be considered as more appropriate because they contains semi synthetic glycerides as excipient with better features than other suppository excipients. | introduction : paracetamol is one of the most used antipyretic- analgesic preparation, which can be found in different pharmaceutical forms and in different doses. due to its wide utilization in the clinical practice, determination of paracetamol in pharmaceutical formulation is of a great importance since that over dosage with paracetamol may cause the hepatic fulminant necroses and other toxic effects. material and methods : study has included two formulations of paracetamol suppositories with doses of 125 mg widely used in the paediatric practice. suppositories prepared according to these two formulations by the melting method and spilling into forms was subject to the quality control by implementing a series of trials and analyses for that aim, such are : reactions of identification, average mass, disintegration time, and homogeneity whilst quantitative determination was performed by applying two methods of instrumental analyze : spectrophotometry in uv zone and cromatography in liquid phase with high pressure.results and discussion : results of these analyses, performed immediately following the preparation and 3 months after the preparation, showed that content of paracetamol in both of two formulations is within the norms of pharmacopoeia. suppositories of paracetamol in doses of 125 mg prepared as per formulation 1 are to be considered as more appropriate because it contains semi synthetic glycerides as excipient which has better features than other suppository excipients. |
nonalcoholic steatohepatitis (nash) is an advanced stage in the spectrum of nonalcoholic fatty liver disease (nafld) first described in 1980. the steep rise in the prevalence of nash and its consequences on liver - related morbidity and mortally have made this condition an area of intense research. nafld is currently prevalent among 30% of the population in affluent countries and nash is diagnosed in 10 - 25% of those affected by nafld. it is estimated that in up to 22% of those affected by nash, the condition leads to cirrhosis. although hcc and cirrhosis are not as common in nash as in conditions such as hepatitis b or c, given the rising prevalence of nash, this condition will be a main future concern in public health. several risk factors have been identified for nash which include obesity, type 2 diabetes mellitus, hypertension and hyperlipidemia, all of which are components of metabolic syndrome. although weight loss is a definite treatment, there is no universally accepted pharmacologic treatment for this condition. the importance of finding a pharmacologic treatment for nash is better understood because not all individuals affected by nash are overweight or have treatable risk factors. moreover, weight loss is not achievable in many patients. several studies have been conducted to evaluate the effectiveness of various medications in treating the hepatic manifestations of nash. another mechanism that seemingly plays a central role in the pathogenesis of nash is insulin resistance and disruption in insulin secretion. the results of a study performed by pagano. have shown that insulin sensitivity is lower in nash patients compared to controls and insulin secretion is higher, which signifies that insulin resistance may be pivotal in the pathogenesis of nash. the prevalence of insulin resistance and the availability and relative safety of insulin - sensitizing agents justifies the necessity of studying their effectiveness in treating nash. biguanides (i.e., metformin) comprise another option. a study by lin. has shown that metformin, but not caloric restriction, greatly reduced hepatomegaly and hepatic steatosis in mice with associated insulin resistance, but had no significant reduction in fasting serum glucose concentrations. however, the effectiveness of metformin in treating human cases of nash and its mechanism of action are still controversial. marchesini. have demonstrated significant improvement in biochemical manifestations of nash in patients treated with metformin. in their study, histological improvements were not observed and it was not clear whether the improvements were achieved through the insulin - sensitizing action of metformin or through its effect on weight. lupi. have shown metformin s protective effect on human pancreatic b cells against fatty acids and its prevention of a disruption in insulin secretion. have conducted a randomized controlled trial and concluded that insulin sensitizers lead to improvements in metabolic, biochemical and histological abnormalities of nash as a result of improved insulin sensitivity. however, a pilot study by loomba. has concluded that metformin lead to improvements in liver histology and alt levels in 30% of patients with nash, probably through its effects on weight loss, which was not related to its insulin sensitizing action or its effect on insulin secretion. however, a few reviews question the effectiveness of metformin. in the current study, we designed a randomized double - blind controlled trial to study the biochemical and histological effects of metformin on biopsy proven cases of nash. subjects with increased liver enzymes who referred to a university clinic (shariati hospital, tehran) during 2003 and 2004 were evaluated for enrollment. liver enzymes were considered to be increased if either alanine aminotransferase (alt) or aspartate aminotransferase (ast) were more than 1.5 times the upper limit of normal on at least two occasions, at least three months apart. patients who took medications known to elevate liver enzymes or experimental medications for nash within the preceding three months were excluded. viral and autoimmune hepatitis and other causes of liver disorders were excluded. other exclusion criteria were : diabetes mellitus, severe medical conditions, pregnancy or intent to become pregnant in the following six months, and not consenting to the study protocol. liver biopsy was performed for all eligible patients and if results were suggestive of nash, patients were recruited. nash was confirmed if over 5% of hepatocytes contained fat droplets and any degree of necroinflammation or fibrosis was noticed. subjects were randomized to receive either metformin, 500 mg twice daily, or an identical - looking placebo. all patients with bmi greater than 25 kg / m were instructed to lose weight. possible adverse drug effects, lipid profi le, and weight changes, as well as compliance were also recorded. another liver biopsy was performed after six months of treatment in subjects who agreed. the liver histology was scored using a modifycation of the system developed by brunt. brunt classifies fatty liver as mild, moderate or severe according to the degree of steatosis, hepatocyte ballooning, lobular inflammation and portal inflammation. this system can occasionally be confusing because the four variables may not always be in agreement. to overcome this problem the sum of these scores was considered as the total pathology grade or nash activity index (nai). fibrosis was staged from zero through four (table 1). this modified brunt system has been validated and proven to have good inter- and intraobserver agreement. secondary outcome variables included changes in histologic variables and adverse effects. assuming a 50% normalization of liver enzymes by metformin, a type one error of 5% and a type two error of 10% the chi square test compared ordinal variables between the two treatment groups and the t test compared continuous variables between groups. paired - samples t - test compared quantitative variables before and after treatment and the wilcoxon signed - ranks test compared changes in histology before and after treatment. the study protocol conformed to the ethical guidelines of the 1975 declaration of helsinki and was approved by the institutional review board and ethics committee of the digestive disease research center of tehran university of medical sciences. thirty - three subjects were included in the study of which 15 were treated by metformin and 18 received placebo. none of the baseline characteristics significantly differed between the two groups (table 2). bmi : body mass index ; ast : aspartate aminotransferase ; alt : alanine aminotransferase ; ldl : low density lipoprotein ; hdl : high density lipoprotein ; five subjects withdrew from the study before the final assessment : 1 female and 3 male subjects from the placebo group and 1 male subject from the metformin group. none of the baseline clinical, biochemical, or histological characteristics of the 5 subjects who withdrew significantly differed from the subjects who completed the study. in the metformin group, 3 subjects complained of diarrhea. three subjects from the placebo group and 1 subject from the metformin group complained of nausea, with complaints of vomiting in 2 subjects from the placebo group. abdominal distention was noted by 2 subjects from the metformin group and 1 subject from the metformin group complained of metallic taste. finally, among the 28 subjects who completed the study, 5 from the placebo group and 1from the metformin group had poor compliance. the frequency of adverse effects was not significantly different between the placebo and metformin groups. among the 28 subjects who finished the study, 16 lost weight, 3 had no change in weight and the remaining 9 gained weight (table 3). overall, the bmi decreased (mean change : 0.54) with marginal significance (p=0.049), but the decrease within the treatment groups was significant. of study subjects, 13 in the metformin and 13 in the placebo group were overweight and received weight loss instructions. the decrease in bmi was not significantly different between overweight subjects who received weight loss instructions and normal weight subjects who received no instructions. liver enzymes (ast and alt) showed significant decreases in both groups (liver enzymes (ast and alt)). ast decreased significantly in both the metformin (p < 0.001) and placebo (p=0.02) groups, as did the level of alt (p<0.001 for both groups). however, there was no significant difference in the magnitude of decrease in liver enzymes between the two treatment groups. changes in other parameters were not significant ; neither within each of the treatment groups nor between them, and neither within the entire group of subjects. the coefficient of regressing change in ast on baseline ast (p=0.006) and the coefficient of regressing change in alt on baseline alt (p<0.001) were significantly positive, indicating that subjects with higher initial ast or alt levels showed greater improvement. biopsy was conducted on all 33 subjects before treatment and 8 subjects after treatment completion (6 subjects in the metformin and 2 in the placebo groups). neither the grade of steatosis, fibrosis stage or nai significantly differed between the two treatment groups, before or after treatment. nai was significantly lower for all 8 subjects after treatment (p= 0.048). weight loss and decrease in bmi were more prominent in the metformin group, but there was no statistically significant difference in the magnitude of decrease between both groups. the insignificant results in either of the groups might be due to inadequate sample size and the low power of the study. alt and ast levels decreased significantly in both treatment groups and in the entire group of subjects, but there was no significant difference in the magnitude of decrease between the two groups. these results again imply that unlike findings reported by marchesini. and idilman. the observed significant decrease in liver enzymes within both groups may be entirely the result of weight loss. the insignificant decrease on other biochemical determinants again shows that metformin has no particular effect on these risk factors. in accord with findings of marchesini. and unlike the report by idilman., neither steatosis, nor histology stage or grade significantly differed between the two groups, either before or after treatment. moreover, no decline was observed in steatosis or histology stage post - treatment among the 8 subjects who underwent biopsies after treatment. however, nai showed significant decreases in all 8 subjects, but the decline was not significant within the treatment groups. these numbers again indicate that insignificant results in either of the groups might be due to inadequate sample size and the low power of the study. with a larger sample size, we could probably detect signify ca nt histologic regression which would be attributed to weight loss. our results show that higher baseline levels of liver enzymes are associated with more prominent decreases in their levels after treatment. these results suggest the hypothesis that weight loss can be associated with improvements in biochemical profiles, especially in cases that baseline liver enzymes are higher. however, since significant weight loss among subjects in our study was not achieved, we could not prove the same association between the improvement in biochemical profile and weight loss. we were unable to show any associations between weight loss instructions or metformin with actual weight loss or biochemical improvements in nash patients. the significant improvement in biochemical profile of nash patients in our study can be solely attributed to weight loss, which is the only common predictor among the subjects. although one might speculate that the observed improvement might be related to some other unmeasured variable that has changed in all our subjects during the study period, we fi nd this unlikely. metformin is demonstrated to be marginally associated with weight loss, but not biochemical improvement. however, weight loss and baseline insulin resistance, but not metformin, are marginally associated with biochemical improvements. the summary of these findings is in accord with results reported by loomba. and a few recent reviews, which state that the association of metformin with biochemical improvement, whether significant or not, is probably mediated through weight loss and the apparent role of insulin resistance on the pathogenesis of nash can probably be manipulated by weight loss, not by metformin. however, as mentioned earlier, our insignificant results may be due to inadequate sample size and the low power of the study. it should be noted that our study was designed to detect a 50% improvement rate, so an improvement of less than 50% might not be detectable. further studies with higher sample size, more compliant subjects and longer treatment duration are mandatory. | backgroundn nonalcoholic steatohepatitis (nash) is a common liver disease that can progress to cirrhosis or hepatocellular carcinoma. it is estimated that up to 3% of the iranian population have this condition. although the pathogenesis of nash is incompletely understood, there is significant evidence pointing to the importance of insulin resistance. metformin is an oral hypoglycemic agent known to improve insulin resistance. this study examines the effectiveness of metformin on biochemical and histological improvement among nash patients in a randomized double - blind controlled trial. methods this study enrolled 33 biopsy - proven nash patients. other causes of liver disorders were excluded. subjects were randomized to receive either metformin, 500 mg twice daily, or an identical - looking placebo. overweight patients were also instructed to lose weight. treatment continued for 6 months. patients were regularly visited and liver enzyme levels recorded. compliance and any adverse drug effects were recorded. resultsin the metformin group, the mean aspartate aminotransferase (ast) level dropped from 61.2 iu / l to 32.7 iu / l and the mean alanine aminotransferase (alt) level dropped from 85.1 iu / l to 50.8 iu / l. the mean ast level in the placebo group dropped from 54.3 iu / l to 37.9 iu / l, whereas the mean alt level dropped from 111.8 iu / l to 55.4 iu / l in the placebo group. the decrease in liver enzymes was significant in both groups, but the magnitude of decrease was not significantly different.conclusionthe improvement observed in liver enzyme levels is totally attributable to weight loss. metformin had no significant effect on liver enzyme levels. |
stroke is the second most common cause of death and one of major causes of disability around the world. stroke may be divided into two major categories by etiology : ischemic stroke (is), which is about 80%, and hemorrhagic stroke, about 20%. environmental factors including hypertension, hyperlipidemia, and smoking are risk factors of stroke, and genetic factors also influence susceptibility to stroke [2 - 4 ]. recently, genetic studies have shown the association between the development of stroke and genetic polymorphisms. reported that paraoxonase 1 (pon1) polymorphism [rs662, gln192arg (a > g) ] contributed to the risk of is.. showed that the synonymous snp [rs2228048, asn389 (c > t) ] of transforming growth factor, beta receptor ii (tgfbr2) might be associated with the development of intracranial hemorrhage (ich) in korean population. park. found that the c alleles of rs3804099 [asn199 (t > c) ] and rs3804100 [ser450 (t > c) ] of toll - like receptor 2 (tlr2) gene were associated with national institutes of health stroke scale (nihss) of is patients. atp - binding cassette, sub - family b (mdr / tap), member 1 (abcb1) is a protein which confers resistance towards multiple cytostatic drugs (multidrug resistance) in tumor cells. abcb1 is also known as multidrug resistance 1 (mdr1) and p - glycoprotein (pgy1). the previous studies about abcb1 have been mainly focused on drug efflux function associated with tumor chemotherapy including colorectal cancer, breast cancer, and brain tumor. however, abcb1 exports endogenous inflammatory mediators such as prostaglandins and leukotrienes, which may aggravate the inflammation and destruction of various size of ischemized tissue, from the initial steps to latent steps of inflammation. in the central nervous system (cns), abcb1 is normally expressed in blood - brain barrier (bbb). in the mouse model of is, the increased abcb1 in brain tissue was able to efflux drugs despite of permeable bbb. in addition, bochud. suggested that abcb1 is related to hypertension via the renin - angiotensin - aldosterone system. although abcb1 may contribute to the pathogenesis or pathophysiology of is, however, there was no case - control study analyzed the relationship between abcb1 and is. in this study, we assessed the association between four snps of the abcb1 gene [promoter snp (rs4148727, -154 t > c), 5'-untranlation region (5'utr) snp (rs3213619, -129t > c), exon snp [rs1128503, gly412(c > t) ], and 3'utr snp (rs3842, a > g) ] and the development, risk factors (hypertension, dyslipidemia, and diabetes mellitus), severity (nihss), and sequelae (modified barthel index, mbi) of is in korean population. is patients were enrolled among participants who diagnosed at the east - west neomedical center and kyung hee medical center (seoul, korea), during 2009 - 2012. the diagnosis of is were confirmed through computed tomography (ct), magnetic resonance imaging (mri), angiography, or duplex sonography by well - trained neurologists. subjects with accidental or iatrogenic stroke, transient ischemic attack, brain tumors, or other cerebrovascular disorders were excluded. controls were enrolled among participants examined through general health check - up program. controls with stroke, ischemic heart diseases, neurological diseases, or any severe underlying diseases this study was conducted according to the guidelines of the helsinki declaration and was approved by the ethics review committee of medical research institute, school of medicine, kyung hee university (seoul, korea). informed consent was obtained from all individuals. if a patient was in incommunicable state, we obtained informed consent from close relatives or legal guardian. all of is patients were divided into subgroups in accordance with the clinical features (table 1). the abnormal range of the clinical features were as following : hypertension, > 140 mmhg in systolic blood pressure (sbp) or > 90 mmhg in diastolic blood pressure (dbp) ; diabetes mellitus, > 126 mg / dl in fasting plasma glucose or > 6.5% hemoglobin a1c (hba1c) ; dyslipidemia, > 250 mg / dl in total cholesterol (tc), > 150 mg / dl in triglyceride (tg), or c ; rs3213619, 5'utr (-129t > c), rs1128503, gly412 (c > t), and rs3842, 3'utr (a > g) ] of the abcb1. peripheral blood sample of all subjects was collected in edta tube, and genomic dna was extracted using dna isolation kit for cells and tissues (roche, indianapolis, in, usa). genotypes of each snp were determined by direct sequencing (macrogen, seoul, korea). pcrs were performed as the following condition : 40 cycles at 94 for 30 sec, 58 for 30 sec, 72 for 30 sec, and 1 cycle at 72 for 5 min for the final reaction. the primer sequences used for pcrs were as following : for rs3842, sense primer ggcacagaaaggcatctatttt, antisense primer caaggcagtcagttacagtcca ; for rs1128503, sense primer cccatctcgaaaagaagttaagg, antisense primer catctcaccatcccctctgt ; for rs3213619, sense primer attcctcctggaaattcaacct, antisense primer ttgggaactgtcccatagtagc ; for rs4148727, sense primer ggtcttcccagtaacctaccaa, antisense primer ttatcccagtaccagaggagga. the pcr products were sequenced by an abi prism 3730xl analyzer (pe applied biosystems, foster city, ca, usa) and sequencing data were analyzed using seqmanii software (dnastar, madison, wi, usa). multiple logistic regression models were applied using following models : codominant1 (major allele homozygotes vs. heterozygotes), codominant2 (major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes+minor allele homozygotes), recessive (major allele homozygotes+heterozygotes vs. minor allele homozygotes), and log - additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes) models. odds ratios (or), 95% confidence intervals (ci), and p - values were determined using snpstats (http://bioinfo.iconcologia.net/index.php?module=snpstats) and spss 18.0 (spss inc., chicago, il, usa). the linkage disequilibrium (ld) block and haplotypes were estimated using haploview version 4.2 (daly lab, cambridge, ma, usa). the age of is group (meanstandard deviation) was 65.712.1 years and control was 63.09.3 years (table 1). table 2 shows the genotype and allele frequencies of the four examined rs4148727, -154t > c ; rs3213619, 5'utr(-129t > c), rs1128503, gly412(c > t), and rs3842, 3'utr(a > g) ] of abcb1 in the control and is groups. the genotype distributions of the four tested snps were consistent with the hardy - weinberg equilibrium in the control group (p>0.05, data not shown). the rs3842 snp was weakly associated with the risk of is (p=0.020, or=0.58, 95% ci=0.36 - 0.92 in codominant1 model and p=0.028, or=0.62, 95% ci=0.40 - 0.95 in dominant model). however, the allele frequencies of rs3842 showed no difference between the is group and the control group. other three snps (rs4148727, rs3213619, rs1128503) were not associated with the development of is (p>0.05). 1). therefore we did not showed the haplotype among the four examined snps. in order to investigate correlation between abcb1 and clinical phenotypes of is, the is patients were divided into two subgroups in accordance with the clinical and biochemical features (n=35 is without hypertension vs. n=81 is with hypertension ; n=86 is without dyslipidemia vs. n=33 is patients with dyslipidemia ; n=72 is without diabetes mellitus vs. n=45 is with diabetes mellitus ; n=56 nihss score t) ] showed weak association with dyslipidemia (p=0.040, or=0.40, 95% ci=0.17 - 0.96 in codominant1 model, p=0.02, or=0.37, 95% ci=0.16 - 0.86 in dominant model, p=0.018, or=0.45, 95% ci=0.23 - 0.90 in log - additive model). the c allele frequency of rs1128503 was lower in dyslipidemia (+) group (25.8%) than that in the dyslipidemia (-) group (41.8%). the difference was statistically significant (p=0.024, or=0.48, 95% ci=0.26 - 0.10). three snps (rs4148727, rs3213619, and rs3842) were not related to dyslipidemia in is (data not shown). one snp of abcb1 [rs3842, 3'utr(a > g) ] showed weak association with diabetes mellitus (fisher 's exact p=0.046, or=0.15, 95% ci=0.02 - 1.22 in codominant2 model, p=0.029, fisher 's exact p=0.049, or=0.15, 95% ci=0.02 - 1.23 in recessive model). three snps (rs4148727, rs3213619, and rs1128503) were not related to diabetes mellitus in is (data not shown). finally, one promoter snp of abcb1 (rs4148727, -154t > c) was marginally associated with nihss in is (p=0.034, fisher 's exact p=0.042, or=3.31, 95% ci=1.09 - 10.01 in codominant1 model, p=0.032, or=2.99, 95% ci=1.05 - 8.48 in dominant model). three snps (rs3213619, rs1128503, and rs3842) were not related to nihss in is (data not shown). the four examined snps showed no association with mbi in is (p>0.05, data not shown). is patients were enrolled among participants who diagnosed at the east - west neomedical center and kyung hee medical center (seoul, korea), during 2009 - 2012. the diagnosis of is were confirmed through computed tomography (ct), magnetic resonance imaging (mri), angiography, or duplex sonography by well - trained neurologists. subjects with accidental or iatrogenic stroke, transient ischemic attack, brain tumors, or other cerebrovascular disorders were excluded. controls were enrolled among participants examined through general health check - up program. controls with stroke, ischemic heart diseases, neurological diseases, or any severe underlying diseases this study was conducted according to the guidelines of the helsinki declaration and was approved by the ethics review committee of medical research institute, school of medicine, kyung hee university (seoul, korea). informed consent was obtained from all individuals. if a patient was in incommunicable state, we obtained informed consent from close relatives or legal guardian. all of is patients were divided into subgroups in accordance with the clinical features (table 1). the abnormal range of the clinical features were as following : hypertension, > 140 mmhg in systolic blood pressure (sbp) or > 90 mmhg in diastolic blood pressure (dbp) ; diabetes mellitus, > 126 mg / dl in fasting plasma glucose or > 6.5% hemoglobin a1c (hba1c) ; dyslipidemia, > 250 mg / dl in total cholesterol (tc), > 150 mg / dl in triglyceride (tg), or c ; rs3213619, 5'utr (-129t > c), rs1128503, gly412 (c > t), and rs3842, 3'utr (a > g) ] of the abcb1. peripheral blood sample of all subjects was collected in edta tube, and genomic dna was extracted using dna isolation kit for cells and tissues (roche, indianapolis, in, usa). genotypes of each snp were determined by direct sequencing (macrogen, seoul, korea). pcrs were performed as the following condition : 40 cycles at 94 for 30 sec, 58 for 30 sec, 72 for 30 sec, and 1 cycle at 72 for 5 min for the final reaction. the primer sequences used for pcrs were as following : for rs3842, sense primer ggcacagaaaggcatctatttt, antisense primer caaggcagtcagttacagtcca ; for rs1128503, sense primer cccatctcgaaaagaagttaagg, antisense primer catctcaccatcccctctgt ; for rs3213619, sense primer attcctcctggaaattcaacct, antisense primer ttgggaactgtcccatagtagc ; for rs4148727, sense primer ggtcttcccagtaacctaccaa, antisense primer ttatcccagtaccagaggagga. the pcr products were sequenced by an abi prism 3730xl analyzer (pe applied biosystems, foster city, ca, usa) and sequencing data were analyzed using seqmanii software (dnastar, madison, wi, usa). multiple logistic regression models were applied using following models : codominant1 (major allele homozygotes vs. heterozygotes), codominant2 (major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes+minor allele homozygotes), recessive (major allele homozygotes+heterozygotes vs. minor allele homozygotes), and log - additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes) models. odds ratios (or), 95% confidence intervals (ci), and p - values were determined using snpstats (http://bioinfo.iconcologia.net/index.php?module=snpstats) and spss 18.0 (spss inc., chicago, il, usa). the linkage disequilibrium (ld) block and haplotypes were estimated using haploview version 4.2 (daly lab, cambridge, ma, usa). all of is patients were divided into subgroups in accordance with the clinical features (table 1). the abnormal range of the clinical features were as following : hypertension, > 140 mmhg in systolic blood pressure (sbp) or > 90 mmhg in diastolic blood pressure (dbp) ; diabetes mellitus, > 126 mg / dl in fasting plasma glucose or > 6.5% hemoglobin a1c (hba1c) ; dyslipidemia, > 250 mg / dl in total cholesterol (tc), > 150 mg / dl in triglyceride (tg), or c ; rs3213619, 5'utr (-129t > c), rs1128503, gly412 (c > t), and rs3842, 3'utr (a > g) ] of the abcb1. peripheral blood sample of all subjects was collected in edta tube, and genomic dna was extracted using dna isolation kit for cells and tissues (roche, indianapolis, in, usa). genotypes of each snp were determined by direct sequencing (macrogen, seoul, korea). pcrs were performed as the following condition : 40 cycles at 94 for 30 sec, 58 for 30 sec, 72 for 30 sec, and 1 cycle at 72 for 5 min for the final reaction. the primer sequences used for pcrs were as following : for rs3842, sense primer ggcacagaaaggcatctatttt, antisense primer caaggcagtcagttacagtcca ; for rs1128503, sense primer cccatctcgaaaagaagttaagg, antisense primer catctcaccatcccctctgt ; for rs3213619, sense primer attcctcctggaaattcaacct, antisense primer ttgggaactgtcccatagtagc ; for rs4148727, sense primer ggtcttcccagtaacctaccaa, antisense primer ttatcccagtaccagaggagga. the pcr products were sequenced by an abi prism 3730xl analyzer (pe applied biosystems, foster city, ca, usa) and sequencing data were analyzed using seqmanii software (dnastar, madison, wi, usa). multiple logistic regression models were applied using following models : codominant1 (major allele homozygotes vs. heterozygotes), codominant2 (major allele homozygotes vs. minor allele homozygotes), dominant (major allele homozygotes vs. heterozygotes+minor allele homozygotes), recessive (major allele homozygotes+heterozygotes vs. minor allele homozygotes), and log - additive (major allele homozygotes vs. heterozygotes vs. minor allele homozygotes) models. odds ratios (or), 95% confidence intervals (ci), and p - values were determined using snpstats (http://bioinfo.iconcologia.net/index.php?module=snpstats) and spss 18.0 (spss inc., chicago, il, usa). the linkage disequilibrium (ld) block and haplotypes were estimated using haploview version 4.2 (daly lab, cambridge, ma, usa). the age of is group (meanstandard deviation) was 65.712.1 years and control was 63.09.3 years (table 1). table 2 shows the genotype and allele frequencies of the four examined rs4148727, -154t > c ; rs3213619, 5'utr(-129t > c), rs1128503, gly412(c > t), and rs3842, 3'utr(a > g) ] of abcb1 in the control and is groups. the genotype distributions of the four tested snps were consistent with the hardy - weinberg equilibrium in the control group (p>0.05, data not shown). the rs3842 snp was weakly associated with the risk of is (p=0.020, or=0.58, 95% ci=0.36 - 0.92 in codominant1 model and p=0.028, or=0.62, 95% ci=0.40 - 0.95 in dominant model). however, the allele frequencies of rs3842 showed no difference between the is group and the control group. other three snps (rs4148727, rs3213619, rs1128503) were not associated with the development of is (p>0.05). therefore we did not showed the haplotype among the four examined snps. in order to investigate correlation between abcb1 and clinical phenotypes of is, the is patients were divided into two subgroups in accordance with the clinical and biochemical features (n=35 is without hypertension vs. n=81 is with hypertension ; n=86 is without dyslipidemia vs. n=33 is patients with dyslipidemia ; n=72 is without diabetes mellitus vs. n=45 is with diabetes mellitus ; n=56 nihss score t) ] showed weak association with dyslipidemia (p=0.040, or=0.40, 95% ci=0.17 - 0.96 in codominant1 model, p=0.02, or=0.37, 95% ci=0.16 - 0.86 in dominant model, p=0.018, or=0.45, 95% ci=0.23 - 0.90 in log - additive model). the c allele frequency of rs1128503 was lower in dyslipidemia (+) group (25.8%) than that in the dyslipidemia (-) group (41.8%). the difference was statistically significant (p=0.024, or=0.48, 95% ci=0.26 - 0.10). three snps (rs4148727, rs3213619, and rs3842) were not related to dyslipidemia in is (data not shown). one snp of abcb1 [rs3842, 3'utr(a > g) ] showed weak association with diabetes mellitus (fisher 's exact p=0.046, or=0.15, 95% ci=0.02 - 1.22 in codominant2 model, p=0.029, fisher 's exact p=0.049, or=0.15, 95% ci=0.02 - 1.23 in recessive model). three snps (rs4148727, rs3213619, and rs1128503) were not related to diabetes mellitus in is (data not shown). finally, one promoter snp of abcb1 (rs4148727, -154t > c) was marginally associated with nihss in is (p=0.034, fisher 's exact p=0.042, or=3.31, 95% ci=1.09 - 10.01 in codominant1 model, p=0.032, or=2.99, 95% ci=1.05 - 8.48 in dominant model). three snps (rs3213619, rs1128503, and rs3842) were not related to nihss in is (data not shown). the four examined snps showed no association with mbi in is (p>0.05, data not shown). in this study, we investigated the relationships between the four snps of abcb1 [rs4148727, -154t > c ; rs3213619, 5'utr(-129t > c), rs1128503, gly412(c > t), and rs3842, 3'utr(a > g) ] and is in korean population. our result suggests that abcb1 might be associated with the development, risk factors (hypertension, dyslipidemia, and diabetes mellitus), and severity (nihss) of is. some studies documented the association between abcb1 polymorphisms (rs3213619, rs1128503, and rs3842) and certain diseases. fujii. investigated the relationship between abcb1 snps (rs2188524, rs3213619, rs1128503, rs2032582, and rs1045642) and major depressive disorder (mdd) in the japanese, they observed that t allele of rs1045642 is risk factor for mdd. reported that haplotype cgc (rs3213619, rs1128503, and rs2032582) may be less susceptible to the leukemogenic effects of indoor insecticide exposures. in present study, we observed that rs4148727 was associated with the severity of is (nihss). the t and c allele frequencies in rs4148727 have been reported to be 0.976 and 0.024 in european, 0.950 and 0.050 in hispanic, 0.917 and 0.083 in asian, and 0.909 and 0.091 in sub - saharan african, respectively (http://www.ncbi.nlm.nih.gov/projects/snp). in our controls, the allele frequency of rs4148727 showed no ethnic difference. to investigate the binding site of transcription factors for the t and c alleles of rs4148727 (-154t > c) the c allele in rs4148727 has an activation protein (ap)-1 transcription factor binding site, whereas the t allele has no ap-1 binding site. regarding that abcb1 is implicated in cancer chemotherapy [9, 10 ], a polymorphism on binding site of ap-1 is an interesting result. ap-1 is related to neuron synapse in regulating synaptic number and strength [24, 25 ]. that is, rs4148727 determines an ap-1 promoter site, which might have enhanced neuronal protection in ischemia, to show weak association with nihss. and the t and c allele frequencies in rs3213619 have been reported to be 0.969 and 0.031 in european, 0.919 and 0.081 in chinese, 0.953 and 0.047 in japanese, and 0.934 and 0.066 in sub - saharan african, respectively. in our controls, the t and c allele frequencies of rs3213619 were 0.947 and 0.053. the t and c allele frequencies in rs1128503 have been reported to be 0.451 and 0.549 in european, 0.709 and 0.291 in chinese, 0.587 and 0.413 in japanese, and 0.124 and 0.876 in sub - saharan african, respectively. the t and c allele frequencies of rs1128503 in our control group were 0.595 and 0.405, those were similar in japanese population. the a and g allele frequencies in the rs3842 snp have been reported to be 0.858 and 0.142 in european, 0.756 and 0.244 in chinese, 0.680 and 0.320 in japanese, and 0.858 and 0.142 in sub - saharan african, respectively. the a and g allele frequencies of rs3842 in our control group were 0.684 and 0.316, those were similar in japanese population. rs3213619 was associated with hypertension in codominant1 model (fisher 's exact p=0.020, or=0.26, 95% ci=0.07 - 0.91) and allele frequencies (fisher 's exact p=0.024, or=0.25, 95% ci=0.28 - 0.78). rs3842 was associated with hypertension in codominant2 model (fisher 's exact p=0.016, or=0.15, 95% ci=0.03 - 0.70) and recessive model (fisher 's exact p=0.003, or=0.11, 95% ci=0.03 - 0.51). as hypertension and non - hypertension groups were already affected by stroke, we observed that minor alleles of both rs3213619 and rs3842 snps might be contributing to the risk of is development in non - hypertensive groups. however study result may still have significance, because poststroke bp rise is associated with prestroke bp level, and bp of hypertensive patients are higher than non - hypertensive patients in the first week of stroke. previous study reported cytochrome p450, family 3, subfamily a, polypeptide 5 (cyp3a5) and abcb1 polymorphisms are associated with hypertension. the 3435 c > t variant of abcb1 was associated with angiotensin stimulated aldosterone level, and cyclosporin a, an abcb1 inhibitor, influences the renin - angiotensin - aldosterone system. these results suggest that association of abcb1 with blood pressure is not only mediated by exogeneous substrates such as ace inhibitors, and may support the association of rs3213619 and rs3842 with hypertension. ninj2 gene showed association with all types of strokes and ischemic stroke, and hdac9 is associated with the risk of large vessel ischemic stroke. however, no recent gwas indicated the abcb1 as risk factor of is. regarding that abcb1 mainly functions as efflux pump, abcb1 may not be directly related to development of is. however, as inflammatory mediators such as prostaglandins and leukotrienes are abcb1 substrate, we hypothesized that abcb1 functions in the bbb may contribute to tissue damaging and severity of is developed by ischemic origin. however, nihss and mbi were not associated with the tested abcb1 snps. in summary, we evaluated the relationships between the four snps of abcb1 (rs4148727, rs3213619, rs1128503, and rs3842) and the development, risk factors, severity, and sequelae of is. and we are the first to report the association between abcb1 snps and the development, hypertension, dyslipidemia, diabetes mellitus, and nihss of is. furthermore, some of subgroups in our study included too small cases to conclude the clear association between candidate snps and is. therefore, the suggested relationship between tested snps and the clinical phenotypes of is still need validation study in larger samples. | neuronal expression of atp - binding cassette, sub - family b (mdr / tap), member 1 (abcb1) has been demonstrated after brain ischemia. to investigate whether abcb1 polymorphisms are associated with the development, risk factors (hypertension, dyslipidemia, and diabetes mellitus), severity (national institutes of health stroke scale, nihss), and sequelae (modified barthel index, mbi) of ischemic stroke (is), four single nucleotide polymorphisms (snps) of the abcb1 gene [rs4148727, promoter, -154t > c ; rs3213619, 5'-untranslation region (5'utr), -129t > c) ; rs1128503, synonymous, gly412 (c > t) ; rs3842, 3'utr, a > g ] were analyzed in 121 is patients and 291 control subjects. snpstats and spss 18.0 were used to obtain odds ratios (or), 95% confidence intervals (ci), and p values. multiple logistic regression models (codominant1, codominant2, dominant, recessive, and log - additive models) were applied to analyze the genetic data. the rs3842 snp was weakly associated with the development of is (p=0.020 in codominant1 model and p=0.028 in dominant model). in the analysis of clinical phenotypes, abcb1 polymorphisms were nominally associated with hypertension (rs3213619 and rs3842, p<0.05), dyslipidemia (rs1128503, p<0.05), diabetes (rs3842, p<0.05), and nihss (rs4148727, p<0.05). interestingly, rs3842 showed statistically strong association between is with hypertension and is without hypertension (fisher 's exact p=0.003, or=0.11, 95% ci=0.03 - 0.51 in recessive model). these results suggest that the abcb1 gene may be associated with the development and clinical phenotypes of is in korean population. |
several definitions have been proposed to explain the concept of stress in the literature. as the first definition, hans selye introduced stress as a non - specific response of an organism in exposure to a demand or a change in the physical situation. eustress, which contains positive effects, which might enhance motivation and efforts among people and which seems to be necessary to have a healthy life. introduced stress as a natural predictable experience with both positive and negative consequences. based on such explanations, it seems that stress might be considered as absence of internal tranquility, which contains different aspects. occupational stress is a relatively modern subject, which frequently has been discussed as a major health - related concern, especially during the recent decades. based on the national institute for occupational safety and health (niosh), occupational stress is defined as any harmful physical or mental response, occurring due to individual 's incompatibility with his / her ability, which might lead to aggressive behaviors, occupational injuries, physical diseases and even death. the international labour organization (ilo) considers occupational stress as the most important health threat for employees, while the world health organization (who) emphasizes health problems, low motivation and low safety as the negative impacts on employees exposed to occupational stress and, consequently, considerable unwanted costs for the employers. the ilo statistics demonstrate that occupational stress might account for 1 - 3.5% of the gross domestic product (gdp) of the countries. the annual cost of occupational stress has been estimated to be more than 300 million dollars for the us and 20 million euros for the european countries. anecdotal reasons highlight the importance of attention to occupational stress as one of the most important health - related problems in the world. many studies have been conducted to evaluate occupational stress among iranian employees ; however, most of them have been conducted on nurses and teachers in iran, rather than being conducted on industrial employees as one of the most at - risk groups. this fact persuaded us to design and conduct this study to evaluate the prevalence and associated parameters of occupational stress among the employees of esfahan steel company (esco), one of the biggest and most important industrial sites in iran. as a cross - sectional design, we applied analysis of a data set, extracted from the self - report information of employees who worked in the operational units of esco, one of the biggest iranian governmental companies. based on the single proportion formula at 95% confidence interval (ci), the sample size was estimated to be composed of 380 respondents, which rose to 440 to increase the confidence level, while 40 were excluded due to their incomplete questionnaires. all the 400 selected participants were male workers who had at least 1 year work experience in esco, and were sampled based on the randomized sampling method from march to july 2008. all the questionnaires were anonymous and we assured participants that their information would be kept confidential. this study was also approved and supervised by the head of the research and development department of esco. two different types of pre - tests (formal and informal) were conducted prior to the main study. the informal pre - test was designed to make any correction in question wording, content and clearness of scenarios, and was administered to 25 subjects similar to the main study participants. with a cronbach 's alpha of 0.87, the formal pre - test was also conducted in march 2008 on 36 esco employees who were not entered in the main study. the results of these two pre - tests helped the researchers to improve the questionnaires to be more easy - to - understand for the participants. the questionnaires were filled out through interviews and no financial incentives were offered to the participants. the main questionnaire included two main parts ; in the first part, socio - demographic data, including age, number of children, marital status, monthly income, educational level, shift work and years of work experience, were asked. an outcome - related questionnaire was designed based on the stress scale extracted from the validated depression, anxiety, stress scale (dass). in the stress scale questionnaire, difficulty in relaxing, nervous arousal, being easily upset / agitated, being irritable / over - reactive and restlessness were assessed based on this scale. all items were rated on a four - point scale, where 0 = does not apply to me, and 3 = applies to me very much or most of the time. as the stress scale questionnaire is designed to be used especially in developed countries (with the cut - off point of 14), based on the receiver operating characteristic (roc) curve, the cut - off point was reconsidered by the authors and found to be 12 for iran. the stress scale questionnaire was translated and validated by a group of public health tutors and professors in shahr - e - kord and esfahan university of medical sciences. the internal reliability of the questionnaire as a dass subscale was found to be high based on a cronbach 's alpha of 0.84. afterwards, perceived stress and likely stressors, including economical problems, family - related problems, work environment, colleagues and task type, were asked. the collected data were analyzed using the statistical package for the social sciences v. 16 (spss inc., chicago, il, usa) for windows. for bivariate analysis, mann whitney and chi - square tests were used. the logistic regression model was examined to determine the associated parameters of stress among the participants. as a cross - sectional design, we applied analysis of a data set, extracted from the self - report information of employees who worked in the operational units of esco, one of the biggest iranian governmental companies. based on the single proportion formula at 95% confidence interval (ci), the sample size was estimated to be composed of 380 respondents, which rose to 440 to increase the confidence level, while 40 were excluded due to their incomplete questionnaires. all the 400 selected participants were male workers who had at least 1 year work experience in esco, and were sampled based on the randomized sampling method from march to july 2008. all the questionnaires were anonymous and we assured participants that their information would be kept confidential. this study was also approved and supervised by the head of the research and development department of esco. two different types of pre - tests (formal and informal) were conducted prior to the main study. the informal pre - test was designed to make any correction in question wording, content and clearness of scenarios, and was administered to 25 subjects similar to the main study participants. with a cronbach 's alpha of 0.87, the formal pre - test was also conducted in march 2008 on 36 esco employees who were not entered in the main study. the results of these two pre - tests helped the researchers to improve the questionnaires to be more easy - to - understand for the participants. the questionnaires were filled out through interviews and no financial incentives were offered to the participants. the main questionnaire included two main parts ; in the first part, socio - demographic data, including age, number of children, marital status, monthly income, educational level, shift work and years of work experience, were asked. an outcome - related questionnaire was designed based on the stress scale extracted from the validated depression, anxiety, stress scale (dass). in the stress scale questionnaire, difficulty in relaxing, nervous arousal, being easily upset / agitated, being irritable / over - reactive and restlessness were assessed based on this scale. all items were rated on a four - point scale, where 0 = does not apply to me, and 3 = applies to me very much or most of the time. as the stress scale questionnaire is designed to be used especially in developed countries (with the cut - off point of 14), based on the receiver operating characteristic (roc) curve, the cut - off point was reconsidered by the authors and found to be 12 for iran. the stress scale questionnaire was translated and validated by a group of public health tutors and professors in shahr - e - kord and esfahan university of medical sciences. the internal reliability of the questionnaire as a dass subscale was found to be high based on a cronbach 's alpha of 0.84. afterwards, perceived stress and likely stressors, including economical problems, family - related problems, work environment, colleagues and task type, were asked. the collected data were analyzed using the statistical package for the social sciences v. 16 (spss inc., chicago, il, usa) for windows. for bivariate analysis, mann whitney and chi - square tests were used. the logistic regression model was examined to determine the associated parameters of stress among the participants. from all 400 participants, 351 (87.7%) were married, 316 (79%) had at least one child, 313 (78.2%) attainted high school or higher education, 245 (61.3%) were shift workers (with rotational duty) and 358 (89.5%) had a monthly salary of more than $ 600. the mean age of the participants was 37 7.4 years and the mean work experience was 13.6 7.5 years [table 1 ]. socio - demographic profile of the esco employees (n=400) although the prevalence of perceived stress was 91.8% among the participants, the results showed that 53% of the participants were found to be stressful. severities of stress were found among 302 (75.8%) as mild or moderate, 53 (13.2%) as severe and 11 (2.8%) as extreme [table 2 ]. perceived stress among the employees of esco (n=400) according to the logistic regression results, a monthly income of less than $ 600 (p < 0.05, or = 1.88, 95% ci = 1.21 - 2.94), family - related problems (p < 0.05, or = 2.75, 95% ci = 1.22 - 6.21), work environment (p < 0.05, or = 3.09, 95% ci = 1.78 - 5.33) and having a second job (p < 0.05, or = 2.68, 95% ci = 1.78 - 6.78) were significantly associated with a higher prevalence of stress among the participants [table 3 ]. predictors of occupational stress among the employees of esco, extracted from logistic regression (n=400) in this study, which was conducted to evaluate the prevalence of occupational stress and its associated factors among the employees of the operational units in esco, one of the biggest industrial units in iran, 53% of the participants were found to be stressful. factors including monthly income, family - related problems, work environment and having a second job showed a significant association with the outcome among the participants. as mentioned earlier, 53% of the participants were defined as stressful in this study. compared with other reports from iran, it seems that occupational stress is more prevalent among the employees of esco ; this ratio was a little higher than 30% among the members of the iranian police force. among managers and matrons of educational hospitals in tehran, in addition, about 40% of the iranian rural health workers reported moderate to severe stress. some exceptions also exist in the literature ; for instance, the prevalence of occupational stress was much higher (67%) among the iranian air force employees, which, given their peculiar occupational situation, seems to be inevitable. the results from the developed countries also show a much higher prevalence of occupational stress among our participants. for example, in an epidemiological survey of 17,000 randomly selected people in the uk, about 20% of the subjects reported having very high or extremely high levels of stress at work. however, as the prevalence of occupational stress is usually evaluated among non - industrial employees, comparison of the results would not be a proper touchstone to assess this outcome among iranian employees. having a monthly income less than $ 600 was significantly associated with the higher prevalence of stress among the participants (or = 1.88, 95% ci = 1.21 - 2.94). association between a lower salary and a higher prevalence of occupational stress has been suggested in the literature, both in iran, and in other countries. a mismatch between the efforts and income level might lead to negative feelings, which cause stress among the employees. a low income also seems to be associated with other negative impacts, such as increased job insecurity, irregular working hours, occupational hazards and low work control, which might increase occupational stress and its negative consequences. family - related problems also increased the prevalence of occupational stress among the employees of esco (or = 2.75, 95% ci = 1.22 - 6.21). the major concern of the esco employees was about their children 's future, especially about their work situation and housing status. however, this association might be directly caused by economic problems. as another variable, work environment showed a significant association with the outcome (or = 3.09, 95% ci = 1.78 - 5.33). as a suggested theory, kudielka. divided work conditions into some components, including job demand, job control, social support and factors related to the effort reward imbalance. based on this theory, rusli. evaluated these components and suggested that one component (job demand) is directly related to stress many other researches in the literature have also suggested an association between work environment and the prevalence of stress among employees. unexpectedly, having a second job was also associated with a higher prevalence of stress among our participants (or = 2.68, 95% ci = 1.78 - 6.78). at the first glance, it seems that having a second job might partly reduce the existing economic problems. however, likely consequent negative impacts such as doubled stressful work conditions weakened the protective role of higher income in exposure to occupational stress among the employees of esco and, consequently, caused this surprising finding. the results of this study showed no significant association between the socio - demographic variables such as age, marital status and educational level with the prevalence of occupational stress among the participants. however, the results of few other studies are not in line with this finding. in general, it seems that the impact of socio - demographics on the occupational stress is strongly influenced by the effects of more important factors such as economic problems. based on the results of this study, we recommend policy makers to pay more attention to non - individual factors, especially to economic problems, rather than socio - demographic variables to get the best feedback in terms of stress reduction among iranian employees. in this regard, it seems that the role of the economic problems is more striking than other factors. lack of research about the occupational stress among employees of industrial units in iran highlights the need for more attention to this population as a more at - risk group in exposure to this outcome. finally, educating the proper methods of coping with stress might be effective to reduce the prevalence of occupational stress among employees in iran. our study had some limitations ; this study had a cross - sectional design and the causative associations were not conclusive. as the data collected were self - reported, consequently, over - report and/or under - report of facts might be likely. and, finally, some variables such as marital status (single / married) and educational level (primary and junior / high school and higher) could be evaluated in a more detailed situation. despite these limitations, this study is one of the few studies that evaluate occupational stress in a large industrial unit in iran. drawing on the results, more than 50% of the employees of the operational units in esco were found to be stressful. monthly income, family - related problems, work environment and having a second job were significantly associated with the prevalence of occupational stress among the participants. nevertheless, existing evidence asserts the importance of further research to evaluate occupational stress among iranian employees, especially among the industrial employees. | background : lack of data on occupational stress among iranian industrial employees persuaded us to design and conduct this study to evaluate the prevalence and associated parameters of occupational stress among male employees of the esfahan steel company (esco), one of the biggest industrial units in iran.methods:in this cross - sectional study, 400 male employees were sampled from the operational divisions of the company. socio - demographic data and stress - related variables were entered into a logistic regression to determine significant associated factors of occupational stress among the participants.results:from all samples, 53% were found as stressful. a monthly salary of less than $ 600 (or = 1.88, 95% confidence interval [ci ] = 1.21 - 2.94), family - related problems (or = 2.75, 95% ci = 1.22 - 6.21), work environment (or = 3.09, 95% ci = 1.78 - 5.33) and having a second job (or = 2.68, 95% ci = 1.78 - 6.78) were significantly associated with the outcome.conclusions:attention to some variables, especially economic problems and the work environment of employees, might play a protective role against the prevalence of occupational stress, not only among the employees of esco but also among all industrial employees in iran. |
dendritic cell - specific intercellular adhesion molecule (icam)-3-grabbing nonintegrin (dc - sign ; also called cd209) is a type ii transmembrane protein with a c - type lectin extracellular domain [1, 2 ]. functionally, dc - sign mediates antigen capture for processing and presentation in the context of major histocompatibility complex class ii molecules [3, 4 ]. dc - sign plays an important role in establishing the initial contact between dendritic cells (dcs) and resting t lymphocytes through its recognition of icam-3 and also mediates dcs trafficking through interactions with endothelial icam-2. in addition, as a pattern recognition receptor (prr), dc - sign function is subverted by numerous pathogens as a strategy to escape from immunosurveillance [6, 7 ]. in the case of human immunodeficiency virus (hiv), dc - sign binds gp120 with high affinity, captures hiv in the periphery, and promotes efficient infection of cells expressing hiv receptors and coreceptors [8, 9 ]. dc - sign also mediates virus uptake by dendritic cells through endocytosis, where hiv remains infectious for prolonged periods of time, evades immune surveillance, and is efficiently transferred to cd4 + t cells in lymph nodes, like a trojan horse. therefore, dc - sign plays a key role in hiv transmission, especially in terms of sexual transmission. dc - sign is preferentially expressed on immature dcs in peripheral tissues and in - vitro - derived monocyte - derived dendritic cells (mddcs) [2, 5 ] and is usually considered as a dc - specific phenotypic marker. it has also been detected on synovial, placenta, and alveolar macrophages, and on a small subset of cd14 + peripheral blood dcs [1012 ]. trumpfheller. analyzed the function of dc - sign in different cells, including monocyte - derived dcs, and three different dc - sign - expressing transfectants termed thp-1, 293, and hos. they found that the function of dc - sign in hiv-1 transmission depends on its cellular context, since only dcs and the thp monocyte cell line, but not 293 and hos, are able to use dc - sign to retain hiv-1 in a highly infectious state for several days. the existing studies indicate that the expression and function of dc - sign is precisely regulated. however, less is known about the mechanism of the regulated expression of dc - sign.. found that the expression of dc - sign on mddcs was induced by interleukine-4 (il-4) through the jak - stat signaling pathway. however, the erk signaling pathway is also considered to be a candidate pathway for dc - sign expression since it is involved in most il-4/il-4 receptor effects [15, 16 ]. given the important role of the functional activities displayed by dc - sign, it is necessary to determine the signaling pathways and factors controlling its expression. since the low level of dcs in human peripheral blood, immature mddc in vitro studies is mostly derived from peripheral blood monocytes in the presence of gm - csf and il-4 [17, 18 ]. the expression of dc - sign is largely dependent on il-4, a cytokine whose action drives monocyte / macrophages into the alternative activation pathway. thp-1 cells, which are widely used as a monocyte / macrophage differentiation model [20, 21 ], can be induced to express functional dc - sign when differentiated by protein kinase c (pkc) activators and il-4. it has been reported that the signaling mechanisms regulating dc - sign expression in monocyte - derived macrophages and dcs also control the expression of this pathogen receptor in differentiated thp-1 cells, implying that the thp-1 cell line represents a useful cellular system for the analysis of the regulated expression and functional activities of dc - sign. here, we used thp-1 cells as the in vitro model of mddcs to study the signaling pathways of il-4-induced expression of dc - sign. we found that multiple signaling pathways were involved in the process of il-4-regulated dc - sign expression, the major of which was the erk signaling pathway. recombined human il-4 was obtained from r&d system (minneapolis, usa) and used at 1000 units / ml. pma was obtained from sigma (st. louis, usa) and used at 10 ng / ml. the nf-b inhibitor helenalin, jak2/3 inhibitor ag490, p38mapk inhibitor sb202190, and the mek1/2 inhibitor pd98059 were purchased from merck (gibbstown, usa) and used at 1 m (hellenalin), 300 g / ml (ag490), 5 m (sb202190), and 50 m (pd98059). mouse anti - human dc - sign monoclonal antibody was obtained from r&d system. the acute monocytic leukemia cell line thp-1 was derived from the american type culture collection (atcc, rockville, usa) and cultured in rpmi 1640 medium supplemented with 10% fetal bovine serum (complete medium), being maintained at 35 10 cells / ml. thp-1 cells were routinely seeded at 5 10 cells / ml in tissue culture dishes, and differentiation was induced by treatment with pma (10 ng / ml) either alone or in combination with il-4 (1000 units / ml). il-4 was added into the cells after treatment with pma for 18 h with complete medium and starvation for 6 h with rpmi 1640 medium supplemented with pma (10 ng / ml). fetal bovine serum was added to 10% 2 h after il-4 addition and differentiation was allowed to proceed for 72 h. for inhibition of signaling pathways, inhibitors of the signaling pathways were added 30 min before il-4 addition and the process of differentiation was as per the above description. for subsequent analysis, differentiated thp-1 cells were detached from tissue culture plates by incubation in pbs 10 mm edta on ice. total rna from untreated or differentiated by pma, pma, plus il-4, or signaling - inhibitor - treated thp-1 cells was extracted using total rna extraction kit (invitrogen) and reverse transcribed into cdna in a total volume of 20 l. real - time polymerase chain reaction (pcr) of dc - sign mrna was performed by amplifying samples of target cdna in a dna engine chromo 4 real - time quantitative pcr system (bio - rad, hercules, usa) using an sybr green kit (roche diagnostics gmbh). oligonucleotides were used for dc - sign mrna amplification (cd209 sense : 5-gggaattcagagtggggtgacatgagtgac-3 and cd209 antisense : 5-ccccaagcttgtgaagttctgctacgcaggag-3) and the inner conference -actin mrna amplification (-actin sense : 5-gggaattcagagtggggtgacatgagtgac-3 and -actin antisense : 5-ccccaagcttgtgaagttctgctacgcaggag-3). the reaction system was a mixture of 10 l sybr green master mix (qiagen), 0.2 l of each oligonucleotides primers, 2 l cdna, and the final volume was taken to 20 l with water. real - time pcr was performed for 40 cycles of denaturation (95c, 45 s), annealing (62c, 30 s), and extension (72c, 30 s), and double - stranded dna was measured at 86c after each cycle. expression of dc - sign on surface of untreated or differentiated by pma, pma+il-4, or signaling - inhibitor - treated thp-1 cells was determined by flow cytometry. the harvested thp-1 cells were washed twice with pbs, incubated with 10 l mouse anti - human dc - sign monoclonal antibody (2550 g / ml) for 1 h, and washed again. secondary antibody was added and incubated for 1 h. flow cytometry analysis was performed with a four - color facscan flow cytometer (becton dickinson). results are expressed as the expression index : percentage of marker - positive cells and the mfi. thp-1 cells differentiated with pma for 18 h were starved for 6 h with rpmi 1640 medium supplemented with pma (10 ng / ml) and then induced with il-4 (1000 units / ml). cells were put on ice and washed by ice cooled pbs to stop the phosphorylation of the kinases and factors of signaling pathways 0 min, 5 min, 10 min, 20 min, 30 min, and 60 min after the addition of il-4. cytoplasmic protein and nuclear protein were extracted using cytoplasmic / nuclear protein extraction kit (pierce). phosphorylated and nonphosphorylated signaling kinases and factors were determined by western blot as described before. 10 g of each sample was subjected to sds - page under reducing conditions and transferred onto an immobilon polyvinylidene difluoride membrane (millipore, bedford, usa). after blocking with 5% nonfat dry milk in 50 mm tris - hcl, ph 7.6, 150 mm nacl, 0.1% tween 20, 1 - 2 g / ml primary antibodies were added and incubated overnight at 4c. after incubation with hrp labeled secondary antibodies for 2 h, the membrane was exposed with the versadoc 5000mp image analysis system (bio - rad). detection of signaling kinases and factors was carried out using specific monoclonal antibodies anti - stat6, anti - erk1/2, anti - nf-bp65, anti - ib, and anti - p38. and phosphorylated kinases and factors were detected using anti - phospho - stat6-tyr641, anti - phospho - erk1/2-thr202/tyr204, anti - phospho - nf-bp65-ser536, anti - phospho - ib - thr19/ser23, and anti - phospho - p38 mapk - thr180/tyr182 (cell signaling technology, beverly, usa). total dna was extracted from thp-1 cells and the whole region of dc - sign promoter was amplified by prc using the forward and reverse primers of p1 and p2, where underlined residues represent additional sequences containing mlu i or bgl ii restriction sites, as shown in table 1. the fragments of dc - sign promoter on both sides of ap-1 were amplified using the forward and reverse primers of p1 and p3, and p2 and p4 separately, and then were linked by prc with a combing primer p5, and forward and reverse primers of p1 and p2, conforming the dc - sign promoter without ap-1 binding site. and the dc - sign promoter without ets-1 binding site was amplified in the same way, using primers of p1, p2, p6, p7, and p8, as shown in table 1. the mixture of pcr reaction consisted of 0.2 l dna template, 2 l forward / reverse primers (10 mm), 4 l mgcl2 (25 mm), 4 l dntp (2.5 mm), 5 l 10pcr buffer, 0.5 l pfu dna polymerase (2 u/l, promega), and the final volume was taken to 50 l with water. pcr was performed for 30 cycles of denaturation (95c, 30 s), annealing (5762c, 1 min), and extension (72c, 1 min). the fragments of dc - sign promoters were double digested with mlu i and bgl ii and gel purified and ligated into mlu i- and bgl ii - digested pgl-3/basic and pgl-3/enhancer luciferase reporter vectors to generate complete dc - sign promoter luciferase reporter plasmids and those without ap-1 and ets-1bingding sites. transfection of dc - sign promoter luciferase reporter plasmids and the inner control prl - tk in hacat and 293 t cells (atcc) was accomplished using trans fast (promega). dc - sign promoter luciferase reporter plasmids and the inner control prl - tk were electransfected into thp-1 cells using amaxa cell line transfection kit (amaxa, germany) in amaxa nucleofector electroporation apparatus (amaxa, germany) with the v-010 electroporation procedure. the transfected cells were cultured for 48 h, and the luciferase activities of dc - sign promoter luciferase reporter plasmids and the inner control prl - tk were detected using the dual luciferase reporter assay kit (promega) in glomax96 microplate luminometer (promega). the relative activity of dc - sign promoter was expressed by the ratio of activity between dc - sign promoter luciferase reporter plasmids and the inner control prl - tk. every test was repeated three times and data was shown as mean se. the statistical significance (p values) of the results student 's t - test was used to compare between two groups while one - way anova was used when comparing more than three groups. we determined the dc - sign mrna and expression on untreated, pma - treated, and pma plus il-4-treated thp-1 cells at different times of differentiation. the results of mrna testing by real - time quantitative pcr showed that pma differentiation for 24 hours increased the level of dc - sign mrna in thp-1 cells up to 30 folds and induction of il-4 greatly enhanced the level of dc - sign mrna. the highest level of dc - sign mrna was detected when induced by pma and il-4 for 24 hours, which was 469 148 times higher than that of untreated thp-1 cells (p < 0.01, see figure 1(a)). therefore, we further detected dc - sign expression on cell surface by flow cytometry. the results showed that pma induced a low level of dc - sign expression on thp-1 cell surface with the percentage of 14.54 3.97% dc - sign+ thp-1 cells and the mean fluorescence intensity (mfi) of 18.12 7.51. il-4 greatly enhanced the percentage of dc - sign+ thp-1 cells, and the mfi at the same time. the highest expression of dc - sign on thp-1 cells differentiated by pma plus il-4, with the percentage of 61.23 15.21% dc - sign+ thp-1 cells and the mfi of 56.80 21.35, was found at 72 hours (24 h for pma and 48 h for pma plus il-4). we found that the majority of the cells were overactivated and aging after differentiated by pma plus il-4 for 96 hours, and the proportion of dead cells increased. although the dc - sign (+) thp-1 ratio was declined, the mfi remained high (figure 1(b)). the stimulation by il-4 on il-4 receptor was mainly transducted through the jak - stat and erk signal pathways [2427 ]. in addition, our previous study suggested that the nf-b signaling pathway may also be involved in the expression of dc - sign. we selected four different alternative pathways (erk, nf-b, jak - stat, and p38mapk) as the target signaling pathways, and detected the dc - sign expression by blocking the corresponding signaling pathways with specific inhibitors. real - time pcr showed that inhibitor of erk pathway (pd98059) blocked the expression of dc - sign mrna by 83.84 4.13%, which was the most obvious among the four inhibitors, followed by the inhibitor of jak - stat pathway (ag490) which decreased dc - sign mrna by 67.16 5.67%. blocking of the nf-b pathway also decreased dc - sign mrna by 40.08 10.12%. blocking of dc - sign mrna by inhibitor p38mapk pathway (sb202190) was not significant (see figure 2(a)). we further detected expression of dc - sign on thp-1 cell membrane using flow cytometry by blocking the signaling pathways with specific inhibitors. dc - sign expression was decreased from dc - sign+ rate of 54.03 7.66% (mfi = 60.53 15.42) on thp-1 cells induced by pma+il-4 to 16.42 5.88% (mfi = 29.26 9.76) on differentiated thp-1 cells treated by pd98059, close to the pma - treated thp-1 cells (15.17 4.47%, mfi = 18.12 7.58.), which mean the nearly total block of il-4 induction. ag490 decreased dc - sign expression by 55.8% with dc - sign+ thp-1 cells of 23.89 5.12% (mfi = 39.67 15.46). hellenalin decreased dc - sign expression by 40% with dc - sign+ thp-1 cells of 32.69 6.69% (mfi = 53.27 18.53). expression of dc - sign on thp-1 cells treated with sb202190 was almost not reduced (see figure 2(b)). in order to get the direct evidence of activation of the signaling pathways the result of western blot test showed that, within the 120 min after addition of il-4, the cytoplasmic levels of phosphorylated erk1/2 of erk pathway, phosphorylated stat6 of jak - stat pathway, and phosphorylated nf-bp65 and i-b of nf-b pathway increased over time from a low concentration to a high concentration, which indicated directly the activation of the three signaling pathways. however, the level of phosphorylated p38 of p38mapk pathway showed no increase in cytoplasm, indicating the inactivity of the p38mapk pathway (figure 3(a)). we further determined whether the phosphorylated erk1/2, stat6 and nf-bp65 enter the nucleus to activate dc - sign promoter directly or through other nuclear factors. the results showed a similar trend of increase of phosphorylated erk1/2, stat6, and nf-bp65 in the nucleus of pma plus il-4-induced thp-1 cells within the first 120 min of il-4 induction (figure 3(b)). liu. reported that there are five nuclear transcription factor binding sites (ap-1, sp-1, ets-1, lyf-1, and nf-b) in the cis acting elements of dc - sign promoter. we studied the role of ap-1 and ets-1 nuclear transcription factor binding sites in the activity of dc - sign promoter, which are the main nuclear factors exploited by erk signaling pathway, using luciferase reporter system. the sequence of complete dc - sign promoter and the mutants with ap-1 or ets-1 binding site deletion were identified by gene sequencing (figure 4). the results showed that dc - sign promoter activity in thp-1 cells was much higher than in hacat cells and 293 t cells. the deletion of ets-1 nuclear transcription factor binding site had significant impact on the dc - sign promoter activity, no matter in hacat cells, 293 t cells, and untreated or pma - treated or pma plus il-4-treated thp-1 cells. the activity of dc - sign promoters without ets-1, no matter the basic or containing enhancers, almost disappeared completely (decreased more than 90%, p < 0.05). the activity of dc - sign promoters without ap-1 decreased partially, from 28.60% to 47.88% in different cell lines, which was most obvious in the thp-1 cell line (p < 0.05, figure 5). thp-1 cells can be differentiated into monocytes / macrophages by pma [30, 31 ]. puig - krger. found that differentiated thp-1 cells can be induced into dcs by il-4 with the expression of dc - sign, and pma stimulation can greatly enhance dc - sign expression. our study further identified il-4-differentiated thp-1 cells as a good in vitro cell model of dc - sign expression. we found that il-4 can significantly induce high expression of dc - sign in both percentage of positive cells and expression density on the cell surface, which demonstrates that dc - sign expression is il-4 dependent. the highest expression of dc - sign on thp-1 cells differentiated by pma plus il-4 was found at 72 hours (24 h for pma and 48 h for pma plus il-4), which was different from the results of puig - krger 's study with the highest expression at 96 h (24 h for pma and 72 h for pma plus il-4). and our study also found that the level of dc - sign mrna was significantly increased, indicating that expression of dc - sign is enhanced by il-4 on the level of mrna. researchers have found that expression of many genes in t lymphocytes is induced by il-4 in an stat-6-dependent manner [26, 27 ]. recent studies have revealed that the jak 2/3 inhibitor tyrphostin ag-490 can prevent dc - sign upregulation on mddcs and differentiated thp-1 cells, suggesting that stat6 activation participates in il-4-induced dc - sign expression [14, 32 ]. il-4-initiated stat6 activation is not sufficient for dc - sign upregulation to take place, because il-4 treatment of proliferating thp-1 cells leads to stat6 activation, but not to dc - sign upregulation, thus suggesting the involvement of additional pathways in the il-4-dependent dc - sign upregulation in thp-1 cells. our study reveals that the erk1/2 inhibitor pd98059 can almost completely inhibit the upregulated dc - sign expression induced by il-4, suggesting that il-4-induced upregulation of dc - sign expression is mainly dependent on the erk mapk signaling pathway. and the jak - stat signaling pathway is also involved in the process for the partial inhibition of il-4-induced dc - sign expression by stat6 inhibitor ag490, which is consistent with the previous studies [14, 32 ]. in addition, in erk mapk signaling pathway, gene activation is mainly regulated through the transcription factors ets-1 and ap-1, and the forming of a heterodimer of ets-1. we further studied the activity of dc - sign promoters without ets-1 or ap-1 transcription factor binding sites and found that the activity of dc - sign promoter without ets-1 transcription factor binding site almost completely disappeared, indicating that the ets-1 transcription factor binding site plays a key role in the activation of dc - sign promoter. the deletion of ap-1 transcription factor binding site can make the activity of dc - sign promoter decreased partly, the reason of which may be the block of heterodimer of ets-1 and ap-1 binding to the cis acting elements of dc - sign promoter. in addition, we detected the phosphorylation of protein kinase in the signaling pathways, and found that the levels of phosphorylated erk1/2 of erk pathway and phosphorylated stat-6 of jak - stat pathway gradually increased over time after il-4 addition, which provides direct evidence for the activation of the signaling pathways. the increased levels of phosphorylated erk1/2 and stat-6 in the nucleus provide further proof of the activation of the signaling pathways. no increased level of phosphorylated p38 kinase is found in either the cytoplasm or nucleus of differentiated thp-1 cells, indicating that the p38 pathway, which also belongs to the family of mapk signaling pathways, like the erk pathway, is not activated by il-4 in the expression of dc - sign. another signaling pathway we found involved in il-4 induced high expression of dc - sign on thp-1 cells is the nf-b pathway, of relevance in a variety of inflammation and immunological responses. our previous study revealed that the deletion of nf-b transcription factor binding site allows the activity of dc - sign promoter to be reduced by half, and the overexpression of nf-b protein can increase the expression of dc - sign on thp-1 cells, suggesting that nf-b signaling pathway may be involved in the expression of dc - sign. another study revealed that dexamethasone, the inhibitor of nf-b signaling pathway, can reduce the expression of dc - sign. combined with the reduced expression of dc - sign by inhibiting nf-b signaling pathway and the increase of phosphorylated nf-b in cytoplasm and nucleus of differentiated thp-1 cells, it can be inferred that the nf-b signaling pathway is also involved in the process of upregulated dc - sign expression. one is pma, which can induce the differentiation of thp-1 cells through activation of pkc and nf-b signaling pathway. the synergistic effect of pma and il-4 found in other studies, which can highly increase the expression of dc - sign on thp-1 cells [14, 28, 32 ], can be attributed to the activation of the nf-b signaling pathway. in summary, our study demonstrates that multiple signaling pathways are involved in the process of il-4-induced high expression of dc - sign on thp-1 cells, in which erk pathway is the main signaling pathway, and jak - stat, and nf-b signaling pathways are also involved. the jak - stat and nf-b signaling pathways may be activated directly by il-4/il-4 receptor, or by the erk pathway indirectly. complex interactions may exist between the signaling pathways, forming a regulatory network (see figure 6). in addition, the regulatory mechanisms of intracellular signaling pathways vary in different cells, and signaling pathways regulating dc - sign expression should be studied in other cells. | dendritic cell - specific intercellular adhesion molecule-3 grabbing nonintegrin (dc - sign) is an important pattern recognition receptor on dendritic cells (dcs), and its expression shows significant cytological and histological specificity, being interleukine-4 (il-4) dependent. the signaling pathways through which il-4 regulates expression of dc - sign are still unclear. we used phorbol 12-myristate 13-acetate- (pma-) differentiated thp-1 cells as the in vitro model of monocyte / macrophage cells to study the signaling pathways involved in il-4-regulated expression of dc - sign. we found that a high expression of dc - sign could be induced by il-4 at the levels of mrna and cell surface protein. upregulated expression of dc - sign was almost completely blocked by the specific inhibitor of erk pathway, and partly reduced by the specific inhibitors of jak - stat and nf-b pathways. the activation of the three signaling pathways was directly confirmed by testing the phosphorylation of protein kinase within the cytoplasm and nucleus over time. the analysis of cis - acting elements of dc - sign promoter showed that the activity of dc - sign promoter without ets-1 transcription factors binding site almost completely disappeared. our results demonstrated that multiple signaling pathways are involved in il-4 induced high expression of dc - sign on thp-1 cells, in which erk pathway is the main signaling pathway and mediated by the ets-1 transcription factors binding site. |
temporomandibular joint (tmj) disorders refer to a heterogeneous group of clinical conditions including pain and limited movement of the tmj. in addition to the increased pain sensitivity and referred pain beyond the affected tmj, both autonomic and endocrine functions can be altered and tmj inflammation can be persistent [13 ]. while the exact mechanisms of tmj pain remain unclear, a growing body of evidence indicates that persistent tmj pain conditions may be mediated by both peripheral and central mechanisms in the trigeminal nervous system. inflammation of the tmj region is a likely triggering factor leading to the pathogenesis of persistent tmj pain [4, 5 ], which may result in increased neuronal excitability in both the peripheral nervous system (e.g., trigeminal ganglion) and the central nervous system (e.g., trigeminal nuclei) [6, 7 ]. transient receptor potential vanilloid 1 (trpv1) and ankyrin 1 (trpa1) are two critical types of trp channels that are strongly implicated in the genesis of somatic inflammatory pain [8, 9 ]. activation of trpv1 and trpa1 can enhance somatic inflammatory pain not only via peripheral sensitization [8, 10, 11 ], but also via central sensitization. trpv1 and trpa1 have also been shown to be involved in persistent pain in the trigeminal system [1316 ]. great pharmaceutical effort has been devoted to developing small - molecule inhibitors of trp channels (e.g., trpv1 and trpa1) but with limited success. it has been recently identified that several families of novel proresolution lipid mediators (prlms), such as resolvins and protectins, derived from omega-3 unsaturated fatty acids and further demonstrated these prlms possess potent anti - inflammatory and proresolution actions in animal models [1719 ]. maresins, derived from docosahexaenoic acid (dha), are newly described macrophage - derived mediators of inflammation resolution. maresin 1 (7r, 14s - dihydroxy - docosa-4z, 8e, 10e, 12z, 16z, 19z - hexaenoic acid) is the first identified maresin family member. maresin 1 shows potent analgesic actions by inhibiting formalin - induced inflammatory pain and chemotherapy - induced neuropathic pain in the somatic system and suppressing trpv1 activity in dorsal root ganglion (drg) neurons. however, the role of maresin 1 in regulating different trp channels, synaptic transmission, and tmj pain in the trigeminal system has yet to be examined. in the present study, i investigated whether and how maresin 1 can effectively modulate trp channels in tmj - related trigeminal primary afferent neurons and tmj inflammation - induced synaptic plasticity in the trigeminal nucleus. all surgical and experimental procedures were reviewed and approved by the institutional animal care and use committee in college of medicine, gachon university. animals were habituated for at least 1 week prior to experiments in a conventional facility with a 12:12 h light cycle (lights on 8.00 am) and had ad libitum access to water and food. tmj - related trigeminal ganglion neurons were identified by tmj injection of a retrograde labeling tracer, dii (d-282, molecular probes, eugene, or, usa), a fluorescent dye [22, 23 ]. briefly, mice were anesthetized with 2% isoflurane. the skin overlying the tmj was shaved, and the injection target was identified by palpating the zygomatic arch and mandible. the retrograde tracer dii (3 l ; 17 mg / ml) was slowly injected into the tmj capsule. the needle (30-gauge) was left in place for ~2 minutes before being withdrawn in order to minimize leakage of dye from the injection site. after 3 days, trigeminal ganglia prepared in 4c hank 's balanced salt solution (hbss ; welgene, daegu, korea) were incubated in 2 ml hbss containing 0.25% trypsin (invitrogen, carlsbad, ca, usa) at 37c for 60 min. the cells were washed, triturated with a flame - polished pasteur pipette, and placed on 0.5 mg / ml poly - l - ornithine (sigma, st. briefly, following whole - cell patch clamp recordings, dii - labeled tg neurons were harvested into patch pipettes with tip diameters of about 1525 m, gently put into reaction tubes containing reverse - transcription reagents, and incubated for 1 h at 50c (superscript iii, invitrogen). the first round of pcr was performed in 50 l of pcr buffer containing 0.2 mm dntps, 0.2 m primers, 5 l rt product, and 0.2 l platinum taq dna polymerase (invitrogen). the protocol included an initial 5 min denaturizing step at 95c followed by 40 cycles of 40 s denaturation at 95c, 40 s annealing at 55c, and 40 s elongation at 72c. the reaction was completed with 7 min of final elongation. for the second round of amplification, the reaction buffer (20 l) contained 0.2 mm dntps, 0.2 m inner primers, 5 l of the first round pcr products, and 0.1 l platinum taq dna polymerase. the amplification procedure for the inner primers was the same as that for the first round. a negative control was obtained from pipettes that did not have cell contents but were submerged in the bath solution. the pcr products were displayed on ethidium bromide - stained agarose gels (2%). dii - labeled tg neurons were visualized using a fixed - stage fluorescence microscope (bx50wi, olympus, japan) with nomarski optics and the sizes of soma were estimated. as previously described, dii - labeled tg neurons were classified into three groups, small - sized (022 m), medium - sized (2337 m), and large - sized (3860 m) neurons. whole - cell voltage- and current - clamp recordings were performed at room temperature to measure currents and action potentials, respectively, with an axopatch-200b amplifier (axon instruments, union city, ca, usa). when filled with the pipette solution, the resistance of the pipettes was 4 - 5 m. the recording chamber (300 l) was continuously superfused (2 - 3 ml / min). series resistance was compensated for (> 80%), and leak subtraction was performed. data were low - pass - filtered at 2 khz and sampled at 10 khz. the pclamp8 (axon instruments) software was used during experiments and analysis. the pipette solution for voltage - clamp experiments was composed of (in mm) 126 k - gluconate, 10 nacl, 1 mgcl2, 10 egta, 2 naatp, and 0.1 mggtp, adjusted to ph 7.4 with koh and osmolarity 295300 mosm. ca - free extracellular solution contained 0 mm cacl2 and 2 mm egta for chelation of ambient ca. the extracellular solution for voltage - clamp experiments contained (in mm) 140 nacl, 5 kcl, 2 cacl2, 1 mgcl2, 10 hepes, and 10 glucose, adjusted to ph 7.4 with naoh and osmolarity 300310 mosm. voltage - clamp experiments were performed at a holding potential of 60 mv. a portion of the subnucleus caudalis (sp5c) was removed from mice (46 weeks old) under urethane anesthesia (1.52.0 g / kg, i.p.) and kept in preoxygenated ice - cold kreb 's solution. the slices were perfused with kreb 's solution (810 ml / min) and saturated with 95% o2 and 5% co2 at 37c. whole - cell patch clamp recordings were made from the superficial layers of the sp5c (lamina i - ii) neurons in voltage - clamp mode at 37c. after establishing the whole - cell configuration, neurons were held at the potential of 70 mv to record sepscs. the internal solution contained (in mm) 135 k - gluconate, 5 kcl, 0.5 cacl2, 2 mgcl2, 5 egta, 5 hepes, and 5 atp - mg, adjusted to ph 7.4 (with koh) and osmolarity 295300 mosm. the perfusion solution contained (in mm) 117 nacl, 3.6 kcl, 2.5 cacl2, 1.2 mgcl2, 1.2 nah2po4, 25 nahco3, and 11 glucose. the resistance of a typical patch pipette is 510 m. membrane currents were amplified with an axopatch 200b amplifier (axon instruments) in voltage - clamp mode. data were stored on a personal computer using pclamp 10 software and analyzed with mini analysis (synaptosoft inc., fort lee, nj, usa). capsaicin and allyl isothiocyanate (aitc), complete freund 's adjuvant (cfa), were obtained from sigma. pertussis toxin (ptx), an irreversible inhibitor of gi - coupled g - protein coupled receptors (gpcrs), was purchased from tocris bioscience (bristol, uk). to determine the involvement of gpcrs in maresin 1 actions, drg neurons were cultured with a ptx (0.5 g / ml) for 18 h. unilateral inflammation was induced by injecting cfa (20 l, 1 mg / ml) into the tmj using a 30-gauge needle. anova or student 's t - test was used to determine the differences using the software origin 6.0 (microcal software, inc., northampton, ma, usa). the trigeminal ganglion (tg) is the location of primary afferent neurons for sensing and relaying nociceptive sensations associated with painful conditions such as dental pain, trigeminal neuralgia, and tmj pain [2527 ]. i labeled the tg neurons by tmj injection of a retrograde labeling tracer, dii, a fluorescent dye, as shown in figure 1(a). three days later, dii labeling was detected in the dissociated tg neurons (figure 1(b)), indicating that trigeminal primary afferent neurons innervating the tmj can be investigated in vitro. interestingly, the proportion of dii - labeled neurons is relatively higher in small - sized neurons (n = 99/180, 55%), compared with medium - sized neurons (n = 45/180, 25%) and large - sized neurons (n = 36/180, 20%) (figure 1(c)). these data demonstrate that dii - labeled small - sized tg neurons are responsible for pain sensation as nociceptors associated with tmj pain [22, 24 ]. by performing patch clamp recordings in dii - labeled small - sized tg neurons perfusion of dii - labeled small - sized tg neurons with capsaicin (100 nm) elicited a marked trpv1 current, and this current was dose - dependently inhibited by maresin 1 (figure 2(a)). notably, maresin 1 inhibited trpv1 currents with a very low ic50 (0.11 nm) in dii - labeled small - sized tg neurons. for comparison, maresin 1 also inhibited trpv1 currents with an ic50 (0.17 nm) in small - sized drg neurons, indicating that the ic50 of maresin 1 for inhibiting trpv1 currents in tg neurons is lower than that in drg neurons (figure 2(b)). in addition, current - clamp recording revealed that maresin 1 also completely blocked capsaicin - induced action potentials (figure 2(c)). notably, pretreatment of dii - labeled small - sized neurons with pertussis toxin (ptx, 0.5 g / ml) for 18 h completely blocked the inhibitory effects of maresin 1 on capsaicin - induced trpv1 currents (figure 2(d)), suggesting a gi - coupled gpcr pathway. interestingly, the highest concentration of maresin 1 (0.35 nm) failed to inhibit the aitc - induced trpa1 current in dii - labeled small - sized neurons (figure 2(e)). the nociceptive markers, trpv1 and nav1.8, are known to contribute to pain transduction in nociceptive neurons [22, 24 ]. therefore, i analyzed expression of trpv1 and nav1.8 mrnas using single - cell rt - pcr following electrophysiological and pharmacological characterization of dii - labeled tg neurons by whole - cell recordings. maresin 1 (0.35 nm) completely blocked capsaicin - induced inward currents in dii - labeled medium- and small - sized neurons (figure 3(a) ; cells # 2, # 3, and # 4), and consistently, these three neurons expressed trpv1 and nav1.8 mrnas (figure 3(b)). however, dii - labeled large - sized neurons (figure 3(a) ; cell # 1) did not respond to capsaicin without trpv1 and nav1.8 mrnas. trpa1 mrnas were also detected with trpv1 and nav1.8 mrnas in small - sized tg neurons, but maresin 1 (0.35 nm) failed to inhibit trpa1 currents (figure 3(a) ; cell # 5). to define the functional role of maresin 1 in tmj pain control, i examined the action of maresin 1 on basal and evoked synaptic transmission in lamina ii dorsal horn neurons of the caudal part of the spinal trigeminal nucleus (sp5c), in which the nociceptive primary afferents form the first intracranial synapses in the trigeminal system. patch clamp recording in lamina ii neurons of sp5c slices showed that maresin 1 did not alter basal synaptic transmission : both the frequency and the amplitude of spontaneous excitatory postsynaptic currents (sepscs) were unaltered after maresin 1 treatment (0.35 nm, figures 4(a) and 4(b)). application of capsaicin (500 nm), a selective trpv1 agonist, to trigeminal nuclei slices evoked a significant increase in the frequency but not amplitude of sepscs in sp5c lamina ii neurons [28, 29 ] (figures 4(a) and 4(b)). of interest, maresin 1 (0.35 nm) completely blocked the sepsc frequency increase by capsaicin (figures 4(a) and 4(b)), suggesting that maresin 1 can also abolish trpv1-evoked synaptic plasticity in the sp5c via possible presynaptic mechanisms. next, i investigated whether tmj injury after complete freund 's adjuvant (cfa) injection increased the frequency and amplitude of sepscs and further tested whether maresin 1 can reverse these epsc changes. tmj inflammation by cfa elicited dramatic increases in both sepsc frequency (64%) and amplitude (22%) in lamina ii neurons of sp5c slices prepared from inflamed mice (1 day) (figures 4(c) and 4(d)). superfusion of the sp5c slices with maresin 1 at a very low concentration (0.35 nm) reversed these increases in sepsc frequency and amplitude (figures 4(c) and 4(d)), indicating that maresin 1 could modulate synaptic plasticity via both presynaptic (sepsc frequency) and postsynaptic (sepsc amplitude) mechanisms. in summary, these results demonstrate that (a) maresin 1, at a very low concentration (0.35 nm), completely blocked capsaicin - induced trpv1 currents in tmj - related nociceptive tg neurons, (b) maresin 1 inhibited trpv1-induced increases in sepsc frequency in the trigeminal nucleus (sp5c), and (c) tmj inflammation by cfa injection induced a greater increase in sepscs in the sp5c, which was abolished by maresin 1. thus, i propose that maresin 1 can effectively attenuate tmj inflammation - induced orofacial pain, by modulating trpv1 function in tmj - related trigeminal nociceptive neurons and synaptic plasticity in the trigeminal nuclei. the mechanisms of tmj pain in the trigeminal system can be different from those of arthritis pain in the somatic system. although there are anatomic and functional similarities between the spinal and trigeminal somatosensory systems, the segmental distribution of the somatic sensory input is less well organized in the trigeminal sensory system. in addition, the distance between the ganglion and its target in the trigeminal system is much shorter than that in the somatosensory system. indeed, my pilot study showed that cfa injection into tmj induced a greater increase in excitatory synaptic transmission in the trigeminal nucleus (sp5c) than that induced in the spinal cord by cfa injection into the hind paw of mice (data not shown). using an unbiased lc - ms - ms based lipidomics approach, dr. serhan 's group uncovered two families of endogenous lipid mediators, including resolvins (e.g., resolvin e1, resolvin d1, and resolvin d2) and protectins (e.g., protectin d1 or neuroprotectin d1) in resolving inflammatory exudates. they are biosynthesized from omega-3 fatty acids such as eicosapentaenoic acid (epa) and dha and show remarkable potency in treating inflammation - related diseases in animal models [32, 33 ]. recent studies have demonstrated that resolvins and protectin potently inhibit somatic inflammatory pain in part by modulating trpv1 and trpa1 activity in dorsal root ganglion (drg) neurons [3336 ] in the somatosensory system. maresin 1 is a newly identified macrophage - derived mediator of inflammation resolution and demonstrates potent anti - inflammatory and potent analgesic actions by inhibiting formalin - induced inflammatory pain and chemotherapy - induced neuropathic pain in the somatic system and suppressing trpv1 activity in drg neurons. however, the role of maresin 1 in regulating different trp channels in the trigeminal system remains unexamined. therefore, i first labeled the dissociated tg neurons innervating the tmj by tmj injection of a retrograde tracer, dii, as an in vitro model to study pain - sensing neurons using patch clamp recordings. interestingly, the proportion of dii - labeled neurons is relatively higher in small - sized tg neurons, compared with medium- and large - sized tg neurons (figure 1(c)). single - cell rt - pcr showed that most dii - labeled small - sized tg neurons express both nociceptive markers, trpv1 and nav1.8 mrnas (figure 3(b)). these data strongly support the notion that the majority, but not all, of the dii - labeled small - sized tg neurons may be considered to be nociceptors. the present study has identified maresin 1 as a highly potent endogenous inhibitor for trpv1 in dii - labeled small - sized tg neurons. it is very remarkable that maresin 1, at very low doses (0.040.35 nm), blocked capsaicin - induced trpv1 currents in dii - labeled small - sized tg neurons (figure 2(a)). of interest, the ic50 of maresin 1 (0.11 nm) for trpv1 inhibition in dii - labeled small - sized tg neurons is lower than that in small - sized drg neurons (figure 2(b)), suggesting that maresin 1 is a more potent inhibitor of trpv1 in the trigeminal sensory system, as compared to the somatosensory system. the inhibitory signaling mechanisms of maresin 1 are largely unknown in the trigeminal system. at present, specific receptors for maresin 1 are yet to be identified. a common pattern emerging with maresin 1 signaling is the requirement for specific gpcrs. remarkably, pretreatment of dii - labeled tg neurons with pertussis toxin (ptx, 0.5 g / ml) for 18 h completely blocked maresin 1 's inhibitory effects on capsaicin - induced trpv1 currents. my results demonstrated that the inhibitory signaling effects of maresin 1 on trpv1 are likely to be mediated by activation of specific ptx - sensitive / gi - coupled gpcrs (figure 2(d)) in dii - labeled small - sized tg neurons. i also demonstrated that capsaicin (500 nm) substantially increased the frequency of sepscs (figures 4(a) and 4(b)) but not the amplitude of sepscs in lamina ii neurons of the trigeminal nucleus, indicating that a trpv1 agonist potentiated glutamate release from presynaptic terminals of primary afferents in the trigeminal system [28, 29 ]. notably, the capsaicin - induced increases in sepsc frequency were blocked by maresin 1 (figures 4(a) and 4(b)). thus, maresin 1 may serve as a potent endogenous inhibitor of trpv1 in the trigeminal system. tissue injury - induced spinal cord synaptic plasticity (i.e., central sensitization) contributes greatly to the development and maintenance of chronic pain [37, 38 ]. inflammation of the tmj region triggers the pathogenesis of persistent tmj pain, because of increased synaptic transmission in the trigeminal nuclei including the subnucleus caudalis (sp5c) [6, 29 ]. the superficial layers of the sp5c (laminae i - ii) are a region of the brain stem and are highly selective for nociceptive processing. in this study, i showed that tmj inflammation by complete freund 's adjuvant (cfa) injection elicited dramatic increases in both sepsc frequency and amplitude in trigeminal nucleus slices prepared from inflamed mice (1 day), as compared to those from control mice (figures 4(c) and 4(d)). of note, maresin 1 (0.35 nm) abolished tmj inflammation - induced increases in sepsc frequency and amplitude (figures 4(c) and 4(d)), indicating that maresin 1 could reduce tmj - inflammatory pain via modulation of synaptic plasticity in the trigeminal system. these findings demonstrate the potent actions of maresin 1 in regulating trpv1 function in tmj - related trigeminal nociceptive neurons and tmj inflammation - induced synaptic plasticity in the trigeminal nucleus. therefore, these new findings suggest that maresin 1 may serve as a novel endogenous inhibitor for treating tmj - inflammatory pain in the orofacial region. | in the trigeminal system, disruption of acute resolution processing may lead to uncontrolled inflammation and chronic pain associated with the temporomandibular joint (tmj). currently, there are no effective treatments for tmj pain. recently, it has been recognized that maresin 1, a newly identified macrophage - derived mediator of inflammation resolution, is a potent analgesic for somatic inflammatory pain without noticeable side effects in mice and a potent endogenous inhibitor of transient receptor potential vanilloid 1 (trpv1) in the somatic system. however, the molecular mechanisms underlying the analgesic actions of maresin 1 on tmj pain are unclear in the trigeminal system. here, by performing tmj injection of a retrograde labeling tracer dii (a fluorescent dye), i showed that maresin 1 potently inhibits capsaicin - induced trpv1 currents and neuronal activity via gi - coupled g - protein coupled receptors in dii - labeled trigeminal nociceptive neurons. further, maresin 1 blocked trpv1 agonist - evoked increases in spontaneous excitatory postsynaptic current frequency and abolished tmj inflammation - induced synaptic plasticity in the trigeminal nucleus. these results demonstrate the potent actions of maresin 1 in regulating trpv1 in the trigeminal system. thus, maresin 1 may serve as a novel endogenous inhibitor for treating tmj - inflammatory pain in the orofacial region. |
osteoarthritis is a multifaceted disease caused by the interaction of multiple genetic and environmental factors (1, 2). osteoarthritis is a joint disease that mostly affects cartilage, which is the slippery tissue that covers the ends of bones in a joint (3). as a matter of fact however, it may also involve younger individuals who occasionally get osteoarthritis, mainly from joint injuries (6). the main risk factors for development of osteoarthritis include older age, overweight and obesity, joint injury, joints that are not properly formed, genetic defects in joint cartilages, and stresses on the joints from certain jobs and playing sports or other physical activities (7, 8). nonetheless, among all the other putative risk factors, age is considered as one of the strongest risk factors for osteoarthritis of all joints (6, 8, 9). from this point of view, the increase in the prevalence and incidence of osteoarthritis with age is deemed a consequence of cumulative exposure to various risk factors and biologic changes that occur with aging that may make a joint less able to cope with adversity, such as cartilage thinning, weak muscle strength, or oxidative damage (7). to date, there is scarce evidence about the magnitude and distribution of osteoarthritis in the adult population of albania, a post - communist country in the western balkans which has been undergoing a rapid socioeconomic and political change in the past two decades. there is evidence of a clear demographic transition in albania, with a six - year increase in the mean age of the population from 2001 to 2011 (10). this demographic change is reflected in an increase of the older population subgroup, where the proportion of individuals aged 65 years and over in albania amounted up to 11% in 2011 (10). furthermore, the rapid process of transition in albania over the past twenty five years has been associated with an intensive process of migration, both internal (from rural areas to urban areas of the country) and external (mostly to the neighboring countries such as greece and italy, but also germany and the united kingdom) (11). migration leads to an increased ageing process (12) which goes in line with an increase in the prevalence of osteoarthritis. nevertheless, there is almost no formal (well - documented) evidence on the prevalence and correlates of osteoarthritis in the adult population of albania to date. in this framework, the aim of our study was to describe the distribution of demographic factors and socioeconomic characteristics among primary health care users diagnosed with osteoarthritis in transitional albania. this study included all individuals diagnosed with osteoarthritis during january 2013 december 2014 in selected primary health care centers in tirana, the capital city of albania. overall, 1179 adult individuals were diagnosed with osteoarthritis (521 men and 658 women) during this time period. the overall mean age of study participants was 59.010.1 years (60.110.6 years in men and 58.19.6 years in women). the diagnosis of osteoarthritis was established in line with the recommendations of the american college of rheumatology criteria for the clinical diagnosis of osteoarthritis (13, 14) : history of the disease : self - reported presence of pain, aching, stiffness, or other symptoms in the joints affected by osteoarthritis ; physical examination : difficulties in flexion / extension and rotation of the joints (range of motions), tenderness, crepitations, or enlargement of the joints ; laboratory findings : erythrocyte sedimentation rate, rheumatoid factor, c - reactive protein and uricemia, and ; radiological findings : joint space narrowing and presence of osteophytes. fisher s exact test was used to compare the distribution of demographic factors and socioeconomic characteristics among male and female participants diagnosed with osteoarthritis. on the other hand, binary logistic regression was used to assess the sex - differences regarding demographic factors and socioeconomic characteristics among primary health care users diagnosed with osteoarthritis in tirana. crude (unadjusted) and subsequently multivariable - adjusted odds ratios (ors) and their respective 95% confidence intervals (cis) were calculated. all the statistical analyses were done by use of the statistical package for social sciences (spss, version 17.0). table 1 presents the distribution of demographic factors and socioeconomic characteristics among primary health care users diagnosed with osteoarthritis in tirana during the period 2013 - 2014. overall, 622 (52.8%) of the patents were less than 60 years of age, whereas 557 (47.2%) of them were 60 years or above. there was evidence of a statistically significant difference in the age - distribution between men and women : the proportion of older men (60 years and above) was higher than in women (52.2% vs. 43.3%, respectively), indicating that osteoarthritis developed at an earlier age in women compared to men (p=0.001). regarding the place of birth, the proportion of men who were born in urban areas was higher in men than in women (69.3% vs. 62.9%, respectively), a difference which was statistically significant (p=0.013). furthermore, the prevalence of unemployment was substantially and significantly higher in women compared to men (28.9% vs. 18.8%, respectively ; p<0.001). on the other hand, there was no statistically significant difference in the proportion of men and women distinguished by their marital status (p=0.344). the proportion of a higher educational attainment (more than 12 years of formal schooling) was considerably higher in men than in women (21.9% vs. 11.7%, respectively), a finding which was highly statistically significant (p<0.001). similarly, the prevalence of a higher income level was significantly higher in men compared to women (22.1% vs. 16.1%, respectively ; p=0.017). likewise, the prevalence of a higher social status was significantly higher in men than in women (15.2% vs. 10.8%, respectively ; p=0.025). distribution of demographic factors and socioeconomic characteristics among primary health care users diagnosed with osteoarthritis in tirana during 2013 - 2014. absolute numbers and their respective column percentages (in parentheses). p - values from fisher s exact test. table 2 presents the sex - differences with regard to the demographic factors and socioeconomic characteristics between primary health care men and women diagnosed with osteoarthritis in tirana during the period 2013 - 2014. in crude (unadjusted) logistic regression models, there was an inverse association of female gender with age (or=0.70, 95%ci=0.56 - 0.88), but a positive relationship with rural birthplace (or=1.33, 95%ci=1.04 - 1.70). furthermore, there was evidence of a positive association between female gender and unemployment (or=1.73, 95%ci=1.28 - 2.34), but not with retirement (or=0.98, 95%ci=0.75 - 1.27) on the other hand, there was a strong inverse association between female gender and educational level (overall p<0.001) : the odds of a higher educational attainment were considerably lower among women with osteoarthritis compared to men (or=0.41, 95%ci=0.29 - 0.58). similarly, the odds of a higher income level and a higher social status were both significantly lower among women than in men diagnosed with osteoarthritis (or=0.61, 95%ci=0.43 - 0.86 and or=0.59, 95%ci=0.40 - 0.87, respectively). sex - differences regarding demographic factors and socioeconomic characteristics among primary health care users diagnosed with osteoarthritis in tirana during 2014 - 2015. odds ratios (or : women vs. men), 95% confidence intervals (95%cis) and p - values from binary logistic regression. overall p - value and degrees of freedom (in parentheses). upon simultaneous adjustment for all the demographic factors and socioeconomic characteristics, female gender was inversely associated with age - group (or=0.67, 95%ci=0.47 - 0.95) and educational level (or=0.39, 95%ci=0.25 - 0.61), but positively related to rural birthplace (or=1.47, 95%ci=1.14 - 1.89) and unemployment (or=1.40, 95%ci=1.02 - 1.92). on the other hand, in multivariable - adjusted models, the association of gender with income level and social status disappeared (table 2). main findings of our study conducted in tirana the albanian capital city include an inverse (negative) association of female gender with the age - group and educational level of primary health care users diagnosed with osteoarthritis. on the other hand, there was evidence of a positive association of female gender with rural birthplace and unemployment of study participants controlling for a wide array of other demographic and socioeconomic factors. our study revealed that women in tirana are more likely to develop osteoarthritis than men. this finding is compatible with many previous reports from the international literature which indicate that females have a higher prevalence of osteoarthritis and also suffer from more severe forms of osteoarthritis compared to their male counterparts (9). the strong positive association of osteoarthritis with menopause has suggested that hormonal factors may play a crucial role in the development of this condition (7). however, it should be noted that results on the effect of estrogen have been controversial in this regard (15 - 17). in the randomized clinical trial referred to as the heart and estrogen / progestin replacement study, in a group of older postmenopausal women with heart disease, no significant difference was noted in the prevalence of knee pain or its resultant disability between participants taking estrogen plus progestin therapy or those taking placebo (9, 18). on the other hand, results from the women s health initiative indicated that, women on estrogen replacement therapy were 15% less likely to require total knee or hip arthroplasty compared to women not taking such therapy, but that estrogen combined with progestin therapy was not associated with the risk of joint replacement (9, 19). our study may have several limitations pertinent to the sample representativeness and the information collected. we included in this study all individuals diagnosed with osteoarthritis who sought medical care in selected primary health care centers in tirana. however, these health centers included in our study are representative to the overall primary health care facilities in tirana. from this perspective, the overall sample of primary health care users diagnosed with osteoarthritis in these health centers included in our study is assumed to be representative of the overall adult primary health care users in tirana. yet, primary health care users may not necessarily reflect the overall adult population of tirana. therefore, our findings can not be generalized to the overall adult population of tirana, but are confined to the primary health care users only. furthermore, at best, findings from this study are generalized to the adult population residing in tirana and not the general population of albania. the instruments for data collection were based on the international protocols and best practices and recommendations for establishment of the clinical diagnosis of osteoarthritis (13, 14). nevertheless, the self - reported data on socioeconomic characteristics of study participants may have been subject to information bias, at least to some extent. notwithstanding the possibility of the aforementioned limitations, our findings provide novel evidence about the distribution of demographic factors and socioeconomic characteristics among adult individuals diagnosed with osteoarthritis in primary health care services in tirana, the capital of albania. future studies should assess the distribution of the major risk factors for osteoarthritis among adult men and women in transitional albania. | aim : the aim of this study was to describe the distribution of demographic factors and socioeconomic characteristics among primary health care users diagnosed with osteoarthritis in albania, a transitional country in the western balkans.methods:this study included all individuals diagnosed with osteoarthritis during january 2013 december 2014 in selected primary health care centers in tirana, the capital city of albania. overall, 1179 adult individuals were diagnosed with osteoarthritis (521 men and 658 women) during this time period. the overall mean age of study participants was 59.010.1 years (60.110.6 years in men and 58.19.6 years in women). the diagnosis of osteoarthritis was based on the history of the disease, physical examination, laboratory findings and radiological findings. binary logistic regression was used to assess the sex - differences regarding demographic factors and socioeconomic characteristics among participants diagnosed with osteoarthritis.results:in multivariable - adjusted logistic regression models, female gender was inversely associated with the age - group (or=0.67, 95%ci=0.47 - 0.95) and educational level (or=0.39, 95%ci=0.25 - 0.61), but positively related to rural birthplace (or=1.47, 95%ci=1.14 - 1.89) and unemployment (or=1.40, 95%ci=1.02 - 1.92) of the patients diagnosed with osteoarthritis.conclusion:our findings provide novel evidence about the distribution of demographic factors and socioeconomic characteristics among adult individuals diagnosed with osteoarthritis in primary health care services in albania. future studies should assess the distribution of the major risk factors for osteoarthritis among adult men and women in transitional albania. |
the first direct detection of gravitational waves (gws), widely expected in the mid 2010s with advanced ground - based interferometers [219, 2 ], will represent the culmination of a fifty - year experimental quest. soon thereafter, newly plentiful gw observations will begin to shed light on the structure, populations, and astrophysics of mostly dark, highly relativistic objects such as black holes and neutron stars. in the low - frequency band that will be targeted by space - based detectors (roughly 10 to 1 hz), gw observations will provide a census of the massive black - hole binaries at the center of galaxies, and characterize their merger histories ; probe the galactic population of binaries that include highly evolved, degenerate stars ; study the stellar - mass objects that spiral into the central black holes in galactic nuclei ; and possibly detect stochastic gw backgrounds from the dynamical evolution of the very early universe. thus, there are very strong astrophysical motivations to observe the universe in gws, especially because the systems and phenomena that can be observed in this fashion are largely orthogonal to those accessible to traditional electromagnetic (em) and astroparticle astronomy. einstein s theory of gravity, general relativity (gr), has been confirmed by extensive experimental tests ; but these have largely been confined to the solar system, where gravity is well approximated by newtonian gravity with small corrections. a few tests, based on observations of binary compact - object systems, have confirmed the weakest (leading - order) effects of gw generation. by contrast, observation of strong gws will provide the first direct observational probe of the dynamical, strong - field regime of gr, where the nature and behavior of gravity can be significantly different from the newtonian picture. gws are prima facie the perfect probe to investigate gravitation, since they originate directly from the bulk motion of gravitating matter, relieving the need to understand and model the physics of other intermediate messengers, typically photons from stellar surfaces or black - hole surroundings. already today we can rely on a very sophisticated understanding of the analytical and numerical techniques required to model gw sources and their gw emission, including the post - newtonian expansion [84, 190 ], black - hole perturbation theory, numerical relativity for vacuum spacetimes, spacetimes with gases or magnetized plasmas, and much more. that these techniques should have been developed so much in the absence of a dialogue with experimental data (except for the binary pulsar) is witness to the great perceived promise of gw astronomy. for a birds - eye view of the field, see the living review by sathyaprakash and schutz, who cover the physics of gws, the principles of operation of gw detectors, the nature of gw sources, the data analysis of gw signals, and the science payoffs of gw observations for physics, astrophysics, and cosmology. this review focuses on the opportunities to challenge or confirm our understanding of gravitational physics that will be offered by forthcoming space - based missions to observe gws in the low - frequency band between 10 and 1 hz. most of the literature on this subject has focused on one mission design, lisa (the laser interferometer space antenna [64, 252, 370 ]), which was studied jointly by nasa and esa between 2001 and 2011. in 2011, budgetary and programmatic reasons led the two space agencies to end this partnership, and to pursue space - based gw detection separately, studying cheaper, rescoped lisa - like missions. esa s proposed elisa / ngo would be smaller than lisa, fly on orbits closer to earth, and operate interferometric links only along two arms. in 2012 elisa was considered for implementation as esa s first large mission (l1) in the cosmic vision program. a planetary mission was selected instead, but elisa will be in the running for the next launch slot (l2), with a decision coming as soon as 2014. nasa ran studies on a broader range of missions, including several variants of lisa to be implemented by nasa alone, as well as options with a geocentric orbit (omega), and without drag - free control (lagrange). the final study report concludes that scientifically compelling missions can be carried out for less, but not substantially less, than the full lisa cost ; that scientific performance decreases far more rapidly than cost ; and that no design choice or technology can make a dramatic reduction in cost without much greater risks. the nasa study noted the possibility of participation in the esa - led elisa mission (if selected by esa) as a minority partner. whatever specific design is eventually selected, it is likely that its architecture, technology, and scientific reach will bear a strong resemblance to lisa s (with the appropriate scalings in sensitivity, mission duration, and so on). thus, the research reviewed in this article, which was targeted in large part to lisa, is still broadly relevant to future missions. such lisa - like observatories are characterized by a few common elements : a set of three spacecraft in long - baseline (mkm) orbits, monitoring their relative displacements using laser interferometry ; drag - free operation (except for lagrange), whereby displacement measurements are referenced to freely falling test masses protected by the spacecraft, which hover around the masses using precise micro - newton thrusters ; frequency correction of laser noise using a variety of means, including onboard cavities and interferometers, arm locking, and a lisa - specific technique known as time - delay interferometry. the predictions of gr that can be tested by space - based gw observatories include the absence of gravitational fields other than the metric tensor ; the number and character of gw polarization states ; the speed of gw propagation ; the detailed progress of binary inspiral, as driven by nonlinear gravitational dynamics and loss of energy to gws ; the strength and shape of the gws from binary merger and ringdown ; the true nature of astrophysical black holes ; and more. some of these tests will also be performed with ground - based gw detectors and pulsar - timing observations, but space - based tests will almost always have superior accuracy and significance, because low - frequency sources are intrinsically stronger, and will spend a larger time within the band of good detector sensitivity. for binary systems with very asymmetric mass ratios, such as extreme mass - ratio inspirals (emris), lisa - like missions will measure hundreds of thousands of orbital cycles ; because successful detections require matching the phase of signals throughout their evolution, it follows that these observations will be exquisitely sensitive to source parameters. the data - analysis detection problem will be correspondingly delicate, but has been tackled both theoretically, and in a practical program of mock data challenges for lisa [37, 39, 450 ]. section 2 provides the briefest overview of einstein s gr, of the theoretical framework in which it can be tested, and of a few leading alternative theories. it also introduces the black - hole paradigm, which augments einstein s equations with a few assumptions of physicality that lead to the prediction that the end result of gravitational collapse are black holes described by the kerr metric. section 4 summarizes the main classes of gw sources that would be observed by lisa - like detectors, and that can be used to test gr. section 5 examines the tests of gravitational dynamics that can be performed with these sources, while section 6 discusses the tests of the black - hole nature and structure. (a conspicuous omission are possible stochastic gw backgrounds of cosmological origin ; indeed, in this article we do not discuss the role of space - based detectors as probes of cosmology and early - universe physics.) newton s theory of gravitation provided a description of the effect of gravity through the inverse square law without attempting to explain the origin of gravity. the inverse square law provided an accurate description of all measured phenomena in the solar system for more than two hundred years, but the first hints that it was not the correct description of gravitation began to appear in the late 19th century, as a result of the improved precision in measuring phenomena such as the perihelion precession of mercury. einstein s contribution to our understanding of gravity was not only practical but also aesthetic, providing a beautiful explanation of gravity as the curvature of spacetime. in developing gr as a generally covariant theory based on a dynamical spacetime metric, einstein sought to extend the principle of relativity to gravitating systems, and he built on the crucial 1907 insight that the equality of inertial and gravitational mass allowed the identification of inertial systems in homogeneous gravitational fields with uniformly accelerated frames the principle of equivalence. einstein was also guided by his appreciation of ricci and levi - civita s absolute differential calculus (later to become differential geometry), arguably as much as by the requirement to reproduce newtonian gravity in the weak - field limit. indeed, one could say that gr was born of almost pure thought. einstein s theory of gr is described by the action 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{gr } } } } = \int { \sqrt { - g } r{{\rm{d}}^4}x},$$\end{document } in which g is the determinant of the spacetime metric and r = gr is the ricci scalar, where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r^{\mu \nu } } = r_{\mu \alpha \nu}^\alpha$\end{document } is the ricci tensor, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{\beta \gamma \delta}^\alpha = \gamma _ { \beta \delta, \gamma}^\alpha - \gamma _ { \beta \gamma, \delta}^\alpha + \gamma _ { \gamma \lambda}^\alpha \gamma _ { \beta \delta}^\lambda - \gamma _ { \delta \lambda}^\alpha \gamma _ { \beta \gamma}^\lambda$\end{document } the riemann curvature tensor, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { \beta \gamma}^\alpha = { g^{\alpha \delta}}({g_{\beta \delta, \gamma } } + { g_{\delta \gamma, \beta } } - { g_{\beta \gamma, \delta}})$\end{document } the affine connection, and a comma denotes a partial derivative. when coupled to a matter distribution, this action yields the field equations 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } \equiv { r_{\mu \nu } } - { 1 \over 2}{g_{\mu \nu}}r = { { 8\pi g } \over { { c^4}}}{t_{\mu \nu}},$$\end{document } where denotes the stress - energy tensor of the matter. since the development of the theory, gr has withstood countless experimental tests [471, 443, 444 ] based on measurements as different as atomic - clock precision, orbital dynamics (most notably lunar laser ranging), astrometry, and relativistic astrophysics (most exquisitely the binary pulsar [293, 471 ], but not only). it is therefore the correct and natural benchmark against which to compare alternative theories using future observations and we will follow the same approach in this article. unlike in the case of newtonian gravity at the time that gr was developed, there are no current observations that gr can not explain that can be used to guide development of alternatives.1 nonetheless, there are crucial aspects of einstein s theory that have never been probed directly, such as its strong - field dynamics and the propagation of field perturbations (gws). furthermore, it is known that classical gr must ultimately fail at the planck scale, where quantum effects become important, and traces of the quantum nature of gravity may be accessible at lower energies. as emphasized by will, gr has no adjustable constants, so every test is potentially deadly, and a probe that could reveal new physics. will s living review and his older monograph are the fundamental references about the experimental verification of gr. in this section, we give only a brief overview of what may be called will s standard model for alternative theories of gravity, which proceeds through four steps : a) strong evidence for the equivalence principle supports a metric formulation for gravity ; b) metric theories are classified according to what gravitational fields (scalar, vector, tensor) they prescribe ; c) slow - motion, weak - field conservative dynamics are described in a unified parameterized post - newtonian (ppn) formalism, and constrained by experiment and observations ; d) finally, equations for the slow - motion generation and weak - field propagation of gravitational radiation are derived separately for each metric theory, and again compared to observations. many of the tests of gravitational physics envisaged for lisa belong in this last sector of will s standard model, and are discussed in section 5.1 of this review. this scheme however leaves out two other important points of contact between gravitational phenomenology and lisa s gw observations : the strong - field, nonlinear dynamics of black holes and their structure and excitations, especially as probed by small orbiting bodies. we will deal with these in sections 5 and 6, respectively ; but let us first delve into will s standard model. the equivalence principle and metric theories of gravitation. einstein s original intuition placed the equivalence principle as a cornerstone for the theories that describe gravity as curved spacetime. as formulated by newton, the principle states simply that inertial and gravitational mass are proportional, and therefore all test bodies fall with the same acceleration (in modern usage, this is known as the weak equivalence principle, or wep). dicke later recognized that in developing gr einstein had implicitly posited a broader principle (einstein s equivalence principle, or eep) that consists of wep plus local lorentz invariance and local position invariance : that is, of the postulates that the outcome of local non - gravitational experiments is independent of, respectively, the velocity and position of the local freely - falling reference frames in which the experiments are performed. weak equivalence principle (wep) : gravitational and inertial masses are equivalent (neglecting self gravity).2. einstein equivalence principle (eep) : local position invariance and local lorentz invariance apply in addition to the wep.3. strong equivalence principle (sep) : eep applies also for self - gravitating objects. turyshev gives a current review of the experimental verification of wep (shown to hold to parts in 10 by differential free - fall tests), local lorentz invariance (verified to parts in 10 by clock - anisotropy experiments), and local position invariance (verified to parts in 10 by gravitational - redshift experiments, and to much greater precision when looking for possible time variations of fundamental constants). although these three parts of eep appear distinct in their experimental consequences, their underlying physics is necessarily related in any theory of gravity, so schiff conjectured (and others argued convincingly) that any complete and self - consistent theory of gravity that embodies wep must also realize eep. eep leads to metric theories of gravity in which spacetime is represented as a pseudo - riemannian manifold, freely - falling test bodies move along the geodesics of its metric, and non - gravitational physics is obtained by applying special - relativistic laws in local freely - falling frames. gr is, of course, a metric theory of gravity ; so are scalar - vector - tensor theories such as brans - dicke theory, which include other gravitational fields in addition to the metric. by contrast, theories with dynamically varying fundamental constants and theories (such as superstring theory) that introduce additional wep - violating gravitational fields [471, section 2.3 ] are not metric. neither are most theories that provide short - range and long - range modifications to newton s inverse - square law. the scalar and vector fields in scalar - vector - tensor theories can not directly affect the motion of matter and other non - gravitational fields (which would violate wep), but they can intervene in the generation of gravity and modify its dynamics. these extra fields can be dynamical (i.e., determined only in the context of solving for the evolution of everything else) or absolute (i.e., assigned a priori to fixed values). the minkowski metric of special relativity is the classic example of absolute field ; such fields may be regarded as philosophically unpleasant by those who dislike feigning hypotheses, but they have a right of citizenship in modern physics as frozen in the additional fields can potentially alter the outcome of local gravitational experiments : while the local gravitational effects of different metrics far away can always be erased by describing physics in a freely - falling reference frame (which is to say, the local boundary conditions for the metric can be arranged to be flat spacetime), the same is not true for scalar and vector fields, which can then affect local gravitational dynamics by their interaction with the metric. this amounts to a violation not of eep, but of the strong equivalence principle (sep), which states that eep is also valid for self - gravitating bodies and gravitational experiments. sep is verified to parts in 10 by combined lunar laser - ranging and laboratory experiments. so far, gr appears to be the only viable metric theory that fully realizes sep. the ppn formalism. because the experimental consequences of different metric theories follow from the specific metric that is generated by matter (possibly with the help of the extra gravitational fields), and because all these theories must realize newtonian dynamics in appropriate limiting conditions, it is possible to parameterize them in terms of the coefficients of a slow - motion, weak - field expansion of the metric. these coefficients appear in front of gravitational potentials similar to the newtonian potential, but involving also matter velocity, internal energy, and pressure. this scheme is the parameterized post - newtonian formalism, pioneered by nordtvedt and extended by will (see for details). of the ten ppn parameters in the current version of the formalism, two are the celebrated and (already introduced by eddington, robertson, and schiff for the classical tests of gr) that rule, respectively, the amount of space curvature produced by unit rest mass and the nonlinearity in the superposition of gravitational fields. in gr, and each have the value 1. the other eight parameters, if not zero, give origin to violations of position invariance (), lorentz invariance (13), or even of the conservation of total momentum (3, 14) and total angular momentum (13, 14). the ppn formalism is sufficiently accurate to describe the tests of gravitation performed in the solar system, as well as many tests using binary - pulsar observations. the parameter is currently constrained to 1 a few 10 by tests of light delay around massive bodies using the cassini spacecraft ; to 1 a few 10 by lunar laser ranging.2 the other ppn parameters have comparable bounds around zero from solar - system and pulsar measurements, except for 3, which is known exceedingly well from pulsar observations. tests in the ppn framework have tightly constrained the field of viable alternatives to gr, largely excluding theories with absolute elements that give rise to preferred - frame effects. the (indirect) observation of gw emission from the binary pulsar and the accurate prediction of its by einstein s quadrupole formula have definitively excluded other theories [471, 422 ]. yet more gr alternatives were conceived to illuminate points of principle, but they are not well motivated physically and therefore are hardly candidates for experimental verification. some of the theories that are still alive are described in the following. the addition of a single scalar field to gr produces a theory described by the einstein - frame action (see, e.g.,), 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde i = { (16\pi g)^{- 1}}\int { [\tilde r - 2{{\tilde g}^{\mu \nu}}{\partial _ \mu}\varphi { \partial _ \nu}\varphi - v(\varphi)]{{(- \tilde g)}^{1/2}}{{\rm{d}}^4}x + { i_{{\rm{matter}}}}({\psi _ { \rm{m}}},{a^2}(\varphi){{\tilde g}_{\mu \nu } }) }, $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde g}_{\mu \nu}}$\end{document } is the metric, the ricci curvature scalar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde r}$\end{document } yields the general - relativistic einstein - hilbert action, and the two adjacent terms are kinetic and potential energies for the scalar field. note that in the action imatter for matter dynamics, the metric couples to matter through the function a(), so this representation is not manifestly metric ; it can however be made so by a change of variables that yields the jordan - frame action, 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = { (16\pi g)^{- 1}}\int { [\phi r - { \phi ^{- 1}}\omega (\phi){g^{\mu \nu}}{\partial _ \mu}\phi { \partial _ \nu}\phi - { \phi ^2}v]{{(- g)}^{1/2}}{{\rm{d}}^4}x + { i_{{\rm{matter}}}}({\psi _ { \rm{m}}},{g_{\mu \nu } }) }, $ $ \end{document } where a() is the transformed scalar field, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_{\mu \nu } } \equiv { a^2}(\varphi){{\tilde g}_{\mu \nu}}$\end{document } is the physical metric underlying gravitational observations, and 3 + 2() = [d(ln a())/d ]. brans - dicke theory corresponds to fixing to a constant bd, and it is indistinguishable from gr in the limit bd. in the ppn framework, the only parameter that differs from gr is = (1 + bd)/(2 + bd). damour and esposito - farese considered an expansion of log a() around a cosmological background value, 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\log a(\varphi) = { \alpha _ 0}(\varphi - { \varphi _ 0 }) + { 1 \over 2}{\beta _ 0}{(\varphi - { \varphi _ 0})^2 } + \cdots, $ $ \end{document } where 0 (and further coefficients) = 0 reproduces brans - dicke with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 0 ^ 2 = 1/(2{\omega _ { { \rm{bd } } } } + 3),\,{\beta _ 0 } > 0$\end{document }, 0 > 0 causes the evolution of the scalar field toward 0 (and therefore toward gr) ; and 0 10 m, a significant fraction of this snr comes from the final merger and ringdown phase. the left - hand panel shows contours in the total - mass - redshift plane for equal - mass binaries, while the right - hand panel shows contours in the total - mass - mass - ratio plane for sources at redshift z = 4. the left - hand panel shows contours in the total - mass - redshift plane for equal - mass binaries, while the right - hand panel shows contours in the total - mass - mass - ratio plane for sources at redshift z = 4. image reproduced by permission from. several detection algorithms for mbh binaries have been studied by a number of different groups, encouraged in part by the mock lisa data challenges. these include markov chain monte carlo techniques, time - frequency analysis, particle - swarm optimization, nested sampling, and genetic algorithms. the most recent mock - data challenge included multiple spinning mbh binary signals in the same dataset, and multiple groups demonstrated their ability to recover these binaries from the lisa data stream. gw observations will directly probe the character of the black holes on scales comparable to the size of the event horizons. how the information about the black holes (such as their spin and mass) is encoded in the waveform is a topic of much focused research (see, e.g., [275, 411, 117, 421 ] for reviews). according to gr, any astrophysical black hole is fully described by just ten numbers, encoding its mass, position, momentum, and spin. correspondingly, a binary is fully described by 20 parameters, three of which, related to its center - of - mass momentum, can not be measured from the gw signal. the precision with which gw observations will be able to extract the parameters characterizing a mbh binary system has been the subject of extensive investigation. the first comprehensive study considered the lisa determination of the sky location, luminosity distance, and mass of mbh systems, using a simple model of lisa s orbital motion around the sun. later studies considered the angular resolution of detectors in precessing - plane configurations like lisa s, as well as ecliptic - plane interferometers that lie flat in the ecliptic as they orbit the sun. more recent studies have considered more complete mbh - binary waveforms with spin effects, higher harmonics, and with merger and ringdown signals, and have looked at the effect of these corrections on lisa s parameter - estimation ability [455, 29, 442, 365, 33, 266, 307 ]. these papers indicate that observations with a lisa - like detector will be able to determine the masses of the two binary components to 0.010.1%, the spins to 0.0010.1%, the sky position of the binary to 110 deg, and the luminosity distance to 0.110%. for elisa, these parameter - estimation studies witness the quality of the information that will be available to astrophysicists for the phenomenological exploration of the astrophysical character of mbh systems. of particular interest is the evolution of black - hole spins and the final spin of merger remnants [80, 242 ], which gw observations should be able to shed some light on. in addition, the set of detected mbh merger systems will provide constraints on the formation history of mbhs [360, 359, 241, 308, 355 ]. in [405, 198 ] it was shown that a detector like lisa would be able to tell with high confidence the difference between ten different models for the growth of structure in the universe. for many of the models, the difference would already be apparent after as little as three months of data collection. a similar differentiation among models would also be possible with elisa, and both detectors will also be able to constrain mbh binaries can also be used as standard candles to probe cosmological evolution. testing relativity using mbh mergers. mbh mergers are good laboratories for testing relativity because of the high snr expected for the signals and the correspondingly large volume within which they can be observed. this makes them good systems for constraining gw polarizations (see section 5.1.1) since relatively weak alternative - polarization signals could in principle be detected. they are also excellent systems for testing gw - propagation effects, such as subluminal propagation speeds and the presence of parity violations, since these effects accumulate with distance and mbh binaries can be seen to very high redshifts (see section 5.1.2). mbh binaries are also well suited for generic tests of gr based on measuring the evolution of inspiral phasing and checking it for consistency with general - relativistic predictions, since the high snr allows a large number of phasing parameters to be measured (see section 5.2). mbh binaries are also the only systems for which the quasinormal ringdown radiation, generated as the highly - distorted post - merger black hole settles down into a quiescent state, will be detectable. although the amount of energy deposited into different ringdown modes is not well understood, the frequencies and decay rates of the ringdown modes are predicted precisely in gr from perturbation theory. the mass and spin of the final black hole can be measured from a single ringdown mode ; if multiple modes are detected, the system can be used to check for consistency with the kerr metric, and therefore provides a probe of black - hole structure. for a comprehensive review of black - hole ringdowns, a more detailed discussion of the use of ringdown radiation to probe black - hole structure may be found in section 6.3. last, the space - based gw observation of mbh binaries offers an unprecedented opportunity to probe the fully - dynamic strong - field regime of gr by comparison of the observed merger signals to the predictions of numerical relativity. this is an area that has not yet been explored in any detail and so we will not discuss it further in this review. however, it is an important subject that should be carefully explored before a space - based gw detector finally becomes a reality. an emri is the gw - emitting inspiral of a stellar - mass compact object, either a black hole, neutron star or white dwarf, into a mbh in the center of a galaxy. a main - sequence star with mean density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar \rho = 10\,{\rm{g c}}{{\rm{m}}^{- 3}}$\end{document } will be tidally disrupted by a black hole of mass 10 m at a distance of 25 schwarzschild radii. at such separations an extra - galactic emri system will not be generating detectable amounts of gravitational radiation in the low - frequency band. the tidal - disruption radius increases with decreasing stellar density and decreasing central - black - hole mass, and the reference values used above are at the upper end of suitable values for main - sequence stars and mhz gw sources. therefore, it is not expected that the inspiral of a main - sequence star will be a candidate for an emri, with the possible exception of such sources in the galactic center, which is sufficiently nearby that radiation from an object orbiting at several tens of schwarzschild radii might be detectable. emris occur in the dense stellar clusters that are found surrounding the black holes in the cores of galaxies, and are triggered by a variety of processes : direct capture : this is the scenario, in which two - body encounters in the stellar cluster gradually perturb the compact - object orbits, changing their angular momentum. this can put a compact object onto an orbit that passes very close to the central black hole. the orbit loses energy and angular momentum in bursts of gravitational radiation emitted near periapse, and if sufficient energy is radiated, the object can be left on an orbit that is bound to the central black hole. it will then gradually inspiral into the central black hole as it loses orbital energy and angular momentum to gw emission.tidal splitting of binaries : galactic - center stellar clusters will also contain binaries, which can similarly be displaced onto orbits that pass close to the central mbh. if this happens, the binary will typically be disrupted, with one object remaining bound to the mbh and the other becoming unbound and being flung out with high velocity. if this happens to a binary containing one or two compact objects, the captured compact object will be left on a fairly tightly bound orbit a few hundred astronomical units from the mbh, and will become an emri on a nearly circular orbit.tidal stripping of giant stars : giant stars typically have a compact core surrounded by a diffuse hydrogen envelope. if such a star passes close to a mbh, the envelope can be partially or completely stripped by the tidal interaction with the mbh. this will deposit the dense core, which is essentially a white dwarf, on a close orbit about the mbh ; the system will eventually become an emri [145, 150],in - situ formation : if a mbh has a massive accretion disc, the disc can become unstable to star formation. stars formed in such a way are biased toward higher masses and will therefore tend to form black - hole remnants. these remnants will be in circular, equatorial orbits around the central mbh, and will inspiral as emris under gw emission. more details on all of these processes and further references can be found in [18, 16 ]. direct capture : this is the standard scenario, in which two - body encounters in the stellar cluster gradually perturb the compact - object orbits, changing their angular momentum. this can put a compact object onto an orbit that passes very close to the central black hole. the orbit loses energy and angular momentum in bursts of gravitational radiation emitted near periapse, and if sufficient energy is radiated, the object can be left on an orbit that is bound to the central black hole. it will then gradually inspiral into the central black hole as it loses orbital energy and angular momentum to gw emission. tidal splitting of binaries : galactic - center stellar clusters will also contain binaries, which can similarly be displaced onto orbits that pass close to the central mbh. if this happens, the binary will typically be disrupted, with one object remaining bound to the mbh and the other becoming unbound and being flung out with high velocity. if this happens to a binary containing one or two compact objects, the captured compact object will be left on a fairly tightly bound orbit a few hundred astronomical units from the mbh, and will become an emri on a nearly circular orbit. tidal stripping of giant stars : giant stars typically have a compact core surrounded by a diffuse hydrogen envelope. if such a star passes close to a mbh, the envelope can be partially or completely stripped by the tidal interaction with the mbh. this will deposit the dense core, which is essentially a white dwarf, on a close orbit about the mbh ; the system will eventually become an emri [145, 150 ], in - situ formation : if a mbh has a massive accretion disc, the disc can become unstable to star formation. stars formed in such a way are biased toward higher masses and will therefore tend to form black - hole remnants. these remnants will be in circular, equatorial orbits around the central mbh, and will inspiral as emris under gw emission.. the intrinsic rate at which emris occur depends on a lot of astrophysics that is rather poorly understood. the rate is strongly influenced by several processes, including : mass segregation : stellar encounters tend on average to lead to equipartition of energy. this means that after a two - body interaction the heavier object tends to be moving slower, and the lighter object tends to be moving faster. if this occurs for two objects around a mbh, the heavier object sinks in the potential of the mbh and the lighter object moves further out. components of the stellar distribution thus become segregated according to their mass. the more massive objects, which tend to be the black holes, end up closer to the mbh and are captured preferentially [18, 188, 313].triaxiality : the centers of galaxies tend to be approximately spherically symmetric, but on larger scales galactic nuclei can be triaxial. orbits in triaxial potentials tend to be centrophilic : they have a tendency to pass close to the center. this process can funnel stars that are further away from the mbh toward the center and hence increase the inspiral rate [233, 364].resonant relaxation : in the standard relaxation picture, each encounter is random and uncorrelated, so stars undergo a random walk. the process is driven by the diffusion of energy which then leads to angular - momentum transfer. however, in a stellar cluster around a mbh, each star will be on a keplerian orbit, which is a fixed ellipse in space. the orbits of two nearby stars will thus exert correlated torques on one another, which can lead to a direct resonant evolution of the angular momentum [375, 376 ]. this process can lead to a much more rapid diffusion of orbits into the regime where they can become emris.the schwarzschild barrier : resonant relaxation relies on the orbits having commensurate radial and azimuthal frequencies, so they remain in fixed planes over multiple orbits. in the strong - field potential of a massive object the schwarzschild barrier refers to the boundary between orbits with and without significant precession. inside this point resonant relaxation a full description of these processes and their relative importance can be found in [18, 16 ]. the range of emri rates per galaxy reported in the literature is 11000 gyr for black holes, and 105000 gyr for white dwarfs, with most recent best - guess estimates of 400 gyr / 20 gyr / 7 gyr for black holes / white dwarfs / neutron stars [235, 19 ]. this means that after a two - body interaction the heavier object tends to be moving slower, and the lighter object tends to be moving faster. if this occurs for two objects around a mbh, the heavier object sinks in the potential of the mbh and the lighter object moves further out. components of the stellar distribution thus become segregated according to their mass. the more massive objects, which tend to be the black holes, end up closer to the mbh and triaxiality : the centers of galaxies tend to be approximately spherically symmetric, but on larger scales galactic nuclei can be triaxial. orbits in triaxial potentials tend to be centrophilic : they have a tendency to pass close to the center. this process can funnel stars that are further away from the mbh toward the center and hence increase the inspiral rate [233, 364 ]. resonant relaxation : in the standard relaxation picture, each encounter is random and uncorrelated, so stars undergo a random walk. the process is driven by the diffusion of energy which then leads to angular - momentum transfer. however, in a stellar cluster around a mbh, each star will be on a keplerian orbit, which is a fixed ellipse in space. the orbits of two nearby stars will thus exert correlated torques on one another, which can lead to a direct resonant evolution of the angular momentum [375, 376 ]. this process can lead to a much more rapid diffusion of orbits into the regime where they can become emris. the schwarzschild barrier : resonant relaxation relies on the orbits having commensurate radial and azimuthal frequencies, so they remain in fixed planes over multiple orbits. in the strong - field potential of a massive object the schwarzschild barrier refers to the boundary between orbits with and without significant precession. inside this point resonant relaxation uncertainties in the event rate come not only from the intrinsic rate of capture, but also from uncertainties in the number density of black holes of suitable mass. at present, only three objects in the range 1010 m are known from kinematic measurements. using galaxy luminosity functions, the luminosity - velocity - dispersion relation and the mbh - mass - velocity dispersion relation, one infers the number density of mbhs in the lisa mass range to be roughly constant per logarithmic mass unit, and equal to a few 10 mpc. however, when black holes merge, the asymmetric radiation of linear momentum over the final few orbits imparts linear momentum (a merger kick) to the remnant black hole. these kicks can be large enough to eject the black hole from its host galaxy. if these black - hole merger kicks are significant for black holes merging in the centers of galaxies, the number of black holes with m 2.8 10 km (or mg 3 10 km (mg 2 10 km (mg a few 10 km seem possible, and would be strengthened by analyzing full catalogs of binary detections at once. instead of the chirping signals from inspiraling binaries, jones proposes a test of the gw dispersion relation using the waves from eccentric galactic binaries, which are emitted at multiple harmonics of the orbital frequencies ; if at least one galactic binary has sufficient eccentricity, jones claims sensitivity comparable to the chirp - dephasing measurements. extend the graviton - mass formalism to more general modified - gravity theories that predict violations of lorentz invariance and modified dispersion relations for gw modes, given by 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^2 } = { p^2}{c^2 } + m_g^2{c^4 } + { \mathbb a}{p^\alpha}{c^\alpha};$$\end{document } both mg and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathbb a$\end{document } can be constrained together, given the corresponding to specific theories, by inspiral - binary observations with ground and space - based detectors. is a conserved quantity, so left and right - circular polarized gravitational radiation propagates alike. many attempts to formulate a quantum theory of gravity require the addition of a parity - violating chern - simons (cs) term to the einstein - hilbert action [14, 7, 363 ] : 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{cs } } } } = { 1 \over { 64\pi}}\int { \theta \,r^{\ast}r{{\rm{d}}^4}x, } \quad \,\,\,{\rm{where}}\quad r^{\ast}r = { 1 \over 2}{r_{\alpha \beta \gamma \delta}}{\epsilon ^{\alpha \beta \gamma \nu}}r_{\mu \nu}^{\gamma \delta};$$\end{document } here r is the riemann tensor, is the levi - civita tensor density, and is a (possibly) position - dependent function that describes the coupling of the cs field to spacetime. this correction creates a difference in the propagation equations for the left- and right - circular gw polarizations, resulting in their amplitude birefringence : one circularly - polarized state is amplified through propagation, while the other is attenuated. this effect is potentially observable with lisa - like detectors for mbh - binary inspirals at cosmological distances (see also), where the amplitude birefringence generates an apparent precession of the orbital plane of the binary. the cs correction accumulates with distance, and is larger for sources at higher redshifts. orbital - plane precession will also arise from general - relativistic spin - orbit coupling, but the scaling of the precession with frequency is different, so the two effects can be distinguished, at least in principle. for an equal - mass binary with redshifted masses of 10 m that is observed plane - on at a redshift z = 15, lisa could constrain the integrated cs contribution at the level of 10. this is several orders of magnitude better than solar - system experiments, which furthermore can only provide local constraints. thus, lisa - like detectors may provide some hints as to the very quantum nature of gravity. in theories that do not satisfy the strong equivalence principle, the internal gravitational binding energies of bodies can create a difference between the inertial dipole moment (i.e., the linear momentum, which is conserved) and the gw - generating gravitational dipole moment. thus, alternative theories of gravity generally admit dipole radiation, but it is forbidden in gr, where the two moments are identical. dipole radiation would be given at leading order by 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_d}\sim{1 \over { { d_l}}}{{\rm{d } } \over { { \rm{d}}t}}(m_1^g{{\bf{x}}_1 } + m_2^g{{\bf{x}}_2})\sim{{{\mu ^i}{\bf{v } } } \over { { d_l}}}\left({{{m_1^g } \over { m_1^i } } - { { m_2^g } \over { m_2^i } } } \right),$$\end{document } where x1,2 are the positions of the binary components, v their relative velocity, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{1,2}^i$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{1,2}^g$\end{document } are their inertial and gravitational masses respectively, is the inertial reduced mass, and dl is the luminosity distance to the observer. for relativistic objects such as neutron stars (ns), the gravitational binding energy can be considerable and so can be the resulting loss of energy to dipolar gws. indeed, the experimental result that the orbital decay of the binary pulsar psr1913 + 16 adhered closely to gr s quadrupole - formula prediction was sufficient to definitely falsify gr alternatives such as bimetric and stratified theories. (amusingly, certain theories even predict that dipole radiation carries away negative energy from a binary.) thus it is factually correct to state that the indirect detection of gws has already provided a strong test of gr. by contrast, the binary pulsar could not falsify scalar - tensor theories in this way, because these are close to gr. for instance, although dipole radiation is predicted by brans - dicke theory and changes the progression of orbital decay, the coupling parameter bd can be adjusted to approximate gr results to any desired accuracy. gr is reproduced for bd, so experimental bounds on brans - dicke are lower bounds. the hulse - taylor binary pulsar does provide a bound on bd, but one that is not competitive with solar - system tests, among which the best comes from the doppler tracking of the cassini spacecraft, which sets bd > 40000 [471, 81 ]. however, other binary systems containing pulsars are known that provide constraints, which are competitive with solar - system constraints. the best constraints on scalar - tensor gravity (and also teves gravity) come from the pulsar - white - dwarf binary j1738 + 0333, which provides the limit bd > 25000. lisa - like detectors can constrain bd by looking for dipole - radiation - induced modifications in the gw phasing of binary inspirals (monopole radiation is also present, but suppressed relative to the dipole), as long as at least one of the binary components is not a black hole : because of the no - hair theorem, black holes can not sustain the scalar field that would lead to a differing and m (as was recently confirmed in full numerical - relativity simulations). this restriction can be circumvented by having non - asymptotically flat boundary conditions for the black hole. if the scalar field is slowly varying far from the black hole (either as a function of time or space) then it can support a scalar field. this scenario was investigated numerically in, which found that accelerated single black holes and black - hole binaries would emit scalar radiation, in the latter case at twice the orbital frequency. if the asymptotic scalar - field gradient that supports the black - hole scalar hair is cosmological in origin, this effect will be negligible, but the possibility does exist in general. except for these considerations, the canonical source for detecting this effect is the inspiral of a neutron star into a relatively low - mass central black hole, although the number of detections of such systems is likely to be very low. early studies [397, 473 ], based on simplified models of the waveforms and of the lisa sensitivity, estimated that for a 1.4 m neutron star inspiraling into a mbh, at fixed snr = 10, the bd bounds would scale as 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { { \rm{bd } } } } > 2 \times { 10 ^ 4}{\left({{{\mathcal s } \over { 0.3 } } } \right)^2}\left({{{100 } \over { \delta { \phi _ d } } } } \right){\left({{t \over { 1\,\,{\rm{yr } } } } } \right)^{7/8}}{\left({{{{{10}^4}{m _ \odot } } \over { { m _ \bullet } } } } \right)^{3/4}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document } (the sensitivity) is a measure of the difference between the neutron - star and mbh self - gravitational binding energies per unit rest mass ; d is the dipole contribution to the gw phasing ; t is the time of observation ; and m is the mbh mass. however, this estimate is reduced by a factor of ten or more when more realistic waveforms are considered that include spin couplings [70, 71 ], spin - induced orbital precession and eccentricity. bounds can also be derived for a massive - scalar variant of brans - dicke theory, and are of order 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({{{{m_s } } \over { \sqrt { { \omega _ { { \rm{bd } } } } } } } } \right)\left({{\rho \over { 10 } } } \right) \lesssim { 10^{- 19}}\,\,{\rm{ev,}}$$\end{document } (where is the mass of the scalar and the detection snr) for the intermediate - mass - ratio inspiral of a ns into a black hole with mass 10 m. these results were obtained using only the leading order correction from the scalar radiation. in the authors extended this calculation to all post - newtonian orders, but in the extreme - mass - ratio limit by using the teukolsky formalism. the conclusion, that constraints on massless scalar - tensor theories from gw observations will, in general, be weaker than those from solarsystem observations, was unchanged. the reason is that scalar - tensor theories are weak - field (infrared) corrections to gr and are therefore largest in the weak field, so the leading order correction captures the majority of the effect. massive scalar - tensor theories were also considered in [110, 495 ]. in those theories, the primary observable consequence is the possible existence of floating orbits at which the scalar flux experiences a condition where gws scatter off the central, massive body, emerging with more energy (extracted from the spin of the central body). the waves transfer that energy to the small orbiting body, increasing its orbital energy. the transition of an emri through such a floating orbit is many orders of magnitude slower than the normal emri inspiral and can last more than a hubble time. if an emri consistent with gr is observed it means that the emri not only did not pass through such a floating orbit during the timescale of the observation but could not have encountered one prior to the observation since it would not then have reached the millihertz band. therefore, an observation of a single emri can constrain the massive scalar - tensor parameter space to many orders of magnitude greater precision than current solar - system observations. a number of other suggestions have been made for low - frequency gw tests of gr that do not quite fit a modified energy - loss description. for instance, dynamical chern - simons theory introduces nonlinear modifications in the binary binding energy and dissipative corrections at the same pn order [426, 483 ] that could be observed in the late inspiral, constraining the characteristic chern - simons length scale to 1010 km, comparable to current solar - system constraints (advanced ground - based detectors could do even better, placing bounds of 10100 km). corrections to the inspiral phasing will also arise if the spacetime outside the central object is not described by the kerr metric or if additional energy is lost into scalar or other forms of radiation. this has been considered for various alternative theories of gravity ; we discuss these results in detail in section 6.2.6. gw tails, which are due to the propagation of gravitational radiation on the curved background of the emitting binary, appear at a relative 1.5pn order (c) beyond the leading - order quadrupole radiation, and their observation would test the nonlinear nature of gr. (this would be a null test of gr, since tails are included in the standard post - newtonian inspiral phasing ; see also the pn - coefficient tests discussed in section 5.2.1. promoting newton s constant, g, to a function of time modifies both a binary s binding energy and gw luminosity, and therefore its phasing. a three - year observation of a 1010 m inspiral would constrain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot g / g$\end{document } to 10 yr. the infinite randall - sundrum braneworld model may predict an enormous increase in the hawking radiation emitted by black holes [164, 436 ]. the resulting progressive mass loss may be observed as an outspiral effect in the quasi - monochromatic radiation of galactic black - hole binaries, as a correction to the inspiral phasing of a black - hole binary and it would also affect the rate of emri events [306, 484 ]. the constraints on the size of extra dimensions coming from observations with lisa will, in general, be worse than those derivable from tabletop experiments. however, decigo observations of bh - ns binary mergers would be able to place a constraint about ten times better than tabletop experiments, assuming a detection rate of 10 binaries per year. as discussed above, quantitative tests of gr against modified theories of gravity evaluate how well the measured signals are fit by alternative waveform families, or (more commonly) by waveform families that extend gr predictions by including one or more modified - gravity parameters, such as bd for brans - dicke theory. to set up these tests we need to work within the alternative theory to derive sufficiently accurate descriptions of source dynamics, gw emission, and gw propagation. an alternative approach is to operate directly at the level of the waveforms by introducing phenomenological corrections to gr predictions : for instance, by modifying specific coefficients, or by adding extra terms. so far these have concentrated on post - newtonian waveforms for circular, adiabatic inspirals, as described by the stationary - phase approximation in the frequency domain : 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h(f) = a{f^{- 7/6}}\exp \left [{ i\psi (f) + i{\pi \over 4 } } \right],$$\end{document } where f is the gw frequency ; a is the gw amplitude, given by geometrical projection factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}_c^{5/6}/{d_l}$\end{document } (with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal m}_c } = { ({ m_1}{m_2})^{3/5}}/{({m_1 } + { m_2})^{1/5}}$\end{document } the chirp mass and dl the luminosity distance) ; and for simplicity we omit the nontrivial response of space - based detectors, as well as the pn amplitude corrections. the phasing (f) is expanded as 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\psi (f) = 2\pi f{t_c } + { \phi _ c } + \sum\limits_{k \in { \mathbb z } } { \left [{ { \psi _ k } + \psi _ k^{\log}\log f } \right]{f^{(k - 5)/3}}}.$$\end{document } for binaries with negligible component spins, the post - newtonian phasing coefficients k are currently known up to k = 7 (3.5 pn order), and in gr they are all functions of the two masses m1 and m2 alone (although \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\psi _ 1 } = \psi _ { 0 - 4}^{\log } = \psi _ 7^{\log } = 0$\end{document }, and 5 is completely degenerate with c, so it is usually omitted) [86, 87, 85, 30 ]. propose a test of gr based on estimating all the k simultaneously from the measured waveform as if they were free parameters, in analogy to the post - keplerian formalism [293, section 4.5 ]. the value and error estimated for each4 k, together with its pn functional form as a function of m1 and m2, determines a region in the m1m2 plane. if gr is correct, all the regions must intersect near the true masses, as shown in figure 5. the extent of the intersection provides a measure of how precisely gr is verified by a gw observation. a fisher - matrix analysis suggests that, for systems at the optimistic distance of 3 gpc, lisa could measure 0 to 0.1% and 2 and 3 to 10%, but that the fractional error on higher - order terms would be at best 1. (51) yields differently shaped regions in the m1m2 plane, which must intersect near true mass values if gr is correct. image reproduced by permission from, copyright by aps. estimating all the binary - inspiral phasing coefficients k of eq. (51) yields differently shaped regions in the m1m2 plane, which must intersect near true mass values if gr is correct. image reproduced by permission from, copyright by aps. however, this setup may understate the power of this kind of test, since most of the estimation uncertainty in the k arises from their mutual degeneracy that is, from the fact that it is possible to vary the value of a subset of without appreciably modifying the waveform. this degeneracy should not impact the degree to which the data is deemed consistent with gr. in a follow - up paper, arun. propose a revised test whereby the masses are determined from 0 and 2, while the other k(as well as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi _ 5^{\log}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi _ 6^{\log}$\end{document }) are individually estimated and checked for consistency with gr. in this case, even for sources at z = 1 (7 gpc), all the parameters can be constrained to 1% (a few % for 4 0.1% for 3), at least for favorable mass combinations. performing parameter estimation for the eigenvectors of the k fisher matrix indicates which combinations of coefficients can be tested more accurately for gr violations. however, it is not clear what significance with regards to testing gr should be ascribed to the accuracy of measuring the k, since we do not know at what level we could expect deviations to appear. by contrast, if we were to find that, say, the n regions in the m1m2 plane do not intersect, we could make the statistically - meaningful statement that gr appears to be violated at the n level. li. [284, 285 ] propose a more satisfying formulation for these tests, based on bayesian model selection, which compares the bayesian evidence, given the observed data, for the pure - gr scenario against the alternative - gravity scenarios in which one or more of the k are modified. the issue of significance discussed above reappears in this context as the inherent arbitrariness in choosing prior probabilities for the k, but del pozzo. argue that this does not affect the efficacy of the model - comparison test in detecting gr violations. (for a comprehensive discussion of model selection in the context of gw detection, rather than gr tests, see also [456, 457, 291 ]. for more recent applications of this formalism to ground - based detectors, see.) in, yunes and pretorius propose a similar but more general approach, labeling it the parameterized post - einsteinian (ppe) framework. for adiabatic inspirals, they propose enhancing the stationary - phase inspiral signal with extra powers of gw frequency : 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h^{{\rm{ppe}}}}(f) = { \tilde h^{{\rm{gr}}}}(f) \times (1 + \alpha { (\pi { \mathcal m}f)^a})\exp \left [{ i\beta { { (\pi { \mathcal m}f)}^b } } \right],$$\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}${{\tilde h}^{{\rm{gr}}}}$\end{document } is given in eqs. while the initial suggestion in is to consider a, b, there are analytical arguments why a and b should be restricted to values a = /3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a = \bar a/3$\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}$b = \bar b/3$\end{document }, which reproduces arun s pn - coefficient scheme for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar b \ge - 5$\end{document}. nevertheless, this representation can reproduce the leading - order effects of several alternative theories of gravity (see table 2). table 2leading - order effects of alternative theories of gravity, as represented in the ppe framework [eq. this table is copied from, except for the two entries labeled with an asterisk. the quadratic curvature ppe exponent given in was b = 1/3, coming from the conservative dynamics. however, it was shown in that the dissipative correction is larger, giving the value b = 7/3 quoted above. the dynamical chern - simons ppe exponent given in was b = 4/3, which was derived using the slow - rotation metric accurate to linear order in the spin. at quadratic order in the spin, the corrections to both conservative and dissipative dynamics occur at lower post - newtonian order, giving b = 1/3. a b brans - dicke07/3 parity violating1 0variable g(t)8/3 13/3 massive graviton01 quadratic curvature07/3 extra dimensions013/3 dynamical chern - simons+3 + 1/3 leading - order effects of alternative theories of gravity, as represented in the ppe framework [eq. this table is copied from, except for the two entries labeled with an asterisk. the quadratic curvature ppe exponent given in was b = 1/3, coming from the conservative dynamics. however, it was shown in that the dissipative correction is larger, giving the value b = 7/3 quoted above. the dynamical chern - simons ppe exponent given in was b = 4/3, which was derived using the slow - rotation metric accurate to linear order in the spin. at quadratic order in the spin, the corrections to both conservative and dissipative dynamics occur at lower post - newtonian order, giving b = 1/3. in, yunes and pretorius are motivated by the possibility of detecting gr violations, but also by the fundamental bias that would be incurred in estimating gw - source parameters using gr waveforms when modified gr is instead correct. in, cornish. reformulate the detection of gr violations described by ppe as a bayesian model - selection problem, similar to the pn - coefficient tests discussed in section 5.2.1. figure 6 shows the bounds, for various fixed b, that could be set with lisa observations of m1,2 10 m binary inspirals at z = 1 and 3. for b corresponding to modifications in higher - order pn terms (which require strong - field, nonlinear gravity conditions to become evident), the bounds provided by lisa - like detectors become more competitive with respect to solar - system and binary - pulsar results (where weak - field conditions prevail). figure 6constraints on phasing corrections in the ppe framework, as determined from lisa observations of 10 m massive - black - hole inspirals at z = 1 and z = 3. the figure also includes the bounds derived from pulsar psr j0737 - 3039, the solar - system bound on the graviton mass, and pn - coefficient bounds derived as described section 5.2.1. the spike at b = 0 corresponds to the degeneracy between the ppe correction and the initial gw - phase parameter. constraints on phasing corrections in the ppe framework, as determined from lisa observations of 10 m massive - black - hole inspirals at z = 1 and z = 3. the figure also includes the bounds derived from pulsar psr j0737 - 3039, the solar - system bound on the graviton mass, and pn - coefficient bounds derived as described section 5.2.1. the spike at b = 0 corresponds to the degeneracy between the ppe correction and the initial gw - phase parameter. (adapted from.) a ppe - like model including dipole radiation in addition to quadrupole radiation but no other modifications to the waveform phasing was described in and was discussed in section 5.1.1 above. the full ppe framework was extended to include all additional polarization states and higher waveform harmonics in. the final form was motivated by considering brans - dicke theory, lightman - lee theory and rosen s theory. in the most general form, eq. 52 is modified to 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{{20}c } { { { \tilde h}^{{\rm{ppe}}}}(f) = { { \tilde h}^{{\rm{gr}}}}(f) \times \exp \left [{ i\beta { { (\pi { \mathcal m}f)}^b } } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + ({ \alpha _ + } { f _ + } + { \alpha _ \times}{f _ \times } + { \alpha _ b}{f_b } + { \alpha _ l}{f_l } + { \alpha _ { { \rm{sn}}}}{f_{{\rm{sn } } } } + { \alpha _ { { \rm{se}}}}{f_{{\rm{se}}}})\quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \times { { (\pi { \mathcal m}f)}^a}\exp \left [{ - i\psi _ { { \rm{gr}}}^{(2) } + i\beta { { (\pi { \mathcal m}f)}^b } } \right ] } \\ { + ({ \alpha _ + } { f _ + } + { \alpha _ \times}{f _ \times } + { \alpha _ b}{f_b } + { \alpha _ l}{f_l } + { \alpha _ { { \rm{sn}}}}{f_{{\rm{sn } } } } + { \alpha _ { { \rm{se}}}}{f_{{\rm{se}}}})\,\,\,\quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \times { { (2\pi { \mathcal m}f)}^c}{\eta ^{{1 \over 5}}}\exp \left [{ - i\psi _ { { \rm{gr}}}^{(1) } + i\delta { { (2\pi { \mathcal m}f)}^b } } \right],\,\, } \\ \end{array}$$\end{document } in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi _ { { \rm{gr}}}^{(l)}$\end{document } is the gr phase of the lth waveform harmonic, = m1m2/(m1 + m2) is the symmetric mass ratio, and fa is the detector response to a gw in polarization mode a. the ppe parameters are ({ a }, { a }, a,, b, c,, d). the authors of considered two further variants of this scheme. one variant restricted the coefficients in the expansion so that they were not all independent, but were related to one another via energy conservation. the second variant included this interdependence of the parameters, and also accounted for modified propagation effects by introducing additional phase - difference yet, this fully extended ppe scheme has not been used to explore the constraints that will be possible with space - based detectors. an analysis using a waveform model with higher harmonics and spin precession, but not alternative polarization states, was carried out in. its authors considered modifications to a subset of the phase and amplitude parameters only, which corresponded to certain post - newtonian orders and could therefore also be interpreted in terms of modifications to the pn phase coefficients as discussed in section 5.2.1. the estimated bounds derived using this more complete waveform model were typically one to two orders of magnitude better than previous estimates for highmass systems, but basically the same for low - mass systems. this is unsurprising, since the effects of spin - precession and higher harmonics will only be important late in the inspiral. high - mass systems generate lower frequency gws and are therefore only observable for the final stages of inspiral, merger and plunge. therefore, late - time corrections are proportionally more important for those systems. for high - mass systems, the authors of estimated that lisa would be able to measure deviations in the phasing parameters to a precision n 0.1, 10, 50, 500, 1000 for n = 1, 0, 1, 3/2, 2 respectively, where n denotes the post - newtonian order, with n the coefficient of f in the waveform phase. using the same model, they also estimated that lisa could place a bound of g > 1 10 km on the graviton compton wavelength when allowing for correlations between the different phase - modification parameters n. an extension of the ppe framework to emri systems requires a model in which orbits can be both eccentric and inclined. to develop this, vigeland. derive a set of near - kerr spacetime metrics that satisfy a set of conditions, including the existence of a carter - constant - like third integral of the motion, as well as asymptotic flatness. the solutions, which were previously found in, are restricted to a physically interesting subset by setting to zero any metric coefficients not required to reproduce known black - hole solutions in modified gravity, and by applying the peeling theorem (i.e., by requiring that the mass and spin of the black hole not be renormalized by the perturbation). the existence of a third integral is not a requirement for black - hole solutions, but in general its absence allows ergodic behavior in the orbits. this is discussed as a potential observable signature for deviations from gr in section 6.2.5. however, data - analysis pipelines designed for gr waveforms may be insensitive to such qualitatively different systems. therefore the existence of a third integral is a practical assumption for interpretation once a gr - like emri has been observed. in, gair and yunes construct gravitational waveforms for emris occurring in the metrics of, based on the analytic kludge model constructed for gr emris. the waveforms provide a ppe - like model for emris that can be used in the same way as the circular ppe framework. parameter - estimation results with these ppe - emri models have not yet appeared in the literature. in, vallisneri provides a unified model - comparison performance analysis of all modified - gr tests that is valid for sufficiently - loud signals, and that yields the detection snr required for a statistically - significant detection of gr violations as a simple function of the fitting factor ff between the gr and modified - gr waveform families. the ff measures the extent to which one can reabsorb modified - gr effects by varying standard - gr parameters from their true values. vallisneri s analysis is valid in the limit of large snr, and may not be applicable to all realistic scenarios with finite snrs. an alternative to modifying frequency - domain inspiral waveforms is offered by cannella. they propose tests based on the effective - field - theory approach to binary dynamics, which expands the hilbert+point - mass action as a set of feynman diagrams. in this framework, gr corrections can be introduced by displacing the coefficients of interaction vertices from their gr values. for instance, multiplying the three - graviton vertex by a factor (1+3) affects the conservative dynamics of the theory in a manner similar to the ppn parameter, but also has consequences on radiation. a similar modification to the four - graviton vertex (parameterized by 4) yields effects at the second post - newtonian order, so it has no analog in ppn. argue that gr - violating values of 3 and 4 would not be detectable with gw signals, but they would instead generate small systematic errors in the estimation of standard binary parameters. however, a thorough analysis of the detectability of such deviations has not been carried out, so this conclusion may be modified in the future. according to gr, black - hole mergers are the most energetically luminous events in the universe, with l 10 l 10 erg / s, regardless of mass : at their climax, they outshine the combined power output of all the stars in the visible universe. nevertheless, second - generation ground - based gw interferometers are expected to yield the first detections of black - hole mergers, but only with rather modest snrs. by contrast, lisa - like gw detectors would observe the mergers of heavier black holes, with snrs as high as hundreds or more throughout the universe, offering very accurate measurements of the merger waveforms. massive - black - hole coalescences may feature significant spins and eccentricity, further enriching the merger phenomenology [80, 380 ]. the powerful merger events correspond to very relativistic velocities and very strong gravitational fields, so much that the pn expansion of the field equations can not be applied, and we must resort to very complex and costly numerical simulations. this makes it challenging to encode the effects of plausible gr modifications in the signal model. the first ppe paper makes such an attempt on the basis of a very crude model of merger - ringdown signals, which would probably be insufficient even to phase - match the gr signals themselves. broad efforts are currently under way to build phenomenological full - waveform (inspiral - merger - ringdown) models [4, 344, 438 ] ; these involve tunable parameters that are adjusted to match the waveforms produced by numerical relativity. such parameters could also be used to encode non - gr effects in the merger - ringdown. however, at this time designing such extensions in a principled way seems daunting. a simpler approach, proposed by hughes and menou, involves the golden binaries for which system parameters can be estimated from both inspiral and ringdown gws. the former encode the parameters of the binary, while the latter encode the parameters of the final black hole formed in the merger. the functional relation of the two sets of parameters can then be compared with the predictions of numerical relativity, providing a null test of the strong - field regime of gr. hughes and menou focus on measuring the remnant s mass deficit, which equals the total energy carried away by gws, so their definition of golden binaries selects those in which the mass deficit can be estimated to better than 5%. for lisa, these systems tend to have component masses between a few 10 m and a few 10 m, and to be found at z 23, making up 110% of the total merger rate depending on black - hole population models. the estimates of are based on rather simple waveform models that omit a range of physical effects, so they could be seen as conservative, given that increased waveform complexity tends to improve parameter - estimation accuracy. a more complete analysis was carried out in, but in the context of ground - based gw detectors rather than space - based detectors. it has become apparent over the last few decades that the centers of most galaxies harbor massive, dark compact objects with masses in the range 1010 m. it is clear that these objects play a very important role in the evolution of galaxies. this is exemplified by the very tight measured correlation (the m- relation) between the mass of the central dark objects and the velocity dispersion of stars in the central spheroid [174, 441 ]. it is generally accepted that the central dark objects are black holes described by the kerr metric, but there is presently no definitive proof of that assumption. the alternatives to the black - hole interpretation include dense star clusters, supermassive stars, magnetoids, boson stars, and fermion balls. support for the blackhole interpretation has arisen as a result of both observational and theoretical work. a short review of the evidence may be found in, although we summarize some key details in section 6.1. as described in section 2.3, the theoretical basis for the belief that these objects are kerr black holes has arisen from proofs that singularities inevitably form during gravitational collapse [341, 351, 224 ] and that the kerr solution is the unique stationary and axisymmetric black - hole solution in gr [248, 114, 379 ]. the uniqueness of the kerr solution is sometimes referred to as the no - hair theorem, since the solution is characterized by just two parameters, the black - hole mass m and angular momentum (per unit mass) a. the field of any vacuum, axisymmetric spacetime in gr can be characterized by a sequence of mass and current multipole moments, which we denote as ml ; and sl ; respectively [204, 217 ]. for kerr spacetimes these multipole moments are all determined by the mass and spin via 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_l } + { \rm{i}}{s_l } = m{({\rm{i}}a)^l},$$\end{document } so these spacetimes require no additional independent parameters or hair. the proof of the uniqueness theorem relies on various assumptions beyond the validity of the einstein equations, so a demonstration of the non - kerr nature of astrophysical black holes could reveal exotic physics within gr. it might also indicate the presence of material in the spacetime outside the black - hole horizon, or a deviation from gr in the true theory of gravity. in this section we discuss the potential of space - based low - frequency gw detectors to probe the structure of massive compact objects and the possible interpretation of these results. short reviews of the prospects for testing relativity with measurements of black - hole hair can be found in [73, 191 ]. the observational evidence for the presence of black holes in the centers of galaxies has come mainly from the studies of active galactic nuclei (agn). these are known to be extremely energetic and also compact typical luminosities of 10 erg s are produced in regions less than 10 m across. the inferred agn efficiency of 10% is much greater than the typical efficiencies of nuclear fusion processes 1%), implying the need for a very deep relativistic potential. x - ray observations show variability on timescales of less than an hour, while observations of iron lines indicate the presence of gas moving at speeds of several thousand km per second. radio observations of water maser discs are consistent with keplerian motion around very compact central objects. in the spiral galaxy ngc 4258 vlba observations have indicated a disc with an inner (detected) edge at 0.1 pc, around an object of mass 3.6 10 m. in addition, about 10% of agns are associated with jets, which move at highly relativistic velocities and persist for millions of years. this requires a relativistic potential that has a preferred axis that is stable over very long timescales. agns are also remarkably similar over several decades of mass, which favors the black - hole explanation, again because kerr black holes are characterized by just m and a. in the milky way, evidence for the presence of a black hole coincident with the sgr a radio source has come from observations of stellar motions. these are completely consistent with keplerian motion around a point source of mass m 4 10 m [206, 205 ]. one star, s2, has been observed for one complete orbit and from this it has been possible to put a limit of 0.066 on the extended fraction of the central mass that could be contained between pericenter and apocenter of the orbit of s2. at perihelion s2 was 100 au from the central object, which provides a fairly tight constraint on its compactness. electromagnetic observations can rule out stellar clusters as the explanation for the massive central objects, but some of the exotic alternatives remain. x - ray emission comes from the very inner regions of accretion discs, but uncertainties in the radius from which the emission is coming and in the mass and spin of the central object limit their utility for probing the structure of the central object. it is also possible to construct very compact boson star spacetimes that could not be ruled out from electromagnetic observations alone. by contrast, gw observations will probe the spacetime structure as the object proceeds through the inspiral and then passes the innermost stable orbit and plunges into the horizon of the central object, if a horizon exists. we discuss the prospects for using gw observations to probe black - hole structure in the following sections 6.2 and 6.3. equation (54) tells us that a kerr black hole is uniquely characterized by two parameters. if we can measure three multipole moments of the spacetime, we can check that they are consistent with eq. if they are not, then the object can not be a kerr black hole. boson stars will typically have more independent multipole moments. in a certain class of models of rotating boson stars, the boson star can be uniquely characterized by three multipole moments [389, 73 ], so a lisa measurement of four multipole moments could also exclude these models as an explanation of the data. gws from emris are complicated superpositions of different frequency components, found at harmonics of the three fundamental frequencies of the orbit : the orbital frequency and the frequencies of precession of the perihelion and of the orbital plane. this complex structure encodes detailed information about the spacetime in which the gws are generated. if the spacetime is assumed to be stationary and axisymmetric, it can be shown that the einstein equations reduce to a single equation, the ernst equation, for a complex scalar function, the ernst potential. by using the ernst potential and expressions due to fodor. that relate this potential to the multipole moments of the spacetime, ryan was able to study the properties of orbits in vacuum and axisymmetric spacetimes that possess an arbitrary set of mass and current multipole moments. however, if such an orbit is given a small radial or vertical perturbation, it will undergo small oscillations at frequencies (the epicyclic frequencies) that correspond to the perihelion or orbital plane precession frequencies of nearly circular and nearly equatorial orbits respectively. these frequencies can be readily computed. for the arbitrary stationary axisymmetric spacetimes considered in one finds 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{{20}c } { { { { \omega _ r } } \over \omega } = 3{{(3m\omega)}^{{2 \over 3 } } } - 4{{{s_1 } } \over { { m^2}}}(m\omega) + \left({{9 \over 2 } - { 3 \over 2}{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^{{4 \over 3 } } } - 10{{{s_1 } } \over { { m^2}}}{{(m\omega)}^{{5 \over 3}}}\quad \quad \quad \quad \quad \quad \quad } \\ { + \left({{{27 } \over 2 } - 2{{s_1 ^ 2 } \over { { m^4 } } } - { { 21 } \over 2}{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^2 } + \left({- 48{{{s_1 } } \over { { m^2 } } } - 5{{{s_1}{m_2 } } \over { { m^5 } } } + 9{{{s_3 } } \over { { m^4 } } } } \right){{(m\omega)}^{{7 \over 3 } } } + \cdots, } \\ { { { { \omega _ z } } \over \omega } = 2{{{s_1 } } \over { { m^2}}}(m\omega) + { 3 \over 2}{{{m_2 } } \over { { m^3}}}{{(m\omega)}^{{4 \over 3 } } } + \left({7{{s_1 ^ 2 } \over { { m^4 } } } + 3{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^2}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+ \left({11{{{s_1}{m_2 } } \over { { m^5 } } } - 6{{{s_3 } } \over { { m^4 } } } } \right){(m\omega)^{{7 \over 3 } } } + \cdots, $ $ \end{document } where is the angular () frequency of the circular orbit being perturbed, r and z are the perihelion and orbital plane precession frequencies, and ml / sl denote the mass / current multipole moments of the spacetime metric, as in eq. (54). the primary conclusion from eqs. (55)(56) is that the various multipole moments enter the different terms in this expansion at different orders in. the precession frequencies and orbital frequency could be extracted from gw observations, so these expansions are, in principle, observable. we can use this information to map the spacetime structure near the central object and verify that the multipole moments are consistent with the no - hair property (54) that we expect for a kerr black hole. bothrodesy or holiodesy5 by analogy with geodesy, in which observations of the motion of satellites are used to probe the gravitational field of the earth. the multipole moments are also encoded in the total orbital energy lost as the orbital frequency changes by a unit logarithmic interval 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{{20}c } { { { \delta e(f) } \over \mu } = { 1 \over 3}{{(m\omega)}^{{2 \over 3 } } } - { 1 \over 2}{{(m\omega)}^{{4 \over 3 } } } + { { 20 } \over 9}{{{s_1 } } \over { { m^2}}}{{(m\omega)}^{{5 \over 3 } } } + \left({- { { 27 } \over 8 } + { { { m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^2 } } \\ { { { 28 } \over 3}{{{s_1 } } \over { { m^2}}}{{(m\omega)}^{{7 \over 3 } } } + \left({- { { 225 } \over { 16 } } + { { 80 } \over { 27}}{{s_1 ^ 2 } \over { { m^4 } } } + { { 70 } \over 9}{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^{{8 \over 3 } } } } \\ { + \left({{{81 } \over 2}{{{s_1 } } \over { { m^2 } } } + 6{{{s_1}{m_2 } } \over { { m^5 } } } - 6{{{s_3 } } \over { { m^4 } } } } \right){{(m\omega)}^3 } + \cdots } \\ \end{array}$$\end{document } a more powerful observable than the three discussed so far is the number of cycles that a trajectory spends near a particular frequency 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \mathcal n}(f) = { { { f^2 } } \over { { \rm{d}}f/{\rm{d}}t } } = { f^2}{{{\rm{d}}e/{\rm{d}}f } \over { { \rm{d}}e/{\rm{d}}t}},$$\end{document } but this is not as clean an observable as the precession frequencies, since it requires computing the rate of energy loss to gws in an arbitrary spacetime. ryan used this formalism in conjunction with a post - newtonian waveform model to estimate lisa s capability to measure the spacetime multipoles. he considered nearly circular and nearly equatorial inspirals, and found that lisa s ability to determine the spacetime multipoles degraded as more multipoles were included in the waveform model. the conclusion was that lisa would be able to make moderately accurate measurements of the lowest three multipole moments, but probably no more. table 3accuracy with which a lisa observation could determine the multipole moments of a spacetime decreases as more multipoles are included in the model, taken from ryan. results are shown for two typical cases, a 10 m + 10 m inspiral and a 10 m + 10 m inspiral ; in both cases the snr of the inspiral is 10./m m / m l max log10(m / m)log10(s1)log10(m2)log10(s3)log10(m4)13.73.5101023.02.91.832.31.91.30.741.51.31.10.11.013.32.8101022.51.00.331.20.10.80.941.00.10.81.21.8 accuracy with which a lisa observation could determine the multipole moments of a spacetime decreases as more multipoles are included in the model, taken from ryan. results are shown for two typical cases, a 10 m + 10 m inspiral and a 10 m + 10 m inspiral ; in both cases the snr of the inspiral is 10. ryan s analysis was restricted to circular and equatorial orbits, but a counting argument suggests that spacetime mapping should still be possible for generic orbits. one complication is that the evolution of the orbital elements must also be inferred from the observation, which spoils the nice form of the expansions (55)(56), since all the multipole moments now enter at each order of the expansion. however, this would also be true if the expansions for circular - equatorial orbits were rewritten as an expansion in some initial frequency, 0, which would more closely represent a band - limited observation. in practice, the lowest - order multipole dominates the lowest term in the expansion and so on, which makes spacetime mapping possible in practice. the ryan formalism neatly illustrates why spacetime mapping with emris is possible, but it is not a very practical scheme. we expect that the massive central objects are indeed kerr black holes and so we really want to consider what imprint small deviations from kerr will leave on the emitted gws. a multipole - moment expansion is not a very practical way to do this, as the kerr metric has an infinite number of nonzero multipoles. several authors have adopted the approach of constructing spacetimes given by schwarzschild - kerr plus a small deviation, and have examined the properties of geodesics in those spacetimes. this was constructed by writing the metric in weyl coordinates and adding a quadrupole perturbation to the potential (in these coordinates, the potential equation reduces to the flat - space laplacian for the (, z) cylindrical coordinates, which facilitates writing solutions). they considered two types of quadrupole perturbation : a torus around the black hole, and the addition of a point mass at each pole. in the second case, the spacetime necessarily contains line singularities running from the point masses either to infinity or to the black - hole horizon, which are needed to support the point masses. the solutions are perturbative, in that the authors kept only the terms that are linear in the deviation from schwarzschild. collins and hughes explored the properties of orbits in these spacetimes by comparing the precession and orbital frequencies of equatorial orbits in the spacetime to orbits with the same orbital parameters in kerr. they found that there were measurable differences in the perihelion precession in the strong field : for instance, at a radius of 20 m for a 2% perturbation of the black hole, the trajectory would accumulate one radian of dephasing in 1000 orbits. collins and hughes coined the term bumpy black hole to describe spacetimes of this type. starting from the hartle - thorne metric [220, 221 ], which is an exact solution to einstein s equations describing the spacetime outside of a slowly, rigidly rotating axisymmetric object, the authors constructed a spacetime with metric of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_{\mu \nu } } = g_{\mu \nu}^{{\rm{kerr } } } + \varepsilon { h_{\mu \nu } } + o(\delta { m_{l \ge 3}},\,\delta { s_{l \ge 2}})$\end{document }, working perturbatively and keeping only the m2 perturbation in the quadrupole mass moment. a comparison of the frequencies of eccentric equatorial geodesics in the quasi - kerr spacetime to the same geodesics in the kerr spacetime indicated that it would take only 100 cycles to accumulate a /2 phase shift for a 1% deviation from kerr. they also computed waveform overlaps and found that, over the radiation - reaction timescale, the overlap of the waveforms for an orbit with a semilatus rectum of ten geometric radii (gm / c) was 20%, 50%, 90%, 98% for inspirals with mass ratio /m = 10, 10, 10, 10 respectively. a third approach to analyzing deviations from the kerr spacetime was considered by gair., who studied geodesic motion in a family of exact spacetimes due to manko and novikov, which include the kerr spacetime for a specific choice of parameters. by using exact solutions of the einstein field equations, they obtained solutions that were valid everywhere, in contrast to the perturbative solutions considered in [125, 207 ], which break down near the central object. however, this scheme offers less control over the multipole moments, since it is possible to choose the lowest multipole that differs from kerr, but then the higher multipoles must also change. studied observable properties of the orbits, including the variation of the precession frequencies of nearly circular and nearly equatorial orbits as a function of orbital frequency, and the loss of the third integral of the motion (see section 6.2.5). these three papers outlined different ways to approach the problem of spacetime mapping in practice. however, none of the analyses were complete as they did not consider inspirals. collins and hughes and glampedakis and babak also ignored waveform confusion by assuming that the orbital elements were the same between the orbits under consideration. glampedakis and babak did discuss the importance of confusion and the role of the inspiral evolution in breaking such degeneracies, but no inspiral results were included in the published paper. observationally, the correct orbits to compare will be those with the same frequencies since we have no way to determine the orbital radius or eccentricity directly. assessing the confusion problem is relatively easy, but including inspiral is very hard in general, since the presence of excess multipole moments in the spacetime leads to changes in the rates of energy and angular momentum loss, which must also be included in the analysis. progress can be made in the presence of small deviations by including only the leading - order contributions to the radiation reaction from the multipole moments. this is an open area of research, although barack and cutler carried out a preliminary assessment using post - newtonian emri waveforms augmented with the leading - order contribution of an excess quadrupole moment to the precession and inspiral rates. the resulting waveforms were an improvement in comparison to ryan s analysis, since they included orbital eccentricity and inclination, and were filtered through an approximation of the lisa response. barack and cutler performed a fisher - matrix analysis of parameter - estimation uncertainties, and hence correctly accounted for the confusion issue. for this simple model, they found that a single lisa observation of the inspiral of a 10 m black hole into a 10 m, 10 m or 10 m black hole could measure the deviation from kerr of the quadrupole moment to an accuracy of (m2/m) 10,10, 10, while simultaneously measuring the mass and spin of the central object to fractional accuracies of 10. this suggests that a lisa - like observatory would be able to perform high - precision tests of the no - hair property of massive compact objects in galactic centers. to put these numbers in perspective, a boson star may have a quadrupole moment that is 100 times that of a kerr black hole with the same mass and spin, so it could easily be excluded. during an emri this can be thought of as a tidal interaction the gravitational field of the small body raises a tide on the horizon that is dragged around through the orbit, leading to dissipation of energy or as energy being lost by gws falling into the black hole. fang and lovelace explored the nature of the tidal - coupling interaction by perturbing a schwarzschild black hole with a distant orbiting moon. they found that the time - dependent piece of the perturbation affected the orbit in an unambiguous way : a time - varying quadrupole moment is induced on the blackhole horizon that is proportional to the time derivative of the moon s tidal field. this quadrupole perturbation extracts energy and angular momentum from the orbit at the same rate that energy and angular momentum enter the horizon. however, the effect of the time - independent piece of the perturbation remained ambiguous. working in the regge - wheeler gauge, fang and lovelace found that this piece vanished, in contrast to a previous result by suen, who used a different gauge. this ambiguity leads to an ambiguity in the phase of the induced quadrupole moment as measured in a local asymptotic rest frame (larf), although the phase of the bulge relative to the orbiting moon is well defined (using a spacelike connection between the moon and the black hole, fang and lovelace found that the horizon shear led the horizon tidal field by an angle of 4m, where is the angular velocity of the moon). the ambiguity of interpretation in the larf makes it impossible to define the polarizability of the horizon or the phase shift of the tidal bulge in a body - independent way. fang and lovelace left open the possibility of developing a body - independent language to describe the response of the central object to tidal coupling, but as yet this has not happened. although the nature of the response of the central object to tidal coupling may be difficult to characterize from gw observations, the total energy lost to tidal interactions is a good observable. ryan s original theorem ignored tidal coupling, but it was later generalized by li and lovelace. they found that the gws propagating to infinity depended only very weakly on the inner boundary conditions (i.e., on the nature of the central object). this means that the spacetime s multipole structure can be inferred from the outgoing radiation field in the usual way, and hence the expected rate of orbital energy loss, assuming no energy loss into the central body, can be calculated. the rate of inspiral measures the actual rate of orbital energy loss, and the difference then gives the rate at which energy is lost to the central object, which is a direct measure of the tidal coupling. li and lovelace estimated that the ratio of the change in energy radiated to infinity due to the inner boundary condition to the energy in tidal coupling scales with the orbital velocity as v. therefore, it should be possible to simultaneously determine the spacetime structure and the tidal coupling through low - frequency gw observations. information about the central object can also come from the transition to plunge at the end of the inspiral. in a black - hole system, we expect gw emission to cut off sharply as the orbit reaches the innermost stable orbit and then plunges rapidly through the horizon. if there is no horizon in the system, the orbit may instead enter a phase where it passes into and out of the material of the central object. this was explored for boson - star models in : kesden. found that persistent radiation after the apparent innermost orbit could be a clear signature of the presence of a supermassive, horizonless central object in the spacetime. this analysis did not treat gravitational radiation or the interaction of the inspiraling body with the central object accurately, but it does illustrate a possible way to identify non - black - hole central objects. something similar might happen if the spacetime were to contain a naked singularity rather than a black hole : in principle, the nature of the emitted waveform after plunge would encode information about the exact nature of the central object. however, this has not yet been investigated. naked - singularity spacetimes may have very - high - redshift surfaces rather than horizons : these spacetimes would be observationally indistinguishable from black holes, unless the inspiraling object happened to move inside the high - redshift surface and then emerged, and the two epochs of radiation could be connected observationally. another example of an object that can be arbitrarily close to a schwarzschild black hole in the exterior but lack a horizon is a gravastar. these are constructed by matching a de sitter spacetime interior onto a schwarzschild exterior through a thin shell of matter, whose radius can be made arbitrarily close to the schwarzschild horizon. it was shown in that the oscillation modes of such a gravastar have quasinormal frequencies that are completely different from those of a schwarzschild black hole. therefore the absence of a horizon would be apparent if ringdown radiation was observed from such a system. in addition, the tidal perturbations that arise during the inspiral of a compact object into a gravastar during an emri can resonantly excite polar oscillations of the gravastar as the orbital frequency passes through certain values over the course of the inspiral. the excitation of these modes generates peaks in the gw - emission spectrum at frequencies that are characteristic of the gravastar, and can also show signatures of the microscopic surface of the gravastar. this process would be apparent both in the amplitude of the detected gws and in the rate of inspiral inferred from the gravitational - waveform phase, since the rate of inspiral will change significantly in the vicinity of each resonance due to the additional energy radiated in the excited quasi - normal modes. although the gravastar model used in [346, 347 ] may not be physically relevant, this work illustrates the more general fact that if the horizon of a black hole is replaced by some kind of membrane, then the modes of that membrane will inevitably be excited during an inspiral and these modes will typically be different to those of a black hole. a change in the inspiral trajectory need not be caused only by differences in the central object, but might arise due to the presence of material in the spacetime, close to the black hole but external to the event horizon. such material could influence the inspiral trajectory, and hence the emitted gws, in two distinct ways : the gravitational field of the matter could modify the multipole moments of the spacetime and hence the orbit as discussed above ; if the orbital path intersected the material, it would cause sufficient hydrodynamic drag on the object to alter the orbit. the influence of the gravitational field of external material was considered in : barausse. constructed a model spacetime that included both a black hole and an external torus of material very close to the central black hole. they examined the properties of orbits in two systems : one with a torus of comparable mass and spin to the central black hole (spacetime a), and one with a torus of low mass, but much greater angular momentum than the central black hole (spacetime b). their comparisons were based on computing equatorial geodesics and then kludge gravitational waveforms in the spacetime and in a corresponding kerr spacetime, and then evaluating the waveform overlap. orbits were identified by matching the radial and azimuthal frequencies between the orbits in the two spacetimes in two ways : altering the orbital parameters, while setting the mass and spin to be the same in the corresponding kerr spacetime ; or altering the mass and spin, while keeping the orbital parameters the same. this approach identified a confusion problem : over much of the parameter space the overlaps were very high, particularly when the second approach was adopted. b than in spacetime a, and overlaps for internal orbits between the black hole and the torus were particularly low. this work suggested that it would not be possible to distinguish between such a spacetime and a pure kerr spacetime in low - frequency gw emri observations. the need to constantly adjust the orbital parameters in order to maintain equality of frequencies would lead to a difference in the evolution of the orbit between the two spacetimes, which might break the waveform degeneracies. the torus model was also not physical, being much more compact than one would expect for agn discs. the effect of hydrodynamic drag on an emri was first considered in and was found to be negligibly small for systems likely to be of interest to space - based gravitational - wave detectors. the problem was revisited in : barausse and rezzolla considered a spacetime containing a kerr black hole surrounded by a non - self - gravitating torus with constant specific angular momentum. the hydrodynamic drag consists of a short - range part that arises from accretion, and a long - range part that arises from the gravitational interaction of the body with the density perturbations it causes in the disc. the accretion onto the small object was modeled as bondi - hoyle - lyttleton accretion6 and the long - range force using collisional dynamical - friction results from the literature [264, 50 ]. the effect of the hydrodynamical drag on the orbital evolution was computed for geodesic orbits and compared to the orbital evolution from radiation reaction for a variety of torus models, varying in mass and outer radius. the conclusion was that, for realistic outer radii for the torus, rout > 10 m, the effect of hydrodynamic drag on the orbital radius and eccentricity was always small compared to radiation reaction. the hydrodynamic drag has a greater relative effect on the orbital inclination, and tends to cause orbits to become more prograde, which is opposite to the effect of radiation reaction. for rout = 10 m, the hydrodynamic drag becomes significant at r 35 m, but this radius is smaller for a more compact torus. thus, this effect will only be important for lisa if we observe a system with a very compact accretion torus, or for systems of low central mass. for the latter, the gws are detectable from orbits at larger radii where hydrodynamic drag can be important. however, the snr of such events will be low, so we are unlikely to see many of them. thus, although it seems that this effect is also marginal, this conclusion is based on considering geodesic orbits, and the possible secular build up of a drag signature over the inspiral has yet to be examined, the influence of an accretion disc on the evolution of an emri embedded within it was explored in [493, 268 ]. one of the channels that has been suggested to produce emris is the formation of stars in a disc, followed by the capture of the compact stellar remnants left after the evolution of those stars [18, 16 ]. the migration of such an emri through the accretion disc could potentially leave a measurable imprint on the gw signal. the literature distinguishes between two types of migration. in type i migration, which generally occurs for lower - mass objects the object excites density waves in the disc, which exert a torque on the object. in general, the torque from material exterior to the orbit is greater than that from material interior to the orbit, which causes the object to lose angular momentum and spiral inward on a timescale that is short relative to the lifetime of the disc. in type ii migration, material enters the gap on the disc s accretion timescale, driving the object and gap inwards on that timescale. [493, 268 ] considered migration in geometrically - thin and radiatively - efficient discs, in which thermal energy is radiated on a much shorter timescale than the timescale over which the material moves inward, so the disc can remain thin. such discs can be described by the shakura - sunyaev -disc model, in which the viscous stress in the disc is proportional to the total pressure at each point in the disc. these discs are known to be unstable to linear perturbations [286, 410, 83, 358 ]. the alternative -disc model, where stress is proportional to the gas pressure only, is stable to perturbations. yunes, kocsis. showed that, over a year of observation, type i migration could lead to 0.01/10 radian dephasings in an emri signal for both -discs and -discs, while type ii migration could lead to dephasings of 10/10 radians. the effects are larger for discs, since these can support higher surface density. for more massive central black holes 10 m the dominant contribution is from bondi - hoyle accretion (see footnote 6 for a description), while for less massive black holes of 10 m the dominant contribution is from the migration. these dephasings were computed for the same system parameters (apart from maximizing over time and phase shifts), so they do not account for possible parameter correlations, but the authors argue that the migration dephasing decouples from gw parameters as the effect becomes weaker with decreasing orbital radius, while relativistic effects become stronger. the relative number of emris that will be produced in discs rather than from other channels is not well understood. however, it will be straightforward to identify such emris, which will be circular and equatorial, to look for and constrain effects of this type. another important question that has not yet been addressed is how to distinguish the effect of external material from a difference in the structure of the central object. if the orbit does not intersect the material, such identification would come from the variation in the effect over the inspiral if the change in the multipole structure comes from material, then at some stage the object would pass inside the matter, and the qualitative effect on the inspiral would be different from that of a central object with an unusual multipole structure. if the orbit does intersect the material, then the spacetime - mapping analysis described above no longer applies, since the geroch - hansen multipole decomposition [204, 217 ] applies to vacuum spacetimes only. if this decomposition could be generalized to nonvacuum axisymmetric spacetimes, then low - frequency gw observation could potentially recover not only the spacetime metric but also the structure of the external material. it would then be possible to verify that this matter obeys the various energy conditions (see section 2.3). an independent indicator of the presence of material in the spacetime would come from the observation of an electromagnetic counterpart to an emri event. for instance, if an inspiraling black hole was moving through an accretion disc, there might be emission from the material that was accreted onto the inspiraling object or from shocks formed in the disc. again, this has not been explored, although it is likely, given the poor sky - position determination of emri events in gw observations, that it would not be possible to conclusively identify such a weak electromagnetic signature in coincidence with a gw event. the presence of exotic matter outside a black hole, in the form of a cloud of axions, was discussed in. the presence of large numbers of light axions would be one consequence of extra dimensions in string theory, so the detection of an axion cloud would provide strong evidence in support of their existence. the axion cloud would modify the motion of an inspiraling black hole in a similar way to regular matter, although estimates for the precision of measurements possible with future gw detectors have not yet been carried out. the passage of an emri through the cloud could also lead to its disruption, which may rule out the cloud as a possible explanation for any observed deviations, but further theoretical work is required to properly quantify these processes. if multiple states with the same orbital momenta l and magnetic moments m but different principal quantum numbers n are occupied, transitions between these states can generate gws with characteristic strain 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 22}}{\alpha ^2}{({\epsilon _ 0}{\epsilon _ 1})^{1/2}}\left({{{10\,{\rm{mpc } } } \over r } } \right)\,\,\left({{{{m_{{\rm{bh } } } } } \over { 2\,{m _ \odot } } } } \right)$$\end{document } for a black hole of mass mbh at a distance r. the pre - factor (01) depends on the axion masses and coupling. the axions can also undergo annihilations, which generate gws with very similar characteristic strains 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 22}}{\alpha ^7}\epsilon \left({{{10\,{\rm{mpc } } } \over r } } \right)\,\,\left({{{{m_{{\rm{bh } } } } } \over { 2\,{m _ \odot } } } } \right).$$\end{document } in both cases the frequency depends on the black - hole mass in the usual way f 1/mbh, with typical values for 2 m black holes of 100 hz and 30 khz respectively. both elisa and ligo could place interesting constraints on the axion parameter space through (non)detections of these events. finally, the self - interactions in the axion cloud could eventually lead to the collapse of the cloud in a explosion [269, 489 ], which would generate gws with strain 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 17}}\left({{\epsilon \over { { { 10}^{- 4 } } } } } \right)\,\,\left({{{100\,{\rm{mpc } } } \over r } } \right)\,\,\left({{{{m_{{\rm{bh } } } } } \over { { { 10}^8}\,{m _ \odot } } } } \right)$$\end{document } and frequency c / gmbh. recent calculations suggest that a bosenova from the milky way black hole at saggitarius a would be marginally detectable by lisa. the bosenova explosion comes about due to a super - radiant interaction between the cloud of particles and the central black hole, which extracts rotational energy from the black hole and transfers it to the cloud of particles. recent results on these super - radiant instabilities can be found in. in the observable signatures of the presence of a cloud of bosons outside a massive compact object was considered. it was shown that the motion of a particle through the cloud would be dominated by boson accretion rather than by gravitational radiation reaction. during this accretion - dominated phase, the frequency and amplitude of the gravitational - wave emission is nearly constant in the late stages of inspiral. the authors also considered inspirals exterior to the boson cloud, and found that resonances could occur when the orbital frequency matched the characteristic frequency associated with the characteristic mass of the bosonic particles. these resonances could lead to significant, detectable deviations in the phase of the emitted gws. perturbations to the emri trajectory could also arise from the gravitational influence of distant objects, such as other stars or a second mbh. at present, it is thought to be very unlikely that a second star or compact object would be present in the spacetime sufficiently close to the emri to leave a measurable imprint on the trajectory, although detailed calculations for this scenario have not been carried out. however, if the mbh that was the host of the emri was in a binary with a second mbh, this could perturb the trajectory by a detectable amount. the primary observable effect is a doppler shift of the gw signal, which arises due to the acceleration of the center of mass of the emri system relative to the observer. it was estimated that, for typical emri systems, the presence of a second black hole within a few tenths of a parsec would lead to a measurable imprint in the signal. the frequency scaling of the doppler effect differs from the scaling of the post - newtonian terms in the unaccelerated waveform, which suggests that this effect will be distinguishable in gw observations. the magnitude of the leading - order doppler effect scales as msec / r, where msec is the mass of the perturbing black hole, and r is its distance. if the second object is within a few hundredths of a parsec, higher - order time derivatives could also be measured from the gw observations. these scale differently with msec and r, which would in principle allow the mass and distance of the perturber to be measured from the emri data. the probability that a second black hole would be within a tenth of a parsec of a system containing an emri is difficult to assess. at redshifts z { p_0}\ } } \,\,p(\omega { \vert}a, m,{\mathcal q}){\rm{d}}{\omega ^{2n}}.$$\end{document } dreyer. deemed that an observation was inconsistent with gr if the actual observed frequencies lay outside such confidence intervals for all possible choices of a, m and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q}$\end{document}. using a toy model for a non - black - hole source, and assuming that two qnms were observed, drawn from four possible excited modes, they estimated that, with a false alarm probability of 1%, the false dismissal rate would be 60% if the snr in the weakest mode was 10, falling to 10% at an snr of 100. assuming a reasonable amount of energy deposition into the qnms, they estimated that this would be satisfied by all lisa mbh merger sources, so the prospects for such tests are good. they computed snrs that improved on those in by using an up - to - date detector noise curve and including black - hole spin. for a source of mass 10 m 0.4. there is a mode degeneracy, which means the snr required blows up when l = l and a 0. the snrs required to resolve both modes are in the range 10010 000. conclude that even under pessimistic assumptions about the amount of energy radiated in ringdown radiation, it should be possible to resolve one qnm and either the damping time or frequency of a second qnm, provided that the first overtone radiates 10 of the total ringdown energy. this will provide enough for a test of the no - hair property of the central object. a stronger test would come from detecting frequencies and damping times for both qnms, but this would require ringdown snrs 1000, which is rather unlikely. the main uncertainty in this analysis was in the excitation coefficients for the various modes, but numerical relativity simulations have now provided some information on the excitation of ringdown modes in a merger. this paper revised downward the snr required to detect either the frequency or damping time of a second mode, which is sufficient for a test of the no - hair theorem, to crit 30 for any binary with mass ratio greater than 1.25. a significant fraction of lisa events should satisfy this criterion (see for example). in, the authors used numerical - relativity simulations of nonspinning black - hole binaries, with a variety of mass - ratios ranging from 1:1 to 1:11, to compute the amplitude to which several different ringdown modes were excited, and hence an estimate of the snr with which lisa would be able to observe them. for all the mass ratios considered, the (3, 3) mode was found to radiate between 2% and 20% of the energy of the (2, 2) mode, and for mass ratios more unequal than 1:2 the (4, 4) also radiated more than 1% of the energy of the (2, 2) mode. for sources at a redshift of z = 1, they estimated a total ringdown snr between 1000 and 20000 for black - hole masses in the range 1010 m and mass ratios from 1:1 to 1:25, with individual snrs in the (2, 2), (2,1), and (3, 3) modes between several hundred and 10 000. the mode snrs do not add in quadrature, but these snrs more than meet the requirements of for lisa to be able to carry out black - hole spectroscopy. the snrs realizable with elisa will be a few factors lower and the systems will tend to have lower masses, for which the intrinsic snrs are also smaller. therefore constraints from elisa will be weaker and it is possible that elisa will not be able to resolve sufficient separate modes, but this has not yet been explored. in, gossan. applied the results of to explore the practicality of using qnm radiation to test relativity. the authors considered mergers with mass ratios of 1:2 and constructed a waveform model comprised of four ringdown modes. they used bayesian methods and approached the problem of testing relativity in two ways : (i) determining the parameters of each ringdown mode separately and checking for consistency ; (ii) comparing the bayesian evidence of the gr model to a non - gr model constructed by allowing for deviations of the mode parameters from the gr predictions. the analysis was carried out for elisa and for the einstein telescope, the proposed third - generation ground - based detector. showed that method (i) could reveal inconsistencies of 1% in the qnm frequency for events of mass 10 m at a distance of 50 gpc and inconsistencies of 10% for systems of mass 10 m at 6 gpc. better constraints will come from systems at smaller distances and will therefore be observed with higher snr. using method (ii), in the case of a signal from a 10 m black hole, 2%, 6%, and 10% deviations in the frequency of the dominant mode would be identifiable at distances of approximately 1 gpc, 5 gpc, and 8 gpc, respectively. deviations of 2%, 6% and 10% in the damping time of the dominant mode would only be detectable at distances of 200 mpc, 700 mpc, and 1.2 gpc, respectively. for a more massive system, of 10 m, a 2% deviation in the frequency / damping time of the dominant mode would be detectable out to 35/25 gpc, but deviations of 5% or more would be detectable at greater than 50 gpc. such massive systems are very rare, however, and the choice of a 1:2 mass ratio means that the qnm radiation is stronger than would be expected for typical elisa events, which are likely to have larger mass ratios. therefore this study was limited in extent, but these preliminary results suggest that qnms could place interesting constraints on gr modifications for the strongest signals detected by elisa. space - based qnm observations could be used directly to put constraints on alternative theories of gravity, but this will require a calculation of the qnms in those alternative theories. it was shown in that the equations governing black - hole perturbations in dynamical cs gravity were different from those in gr, with the consequence that the qnm spectrum would also be different. the authors did not compute qnms, but qnm frequencies for non - spinning black holes in dynamical cs gravity were computed in. the qnm spectrum was found to be different, due to the coupling of the black hole oscillations to the scalar field, but the detectability of these deviations by a gravitational - wave detector was not estimated. therefore, it is not yet clear at what level ringdown radiation will be able to constrain the cs coupling parameter. if a lisa - like observatory observes a mbh inspiral and does not detect qnm ringing from the merger remnant where radiation would be expected, this might indicate a violation of the cosmic - censorship hypothesis. qnm ringing arises as a result of gws becoming trapped near the horizon of the black hole. if a horizon was absent, for instance if a super - extremal (a / m > 1) kerr metric was formed, we would not expect to observe the qnm radiation. this would be evidence for the existence of a naked singularity, and a counterexample to cosmic censorship, although not an indication of a problem with gr. although there are still many open questions, current research clearly indicates that low - frequency gw detectors will be able to make strong statements about the structure of the massive compact objects in the centers of galaxies by observing emris and by detecting ringdown radiation following supermassive black - hole mergers. at the very least, it will be possible to test that an observation is consistent with an inspiral into a kerr black hole, and to state quantitatively how so large a deviation from kerr could have been masked by instrumental noise. if a system differed significantly from kerr, we would identify the deviation and should be able to quantify its nature. how well we would be able to distinguish between different types of deviation (e.g., external matter vs. a different multipole structure of the central object) is still not totally clear, but there are ongoing efforts in this direction. the tests of black - hole structure outlined in this section will rely on the detection of small differences between the observed gws and the gws expected in gr. these waveforms can be computed using black - hole perturbation theory, by evaluating the gravitational self - force, but despite extensive work in this area the calculation of the self - force is not yet complete even at first order in the mass ratio. the first - order gravitational self - force has been computed for circular and generic orbits in the schwarzschild spacetime and the scalar self - force is known for circular equatorial orbits in the kerr spacetime. recently, the first self - force - driven inspirals have been computed, under an adiabatic approximation for the gravitational self - force, and self - consistently for the scalar self - force.9 emri models will require knowledge of the gravitational self - force for generic orbits in kerr, which is still not available at first order. it has also been recognized that the radiative part of the second - order self - force will also be required to derive emri waveform phase to sufficient accuracy. a formalism for the evaluation of the second - order self - force has been developed, but not yet implemented. complete reviews of the current status of the self - force program can be found in [45, 362 ]. additional complications in emri modeling arise from the need to accurately follow the transition of the inspiral through transient resonances. although many of these issues will have been resolved by the time data from a space - based gravitational - wave interferometer is available, they must be borne in mind in the interpretation of future results on tests of gr. low - frequency gw detectors will provide information on black - hole structure to a much higher precision than can be achieved by other techniques. it has been suggested that observations of imris with advanced ligo will have some power to do similar tests. this data will be available ten or more years prior to any space - based experiment. however, imri - based tests will be much weaker, since many fewer cycles of the gws will be detected in a single observation due to the larger mass ratio (10 10) ; furthermore, event rates are highly uncertain and accurate modeling of imris is far behind emri modeling [239, 240 ]. an imri observation would be able to detect a deviation from kerr of fractional order unity in the quadrupole moment of a source, compared to the 10 fractional accuracies achievable with lisa emris. psaltis provides a thorough review of tests of strong - field gravity using electromagnetic observations. x - ray interferometers (e.g., the black hole imager) will soon have the angular resolution to directly image the horizon of extragalactic black holes at distances of 1 mpc. the accretion flow for the milky way black hole, sgr a, can already be imaged directly down to its innermost edge. however, interpretation of the observations is complicated by the need to simultaneously constrain the disc properties. observations at multiple wavelengths will be required to fit out the disc and directly image the shadow of the black hole on the disc.evidence for the existence of horizons also comes from observations of quiescent x - ray binaries. an x - ray binary typically comprises a star filling its roche lobe and transferring matter to a compact - object companion. that compact object can either be a neutron star or a black hole. if the object is a neutron star then most of the gravitational potential energy of the accreting matter has to be radiated away, but for black holes a significant amount of this energy can be advected through the black - hole horizon and is therefore not radiated to infinity. among systems in the same state of mass transfer, those containing neutron stars are expected to be systematically more luminous than those containing black holes, as has been observed in our galaxy.electromagnetic observations will really indicate the existence of a high - redshift surface in the system, and not necessarily an actual horizon. if a system contained a naked singularity with a high - redshift surface, but not a true event horizon, this would not be evident from the electromagnetic observations alone. by probing the multipole structure and verifying consistency with the no - hair theorem the highest - temperature emission from a disc comes from the innermost stable circular orbit (isco), and the flux at that temperature is proportional to the isco radius squared. if the inferred spin was found to exceed one, this might indicate failure in the black - hole model. such spin determinations have typical errors of 10%, much greater than lisa s expected errors of 0.01%.indirect inference of the location of the inner edge of the accretion disc could also come from the interferometric observations mentioned above in the context of horizon detection. in the authors use radio interferometry to resolve the base of the jet in the galaxy m87, finding it to be 5.5 0.4 schwarzschild radii. the jet - base radius is interpreted as being greater than or equal to the radius of the innermost edge, so this system provides evidence for prograde accretion onto a spinning black hole. observations of jets in other nearby galaxies may follow in the future, but the distance to which these structures could be resolved is quite small.accretion-disc mapping. particles in the accretion disc around a black hole move on circular geodesics of the metric. if the orbits in the disc could be mapped, this would allow spacetime mapping along the lines of ryan s theorem. two possible probes of accretion - disc structure have been identified : quasi - periodic oscillation (qpo) pairs and iron k lines. qpos have been used to constrain possible values of black - hole masses and spins, but there are uncertainties in the radius at which they originate, and in the resonance that gives rise to the pairs of lines. iron lines show broadening due to differential gravitational redshift and doppler shift at different points in the disc, but again their interpretation depends on unknown details of the disc geometry. in principle, the time variability of an iron line after a single flare event could constrain both the geometry and map the disc. future observations with instruments such as ixo / athena will have the time - resolution to attempt this. horizon detection. x - ray interferometers (e.g., the black hole imager) will soon have the angular resolution to directly image the horizon of extragalactic black holes at distances of 1 mpc. the accretion flow for the milky way black hole, sgr a, can already be imaged directly down to its innermost edge. however, interpretation of the observations is complicated by the need to simultaneously constrain the disc properties. observations at multiple wavelengths will be required to fit out the disc and directly image the shadow of the black hole on the disc. evidence for the existence of horizons also comes from observations of quiescent x - ray binaries. an x - ray binary typically comprises a star filling its roche lobe and transferring matter to a compact - object companion. that compact object can either be a neutron star or a black hole. if the object is a neutron star then most of the gravitational potential energy of the accreting matter has to be radiated away, but for black holes a significant amount of this energy can be advected through the black - hole horizon and is therefore not radiated to infinity. among systems in the same state of mass transfer, those containing neutron stars are expected to be systematically more luminous than those containing black holes, as has been observed in our galaxy. electromagnetic observations will really indicate the existence of a high - redshift surface in the system, and not necessarily an actual horizon. if a system contained a naked singularity with a high - redshift surface, but not a true event horizon, this would not be evident from the electromagnetic observations alone. by probing the multipole structure and verifying consistency with the no - hair theorem the highest - temperature emission from a disc comes from the innermost stable circular orbit (isco), and the flux at that temperature is proportional to the isco radius squared. if the inferred spin was found to exceed one, this might indicate failure in the black - hole model. such spin determinations have typical errors of 10%, much greater than lisa s expected errors of 0.01%. indirect inference of the location of the inner edge of the accretion disc could also come from the interferometric observations mentioned above in the context of horizon detection. in the authors use radio interferometry to resolve the base of the jet in the galaxy m87, finding it to be 5.5 0.4 schwarzschild radii. the jet - base radius is interpreted as being greater than or equal to the radius of the innermost edge, so this system provides evidence for prograde accretion onto a spinning black hole. observations of jets in other nearby galaxies may follow in the future, but the distance to which these structures could be resolved is quite small. particles in the accretion disc around a black hole move on circular geodesics of the metric. if the orbits in the disc could be mapped, this would allow spacetime mapping along the lines of ryan s theorem. two possible probes of accretion - disc structure have been identified : quasi - periodic oscillation (qpo) pairs and iron k lines. qpos have been used to constrain possible values of black - hole masses and spins, but there are uncertainties in the radius at which they originate, and in the resonance that gives rise to the pairs of lines. iron lines show broadening due to differential gravitational redshift and doppler shift at different points in the disc, but again their interpretation depends on unknown details of the disc geometry. in principle, the time variability of an iron line after a single flare event could constrain both the geometry and map the disc. future observations with instruments such as ixo / athena will have the time - resolution to attempt this. electromagnetic observations are generally hampered by a lack of knowledge of the physics of the material that is generating the radiation. gw systems are, by contrast, very clean since the same theory describes the spacetime and the radiation generation. while there is some hope that future electromagnetic observations will perform crude spacetime mapping, lisa emri observations will improve any previous constraints by orders of magnitude. although the bibliography of this review lists more than 500 references, fundamental tests of gravity remain the least explored sector of gw astronomy. it is also one where new discoveries may be hardest to come by even if they would be momentous if they do. space - based gravitational - wave detectors will improve certain existing constraints on alternative theories ; even more important, they will provide novel and unique opportunities to precisely characterize the poorly explored nonlinear, dynamical sector of gravitation, as well as the properties of gravitational radiation fields. these measurements will provide a definite confirmation that einstein s theory, born from insight and beauty, applies in the most extreme regimes ; or they may expose new phenomena, leading to new models for gravitational physics. the research that we have reviewed spans a broad assortment of gw observations, which probe many disparate aspects of gravitational phenomenology. some of these investigations have yet to be refined to the same level of formal rigor as other, more astrophysical applications of gw astronomy. nevertheless, they paint an exciting picture of the expected fundamental - physics payoff of space - based detectors. we expect that this will remain an active and intriguing research area for many years : thus, it is appropriate that this review should be living, and we welcome the suggestions of our readers in improving it and keeping it up to date. | we review the tests of general relativity that will become possible with space - based gravitational - wave detectors operating in the 105 1 hz low - frequency band. the fundamental aspects of gravitation that can be tested include the presence of additional gravitational fields other than the metric ; the number and tensorial nature of gravitational - wave polarization states ; the velocity of propagation of gravitational waves ; the binding energy and gravitational - wave radiation of binaries, and therefore the time evolution of binary inspirals ; the strength and shape of the waves emitted from binary mergers and ringdowns ; the true nature of astrophysical black holes ; and much more. the strength of this science alone calls for the swift implementation of a space - based detector ; the remarkable richness of astrophysics, astronomy, and cosmology in the low - frequency gravitational - wave band make the case even stronger. |
the association between thyroid function and dyslipidemia is well demonstrated not only in overt hypothyroidism, but also in low normal thyroid function with tsh level within normal range. patients with hypothyroidism are known to have significantly higher serum total cholesterol, low - density lipoprotein cholesterol (ldl - c), and triglyceride levels compared to healthy population. in addition, carotid intima - media thickness and flow - mediated vasodilatation, which are surrogated markers of atherosclerosis and vascular endothelial dysfunction, are also known to be associated with thyroid function, even when tsh level is within normal reference range [25 ]. although dyslipidemia and atherosclerosis are both well - defined major risk factors of cardiovascular diseases, whether patients with high or high normal tsh level are definitely at an increased risk of cardiovascular diseases is still under controversy [6, 7 ]. the possible influence of various clinical factors related to cardiovascular diseases, such as age, gender, and body mass index (bmi), is one of the reasons contributing to the uncertainty of the association [6, 8 ]. this suggests that a variety of influencing factors must be taken into account when approaching dyslipidemia and furthermore, cardiovascular risk factors accompanied by hypothyroidism. when performing postoperative radioiodine adjuvant therapy or diagnostic radioiodine whole body scan in patients with differentiated thyroid cancers (dtc), a tsh elevation higher than 30 mu / l achieved through thyroid hormone withdrawal (thw) (withdrawal of levothyroxine (l - t4)) is required in most cases. thw inevitably results in acute thyroid hormone deficiency in patients who have undergone total thyroidectomy, and abnormal lipid profiles, such as hypercholesterolemia, are accompanied in majority of such cases. several previous studies have investigated hypothyroid symptoms and metabolic effects following thw in patients with dtc ; however, none has elucidated the factors influencing post - thw dyslipidemia. a subgroup of such patients actually does not develop dyslipidemia after thw [1013 ]. this study retrospectively analyzed the association between serum cholesterol level and various clinical factors in the setting of tsh elevation in order to better clarify the clinical pattern of dyslipidemia occurring after thw in dtc patients and, as a result, found that bmi is an independent factor determining post - thw serum cholesterol level. a total of 142 consecutive patients diagnosed with papillary thyroid carcinoma with intermediate recurrence risk based on 2009 american thyroid association (ata) guideline and who underwent postoperative 1.1 gbq radioiodine (131-i) remnant ablation (rra) in endocrinology cinic, severance hospital, seoul, korea, from january 2010 to march 2012, were retrospectively reviewed. among them, the data of 61 patients who consented to the measurement of lipid profiles following thw were selectively included in the analysis. the lipid profiling was performed simultaneously with stimulated thyroglobulin (tg) assay when the patients visited the endocrinology clinic after a 3-week thw regime. all subjects who had histories of diabetes mellitus, use of lipid - lowering drugs, or underlying thyroid dysfunction were excluded in the analysis. the medical records of the patients were reviewed, and the preoperative baseline characteristics, including age, gender, bmi, various biochemical parameters measured before and after surgery, and postoperative rra, were retrospectively analyzed. this retrospective study was approved by our institutional review board, and informed consent was not required. as per postoperative follow - up protocol for dtc in our institution, the patients received low - dose rra 3 months after undergoing total thyroidectomy, and tsh - suppressive dose of l - t4 (150 g / d) was administered for 3 months after the surgery. after 4 weeks of thw, thyroid function test (tft) and lipid profiling were performed in conjunction with measurement of tg, anti - tg antibody immediately before the oral administration of 1.1 gbq i-131. a postablation whole body scan was performed 48 h after administration of i-131, and, thereafter, tsh - suppressive dose of l - t4 was begun again in all patients. following postoperative rra, the patients visited our endocrinology outpatient clinic approximately every six months during immediate postoperative follow - up periods for surveillance of recurrence and persistence of the disease. the mean duration from the day of rra to the first and second follow - up visit days was 5.9 1.0 and 15.9 3.7 months, respectively, and a tsh - suppressive does of l - t4 (150 g / d) was maintained in all patients during this period. in the first follow - up visit after rra (p2), tft and lipid profiling were performed in all 61 subjects, and, in 41 subjects, the measurements were repeated in the second follow - up visit after rra (p3). baseline tft and biochemical parameters including plasma glucose, uric acid, total cholesterol, triglycerides, and high - density lipoprotein cholesterol (hdl - c) were measured immediately before total thyroidectomy. tft and lipid profiles were measured after thw for postoperative rra and at one or two follow - up visits thereafter. after 12 h of fasting, all blood samples were obtained from the patients and stored at 70c for subsequent assays. serum tsh and free t4 and t3 were measured by chemiluminescent microparticle immunoassay (architect system, abbott ireland diagnostic division, lisnamuck, longford, co. longford, ireland). plasma glucose, uric acid, triglycerides, total cholesterol, and hdl - c levels were assayed using a routine hitachi 7600 autoanalyzer (hitachi instruments service, tokyo, japan). low - density lipoprotein cholesterol (ldl - c) levels were calculated using the friedewald formula. the differences between the mean level of baseline total cholesterol of subjects and that of each serial time point were validated through paired sample t - test. spearman correlations were used to examine the relationships between total cholesterol level after thw and age, gender, bmi, tsh level, ldl - c level after thw, and total cholesterol at other time points. comparison of categorical variables between bmi groups was performed using chi - square test and fisher 's exact test (two - tailed), as is appropriate. to investigate the association between bmi and other clinical parameters including age, gender, and post - thw total cholesterol more accurately, we employed a logistic regression analysis. p values < 0.05 were considered statistically significant for all tests. all calculations and statistical analyses were performed using ibm spss software package for windows (version 20.0 ; ibm corp. the mean age of 61 subjects (m : f = 14 : 47) was 49 11 yr, and the mean bmi was 23.9 3.3 we first compared tft and lipid profiles measured across four time points (p0 - 3) : immediate preoperative period (p0), the day of 131-i administration after thw (p1), the first follow - up visit after rra (p2), and the second follow - up visit after rra (p3) (table 2). the mean total cholesterol level after thw (p1) significantly increased compared to that at preoperative baseline (p0) (249.1 58.0 mg / dl versus 194.8 39.8 mg / dl, p < 0.001), but at follow - up visit after rra and lt-4 readministration (p2), it returned to baseline level (p0) (figure 1). plasma ldl - c also increased to 167.7 52.0 mg / dl at p1, during which tsh elevation was also observed, but returned to within normal range thereafter. plasma tg and hdl - c did not exceed the normal range at p1, p2, or p3. all the serum triglycerides levels measured at different three time points were below 400 mg / dl. the maximal value of triglycerides in study subjects was 340 mg / dl (p1), 239 mg / dl (p2), and 298 mg / dl (p3), respectively. a simple correlation analysis between post - thw total cholesterol (p1) and various clinical parameters, including bmi, age, gender, elevated tsh level after thw (l - t4-off), and preoperative baseline total cholesterol level, was performed (table 3). as a result, preoperative baseline total cholesterol showed significant correlation with post - thw cholesterol, as expected ; however, age, gender, or elevated tsh level after thw itself was not correlated with post - thw total cholesterol (p1). interestingly, a significant correlation between preoperative bmi and post - thw total cholesterol was found (r = 0.263, p = 0.041). post - thw total cholesterol (p1) was also associated with post - thw calculated ldl - c. the level of total cholesterol measured at follow - up visit showed correlation only at the first follow - up visit after rra (p2). to further clarify the association between bmi and total cholesterol in thyroidectomized patients, the correlation between total cholesterol and bmi at each of the four time points was evaluated (figure 2). while bmi was not correlated with total cholesterol level at baseline (p0), the correlation appeared not only at p1, but also at p2, which then disappeared at the second follow - up visit completed at 15.9 3.7 months after rra. we stratified the patients as either normal weight (n = 21) or overweight group (n = 40) based on the criteria of bmi of 23 kg / m, and their demographics, baseline biochemical parameters, and the changes of lipid profiles after thw were compared (table 4). the overweight group was older than normal weight group (53.1 9.2 vs. 39.9 10.5 yr, p = 0.001), but there was no gender difference between the two groups. intriguingly, the two groups did not show a significant difference in total cholesterol measured at baseline (p0) or at post - rra follow - up visits (p2 and p3). the two groups significantly differed only in post - thw total cholesterol and ldl - c level (p1). in other words, the overweight group had the greater post - thw total cholesterol and ldl - c level compared to normal - weight group. finally, we performed multiple logistic analyses with post - thw total cholesterol level as a dependent variable (table 5), which found bmi to be an independent determining factor of post - thw total cholesterol level along with preoperative baseline total cholesterol level and elevated tsh level following thw (p = 0.012). due to the rising incidence of thyroid cancers, much interest has been raised regarding the possible metabolic effects or long - term health outcomes resulting from thyroid hormonal derangement following thyroidectomy. patients who undergo total thyroidectomy are potentially exposed to long duration of subclinical or mild hyperthyroidism due to a long - term tsh suppressive therapy ; however, thw regimens required for diagnostic or therapeutic radioiodine whole body scan may induce acute thyroid hormone deficiency. previous studies have investigated the metabolic effects caused by transient severe hypothyroidism due to thw on peripheral target organs [10, 11 ]. transient alterations of lipid metabolism are one of the hallmark clinical features of such thw - induced effect, and several studies have also focused on how changes in lipid profiles under such circumstances affect the vascular function. however, in clinical situations, the spectrum of thw - induced changes in lipid profile is wide : in some patients, the change is very mild, while in others, severe hypercholesterolemia is observed. nonetheless, there has been no study directly analyzing the various associations between thw and transient dyslipidemia or elucidating the clinical factors influencing the association. although the effects of thyroid hormone on cholesterol metabolism are not yet fully known, it is accepted that changes in thyroid function exert a wide range of influence on the overall cholesterol metabolism, including its synthesis, metabolism, and mobilization. overt hypothyroidism is associated with tissue expression and altered activities of various regulators participating in lipid metabolism, including hepatic hydroxymethylglutaryl coenzyme a (hmg coa) reductase, ldl - c receptors, sterol regulatory element - binding protein-2 (srebp-2), cholesteryl ester transfer protein (cetp), hepatic lipase, lipoprotein lipase, and atp binding cassette transporter a1 (abca1) transporter [1620 ]. consequently, hypothyroid patients generally show elevated total cholesterol and ldl - c level, as well as normal to elevated hdl - c, tg, and very low density lipoprotein (vldl). there has been abundant clinical evidence on the relationship between hypothyroid status, abnormal lipid profile, and the related health outcomes such as atherosclerosis and metabolic syndrome [2227 ]. on the contrary, relatively few studies have investigated the metabolic effects of acute thyroid hormone deficiency in dtc patients who have to undergo thw for postoperative 131-i therapy or for measurement of stimulated serum tg and radioiodine whole body scan to evaluate residual / recurrent diseases [10, 11, 13 ]. in a prospective study on 15 female dtc patients, chang. have reported that short - term hypothyroidism due to thw and the consequent worsening of metabolic parameters does not affect the measurements of surrogate marker for endothelial function, such as brachial artery diameter. nonetheless, the recent surge in number of patients with dtc, combined with mostly low mortality risk of the cases of dtc, suggests the importance of maintaining an optimal thyroid functional status in athyreotic patients through the follow - up periods with a suppressive or replacement dose of thyroid hormone, both in terms of oncologic safety and favorable metabolic health outcome. therefore, the clinical features and metabolic effects of transient severe hypothyroid status following thw in dtc patients require further clarification. the occurrence of hypercholesterolemia in hypothyroid patients can not be explained with a single mechanism. a combination of results of several clinical studies has found that tsh and thyroid hormone level are both independently associated with total cholesterol level ; furthermore, various clinical factors, including gender, age, fasting plasma glucose, bmi, and smoking status, are also thought to exert independent effects on total cholesterol levels. in this study, we have reported that bmi is significantly associated with total cholesterol level measured after 4 weeks of l - t4 withdrawal in transiently hypothyroid, athyreotic patients independently of baseline total cholesterol level, elevated tsh level after thw, age, and gender. to our knowledge, this is the first study to have investigated the association between bmi and short - term hypothyroidism - induced hypercholesterolemia occurring during the course of postoperative surveillance in dtc. in this study, the mean post - thw total cholesterol and ldl - c level exceeded the normal range only in overweight group of patients, defined as bmi of 23 kg / m or higher. the patients with normal body weight showed normal levels of total cholesterol and ldl - c. furthermore, while total cholesterol level returned to preoperative baseline level at the follow - up visits after rra and resumption of l - t4 replacement, the correlation between bmi and total cholesterol level was maintained until the first follow - up visit. since only two patients in our patients group satisfied the criteria of obesity suggested by the world health organization (bmi greater than 30 kg / m), we have instead employed a proposed bmi cutoff for overweight (23 kg / m) which takes into account the health risk for asian population [28, 29 ]. in a study of 18 dtc patients who underwent thw, huang. reported insulin resistance and bmi to be the factors predictive of severity of hypothyroid symptoms after thw. our study may be in line with this previous report, and we suspect that overweight athyreotic patients can be more vulnerable not only to hypothyroid symptoms but also to hypothyroidism - induced abnormality of lipid profile after thw. other past clinical studies have reported that insulin sensitivity may act as a modulator in the relationship between tsh and serum lipid level in healthy euthyroid subjects and female diabetic patients and insulin - resistant subjects are more susceptible to the higher risk of hypercholesterolemia as tsh level increases [30, 31 ]. the results of this study imply that increased hepatic cholesterol synthesis may be observed in dtc patients with high bmi due to relatively elevated insulin resistance. hypothyroidism simultaneously suppresses hepatic cholesterol synthesis and hepatic ldl - c receptor expression : the reduced clearance of ldl - c overpowers the effects of low hepatic cholesterol synthesis in hypothyroid status, resulting in hypercholesterolemia. as such, we suggest that the possible increase of baseline hepatic cholesterol synthesis following increased bmi may be the underlying reason behind the association between elevated bmi and post - thw hypercholesterolemia. however, the probable explanation for the association between bmi and post - thw hypercholesterolemia is just one of many assumptions since there was no definite evidence for the difference of insulin resistance among the subjects in this study. we could not obtain any surrogate parameters regarding metabolic status except bmi and baseline routine chemistry results. we aimed to elucidate the difference in fasting serum glucose level or uric acid level according to bmi, but it was not significant as the number of subjects was relatively small (table 4). a larger scaled study is also required to explain the exact mechanism of the association between bmi and post - thw hypercholesterolemia. first of all, this was a retrospective study analyzing relatively few subjects, and, therefore, a multifaceted study evaluating the effect of hypothyroidism on various aspects of lipid metabolism, including cholesterol synthesis, clearance, and efflux, must be performed in the future to further clarify the exact molecular mechanism of the effect of bmi on dyslipidemia caused by thyroid hormone deficiency in athyreotic patients. we also could not include thorough lipid profile data at time point p0 except total cholesterol level, because only routine chemistry exam was performed for preoperative blood test (p0) according to the surgical preparation protocol of our institute. the data about thyroid function tests and lipid profile at a time point between surgery and the start of thyroid hormone withdrawal also could not be analyzed in this retrospective study because they were not routinely performed in our institute. there might be some difference between baseline preoperative lipid profile and the lipid profile immediately before the start of thyroid hormone withdrawal which must be accompanied with suppressed thyrotropin level by supraphysiological levothyroxine replacement. furthermore, the most lipid abnormalities accompanied by short - term acute hypothyroidism are reversible with l - t4 replacement, so the long - term clinical implication of this study should also be evaluated through additional prospective studies. nonetheless, this study found a statistically significant association between bmi and post - thw hypercholesterolemia despite a small sample size of 61 patients ; since thw is being more widely employed (both diagnostically and therapeutically) as part of postsurgical management of thyroid cancer, attention needs to be paid to post - thw lipid profile abnormality and its long - term health outcome, at least in patients with underlying metabolic derangements, such as overweight and insulin resistance. in conclusions, bmi is an independent determining factor for serum cholesterol level after thw in thyroidectomized patients. overweight patients who have undergone total thyroidectomy may be more susceptible to the adverse metabolic effects of thyroid hormone deficiency. these patients must be considered to be in higher risk of dyslipidemia when exposed to hypothyroid and athyreotic status. maintaining bmi and thyroid hormonal status in appropriate range needs to be stressed more in this group during the postoperative follow - up periods of thyroid cancers. | thyroid hormone withdrawal (thw) for postoperative radioiodine adjuvant therapy or diagnostic radioiodine whole body scan in patients with differentiated thyroid cancers results in acute thyroid hormone deficiency and abnormal lipid profiles. to better clarify the clinical pattern of dyslipidemia occurring after thw, we retrospectively analyzed the association between serum total cholesterol level after thw and various clinical factors in a total of 61 patients who underwent total thyroidectomy due to papillary thyroid cancers from january 2010 to march 2012, in severance hospital, seoul, korea. preoperative baseline total cholesterol was significantly correlated with post - thw total cholesterol level ; however, age, gender, or elevated tsh level after thw itself was not correlated with post - thw total cholesterol level. a significant correlation between preoperative measured bmi and post - thw total cholesterol level was found (r = 0.263, p = 0.041). in multiple logistic analysis, bmi was an independent determining factor of post - thw total cholesterol level (p = 0.012). |
we investigated the in vitro susceptibility of various cell cultures and embryonated eggs to usutu virus infection. human (hela), green monkey (vero), equine (ed), bovine (mdbk), porcine (pk-15), rabbit (rk-13), canine (mdck, dk), feline (cr), hamster (bhk-21, bf), rat (c6), and turtle (th1) permanent cell lines, as well as primary horse kidney (eqk), chicken embryo fibroblast (cef), and goose embryo fibroblast (gef) cell cultures were tested. cells were propagated in earle 's minimal essential medium (mem) (gibco invitrogen, paisley, uk) containing l - glutamine, antimicrobial drugs, and 10% fetal calf serum (fcs). the cells were regularly subcultured by employing standard techniques. to 1-day - old confluent monolayers of the permanent cell lines and primary cell cultures, grown on the surface of chamber slides, the austrian usutu virus strain vienna 2001-blackbird (genbank accession no. the virus was originally isolated in vero cells in 2001 from the brain homogenate of a blackbird found dead in the area surrounding vienna. the second virus passage was used for the experiments ; 50% tissue culture infective dose (tcid50) was determined, and aliquots of the virus were stored frozen at 80c until used. the virus was added to the cells, which were then incubated at 37c for 1 h. thereafter, the inoculum was removed, the cell cultures were washed once with phosphate - buffered saline (pbs), and mem containing 2% fcs, l - glutamine, and antimicrobial drugs were added. for all cell types, controls were cultivated simultaneously and treated in the same way as the infected cultures with the exception that mem was used for inoculation. all cell cultures were incubated at 37c for 3 to 5 days ; then the medium was removed and the monolayers were fixed with chilled (20c) acetone. the cells were stained with hematoxylin - eosin (he) and examined microscopically. in parallel, immunohistochemical (ihc) testing was carried out on the cell cultures by using the avidin - biotin complex technique, with a polyclonal antiserum raised in mice against wnv antigens, for which cross - reactivity with usutu virus had been demonstrated previously (1). the number of antigen - positive cells was evaluated microscopically and scored (see table). ihc, immunohistochemical ; scoring criteria : (+), 1%5% positive cells ; +, 6%25% positive cells ; + +, 26%50% positive cells. embryonated chicken eggs (strain lsl white, which was derived from the strain white leghorn), originating from a specified pathogen free (spf) herd (valo eggs, lohmann, cuxhaven, germany), were injected into the allantoic sac with 6 x 10 tcid50 of usutu virus at the age of 10 days. the eggs were incubated together with mock - infected controls at 37.5c for further 4 days and were checked daily by transillumination. on day 4 postinfection the eggs were opened, and the embryos were fixed in 4% buffered formaldehyde solution. histologic sections were made from paraffin - embedded organs of the embryos, and the slides were analyzed by light microscopy after he and ihc staining, respectively, as described above. three to 4 days after inoculation, pronounced cpes were observed in usutu virus infected vero and pk-15 cell cultures as well as in gef cells. the first foci of cell rounding and subsequent shrinkage of the cells were observed on day 2 or day 3 postinfection, when groups of 4 to 8 cells, but also single cells, showed rounding and degeneration ; within 1 day the affected cells lost their adherence to the bottom of the flask and floated in the medium. within typical usutu virus cpe is shown in he - stained vero cells in figure 1. the mock - infected vero, pk-15, and gef control cell cultures did not show any cpe. the other investigated cell types inoculated with usutu virus did not develop visible cpe within a period of 5 days, and they were also negative by microscopy after he staining. however, by ihc with cross - reactive wnv - antiserum, focal virus multiplication was detected in all cell cultures, independent of animal species and tissue type, except chicken embryo fibroblast cells (figure 2). the percentage of usutu virus antigen positive cells varied from 1% (dk) to 50% (gef) (table). in the case of hela cells, different clones adapted to the propagation of human rhinoviruses (hela rhino) and herpes simplex viruses (hela hsv), respectively, were also tested, but they gave the same results as the commonly used (atcc) hela cells by he and ihc staining. cytopathic effect (cpe) of vero cells caused by usutu virus infection, 4 days postinfection (hematoxylin - eosin staining). immunohistochemical (ihc) tests were performed by using a polyclonal antibody to west nile virus, which cross - reacts with usutu virus. a) vero control ; b) vero infected ; c) cr [define ] (feline) control ; d) cr infected ; e) goose embryo fibroblast (gef) [define ] control ; f) gef infected ; a, b) bar = 50 m ; c f) bar = 100 m. the usutu virus infected chicken embryos did not show any lesions when investigated by gross and histopathologic examination after 4 days of incubation and were negative by ihc as well. to rule out the slight possibility that the usutu virus strain used for inoculation underwent a change in cell tropism during the 2 passages in vero cells, cef, vero, pk-15, mdck, and dk cells, as well as embryonated chicken eggs, were reinfected with the original usutu virus isolate (before passaging) ; the results were identical to the results obtained with usutu virus passaged twice before use. the appearance of cpe in flavivirus - infected cell cultures depends on the virus and host cell type, as well as on moi levels and incubation time employed (8). in many cases, the presence and multiplication of flaviviruses do not inhibit significantly the host cell macromolecular synthesis, resulting in noncytopathic persistent infections (9,10). pathogenesis and virulence of flaviviruses are influenced in vivo by several virus- and host - dependent factors, including the role of defective interfering particles, viral receptors, neurovirulence, immune - response (e.g., antibody - dependent enhancement), and host resistance genes (8). although some of these processes are not yet fully understood, the basic requisite of any pathogenic effect is the host susceptibility to the virus infection. this study demonstrates that usutu virus can infect cell cultures of various tissue types derived from a wide variety of animal species, including cell lines of human origin. since only vero, pk-15, and gef cells develop cpe after usutu virus infection, these cell lines and cell culture are the most appropriate ones for diagnostic purposes (e.g., virus isolation, plaque reduction neutralization test). as demonstrated by ihc, considerable differences have been found in the susceptibility of the various cell lines and cultures to usutu virus infection and in the extent of spread of the infection ; even cell lines derived from the same animal species and organ varied significantly in their susceptibility to usutu virus infection, e.g., mdck cells strongly support usutu virus multiplication, while dk cells are far less susceptible. both of these cell lines, however, have been derived from dog kidneys. on the other hand, the differences between the 2 canine kidney cell lines might also be the consequence of different random mutations (e.g., in genes of the interferon or other innate defense systems) that allowed the cells to immortalize. since in austria, usutu virus infects wild birds and causes high death rates, especially in blackbirds, one would think that birds are most susceptible hosts for the virus. therefore, the finding that both the chicken embryo fibroblast monolayers and the chicken embryos are apparently resistant to usutu virus infection was unexpected. usutu virus, however, is not the only flavivirus with such contradiction in host spectrum. ilheus virus, a south american mosquitoborne flavivirus belonging to the ntaya virus group (7), also naturally affects wild birds and produces plaques in primary rhesus kidney cells and various established cell lines (vero, ps, bhk-21, and llc - mk2), but not in avian cells (8). preliminary results of our chicken experiments with usutu virus also support that idea that the domestic chicken is resistant to the infection, even when young. further investigations involving different bird and mammal species will be necessary to show the most important host species, natural reservoirs, and vectors of usutu virus and to estimate its epidemiologic impact and possible threat to domesticated animals and to the human population. | we investigated the susceptibility to usutu virus (flavivirus) of 13 permanent cell lines, 3 primary cell cultures, and chicken embryos. vero, pk-15, and goose embryo fibroblast cells developed cytopathic effects ; however, viral multiplication was detected in all mammalian cell types by immunohistochemical tests. chicken embryo fibroblast cells and chicken embryos were resistant. |
peritransplant renal failure is an indicator of poor prognosis after liver transplantation [1, 2, 3, 4 ]. end - stage liver and kidney disease (elkd) on hemodialysis is an indication for deceased donor simultaneous liver - kidney transplantation (lkt) [4, 5 ]. on the other hand, living donation of liver and a kidney is generally too challenging and invasive to be performed, and only a few cases of living donor simultaneous or sequential lkt for elkd have been reported [6, 7, 8, 9, 10 ]. in living donor transplantation settings, the safer alternative approach is living donor liver transplantation (ldlt) and continuation of hemodialysis. however, ldlt for a patient on hemodialysis has never been reported, and thus the indications for ldlt in such patients are also unknown. here the recipient was a 63-year - old japanese male who had been suffering from hepatitis c cirrhosis since the age of 37 years. he had undergone endoscopic variceal ligation for esophageal varices at the age of 58 years. his liver function had gradually become decompensated and he had developed hepatic encephalopathy 5 months before admission. he had had cryptogenic chronic nephritis from the age of 19 years and started hemodialysis at the age of 60 years. he had no other complications such as diabetes, hypertension or hemodialysis - related complications. his height was 159 cm and his weight was 53 kg before hemodialysis and 51 kg after hemodialysis. his child - pugh score was 10 (grade c) with a total bilirubin level of 0.9 mg / dl, an albumin level of 3.4 g / dl and a prothrombin time of 68% (international ratio 1.25). he was seropositive for hepatitis c virus (hcv) antibodies but seronegative for hcv rna. the levels of blood urea nitrogen and creatinine were 61 and 9.01 mg / dl, respectively. a computed tomography scan revealed the presence of liver cirrhosis, splenomegaly and developed collateral vessels such as splenorenal shunt, recanalized paraumbilical vein and gastric varices. the bilateral kidneys were very atrophic, which was consistent with irreversible renal failure (fig. the donor was his healthy 58-year - old wife with identical blood type to the recipient. the right lobe graft was procured using a typical method described elsewhere [1, 11, 12, 13, 14 ]. the actual graft weight was 546 g, which accounted for 50.4% of the recipient 's standard liver volume. in the recipient, intraoperative continuous hemodiafiltration (chdf) without water removal a total hepatectomy and implantation were performed under stable hemodynamics using an extracorporeal veno - venous bypass. the v5, right inferior hepatic vein and right hepatic vein of the right lobe graft were reconstructed to have a co - orifice using the left internal jugular vein and explanted portal vein grafts of the recipient at the backtable according to our usual method [15, 16 ]. the anhepatic, cold ischemic and warm ischemic times were 140, 169 and 65 min, respectively. the blood loss was 2,000 g, for which 10 units of red cell concentrate, 10 units of fresh - frozen plasma and 30 units of platelet concentrate were transfused. chdf was continued until postoperative day 4, at which point chdf was converted to hemodialysis. the amount of water removal was appropriately adjusted according to blood pressure, central venous pressure and body weight. the drained ascites was below 500 ml / day and all abdominal drains were removed by postoperative day 6 except for the biliary stents (table 1). other than the renal replacement therapy and dose modulation of renal excretory drugs such as acyclovir, the perioperative management of the recipient was typical, as previously described [1, 11, 12, 13, 14 ]. immunosuppression was induced with intravenous methylprednisolone and then switched to oral prednisolone, cyclosporin a and mycophenolate mofetil. he left the intensive care unit on postoperative day 5 and was discharged on postoperative day 36 with good hepatic function. to the best of our knowledge, this is the first report of ldlt for a patient on chronic hemodialysis. the posttransplant 2-year survival rates are 75.9% for deceased donor lkt and 70.8% for deceased donor isolated liver transplantation for elkd on hemodialysis. on the other hand, however, ldlt for a patient on hemodialysis is potentially risky, and most surgeons hesitate to perform the procedure. in fact, ldlt for a patient on hemodialysis has never been reported, and thus the indications are unknown. in the present case, there were three indications for ldlt. undetectable serum hcv rna before liver transplantation has been shown to decrease the rate of posttransplant disease recurrence [17, 18 ]. reported that patients with sustained viral response for interferon therapy, as determined by a sensitive assay (lower limit < 600 iu / ml), had no virological recurrence, histological recurrence or graft failure. the present patient was determined to be seronegative for hcv rna by an even more sensitive assay (lower limit < 15 iu / ml). therefore, he was expected to have a good prognosis without hepatitis c recurrence after ldlt. non - diabetic patients on hemodialysis show much better survival rates than diabetic patients on hemodialysis. third, the patient had only been on hemodialysis for 3 years and had no other complications. taking these three factors into consideration, ldlt was indicated for this patient. simultaneous or sequential lkt from the donor was not indicated for two reasons. first, liver - kidney donation from a single donor has not been established and is very invasive, especially for the relatively old donor in this case (58 years of age). second, the recipient was expected to continue hemodialysis because he had only been on hemodialysis for 3 years. the intraoperative and postoperative points were use of chdf, care of in - out balance and drug dose modulation. the other managements did not need to be specialized. in conclusion, to the best of our knowledge, this is the first report of ldlt for a patient on chronic hemodialysis. | end - stage liver and kidney disease (elkd) is an indication for deceased donor simultaneous liver - kidney transplantation. although a few cases of living donor liver - kidney transplantation have been reported, the invasiveness remains to be discussed. living donor liver transplantation (ldlt) is an alternative choice for elkd, but has never been reported. here, we report a case of successful ldlt for a patient with elkd on hemodialysis. the patient was a 63-year - old male and had decompensated hepatitis c cirrhosis with seronegativity for hepatitis c virus. he had non - diabetic end - stage renal failure and had been on hemodialysis for 3 years. he was in good general condition except for hepatic and renal failure. the living donor was his 58-year - old healthy wife. a right lobe graft was transplanted to the recipient under continuous hemodiafiltration (chdf) and extracorporeal veno - venous bypass. chdf was continued until postoperative day 4, at which point chdf was converted to hemodialysis. his posttransplant course was good and he was discharged on postoperative day 36. to the best of our knowledge, this is the first report of ldlt for a patient on chronic hemodialysis. therefore, being on hemodialysis is not a contraindication for ldlt. ldlt is feasible for a patient with elkd on hemodialysis. |
this can be an unusual cause of primary male infertility as a result of abnormalities in sexual function. this report describes a 40 year old man who presented to us on account of inability to impregnate his wife after 2 years of marriage. history revealed poor stream of urine since childhood and passage of scanty ejaculate during intercourse. in this report, it was found that adult posterior urethral valve though uncommon may be a cause of male infertility. posterior urethral valve (puv) is a congenital malformation found in males that is characterized by the presence of abnormal obstructing membranes in the posterior urethra. the exact embryology of the condition is not completely understood but it is believed to arise mainly as a result of an anomalous insertion of the mesonephric duct into the primitive fetal cloaca (1). this leads to an obstruction in the flow of urine through the urethra with the classic presentation of poor stream at birth. it is the most common cause of bladder outlet obstruction in male children and can be complicated by urinary tract infection (uti), structural changes in the urinary tract and impaired renal function (2, 3). the diagnosis of the condition may be suggested prenatally following a maternal abdominal scan in the presence of hydronephrosis and oligohydramnios. postnatally, diagnosis is made usually with the aid of micturating cystourethrogram during evaluation of voiding dysfunction (2). in cases with mild obstruction however, puv in the adult male is reported to be an unusual finding (5, 6). this may occur in patients with mild obstructing valves who do not develop severe complications of the disease. this presentation may be an unusual cause of urinary and sexual symptoms (7). in this paper, an attempt was made to present the management of a 40 year old man with primary infertility due to puv causing retained ejaculate during sexual intercourse. a 40 year old presented to the urology unit of the university of benin teaching hospital, nigeria in april, 2013 with complaint of inability to impregnate his wife after 2 years of marriage. the couple lived together with regular sexual intercourse (2 - 3 times weekly). however, there was associated passage of scanty and sometimes absent ejaculate during intercourse with trickling of seminal fluid few minutes thereafter. there was no history of urethral trauma but the patient had several episodes of uti which was treated in a community hospital. an assessment of urethral stricture leading to primary male infertility was initially entertained. however, contrast studies by means of a retrograde urethrogram and a micturating cystourethrogram showed a dilated posterior urethra suggestive of puv with associated bladder trabeculations (figure 1). the patient was not able to produce sufficient semen for proper seminal fluid analysis (sfa), on the two occasions the test was requested. urine culture did not yield any growth of organisms while serum urea, electrolytes and creatinine values were within normal limits (na 138 meq / l, k 4.2 meq / l, urea 30 mg / dl and creatinine 1.1 mg / dl). he subsequently had urethrocystoscopy which confirmed type 1 of puv that was ablated during the procedure using a diathermy bugbee electrode. after removal of catheter the next day, there was significant improvement of the urine stream with a peak flow rate of 15 ml / s. micturating cystourethrogram of the patient showing dilated posterior urethra four weeks later during follow up visit, the patient reported having good volume of ejaculate during intercourse with further improvement in urine stream. his sfa report after 6 weeks of valve ablation was satisfactory, with values within normal ranges (volume ; 3.5 ml, count ; 38x10/ml, motility ; 55%, morphology ; 40%). most cases of puv are usually diagnosed either before birth with the aid of a prenatal scan or soon after birth following evaluation of a male child with poor urinary stream (2). though cases with mild obstructing valves may present later in child - hood, the diagnosis of the condition is uncommon in adulthood (5, 6). presentation in adulthood may occur due to the presence of a mild form of the disease devoid of the life threatening complications noted in children with the severe form. in nigeria, many patients with the disease still present late (8). this may be facilitated by ignorance of the symptoms of the disease by parents, guardians and affected individuals as well as paucity of specialized health - care. our patient presented on account of inability to impregnate his wife and was found to have associated poor urinary stream and passage of scanty ejaculate in the course of evaluation. the diagnosis was suggested with radiological finding of a dilated posterior urethra and confirmed on urethrocystoscopy. the patient had recurrent uti but there was no associated impairment in renal function seen in severe cases (3, 4). obstruction to antegrade flow at the posterior urethra occurs not only with urine but with semen after normal ejaculation. as such, sufficient amount of semen may not be introduced within the vagina during intercourse leading to a reduction in the likelihood of fertilization. the patient was not able to impregnate his wife after 2 years of marriage despite regular sexual intercourse. this may have been due to retention of significant amount of seminal fluid within the posterior urethra. satisfactory outcome following valve ablation was achieved with improvement in urine stream and ejaculate volume. he had normal sfa values 6 weeks after valve ablation and was able to impregnate his wife in the course of follow up. patients presenting in the fertility clinic with a history of scanty ejaculate and reduced urinary stream should be evaluated in this regard. | backgroundposterior urethral valve presenting in adulthood is uncommon. this can be an unusual cause of primary male infertility as a result of abnormalities in sexual function.case presentationthis report describes a 40 year old man who presented to us on account of inability to impregnate his wife after 2 years of marriage. history revealed poor stream of urine since childhood and passage of scanty ejaculate during intercourse. a micturating cystourethrogram revealed dilated posterior urethra in keeping with posterior urethral valves. endoscopic valve ablation was done with subsequent improvement in ejaculate volume and urine stream. his spouse achieved pregnancy thereafter.conclusionin this report, it was found that adult posterior urethral valve though uncommon may be a cause of male infertility. restoration of fertility potential can be achieved following valve ablation. |
prevalence of pediatric obesity dramatically increased in the last decades especially in western countries 1 but also in developing countries 2 3. according to the national health and nutrition examination surveys (nhanes), among 6- to 11-year - old children, prevalence of obesity raised from 4.2 % in the 1960 s, to 19.6 % in 2007 2008, nearly a 5-fold increase in 40 years 1. as a direct consequence, prevalence of the most relevant comorbidities such as hypertension and type ii diabetes increased from 2.8 % of the adult population in 1980 to 6.4 % in 2011 4. bariatric surgery in adolescents has gained wide popularity in the last decades and represents an attractive option in patients affected by chronic metabolic syndrome and bmi > 40 5 6 7. nevertheless, it should be avoided in younger prepubertal children and less invasive forms of treatment favoured in this populationt. endoscopic treatment by intragastric balloon should be taken into account in treatment of pediatric morbidly obese patients. it does nt modify gastrointestinal anatomy and can be safely carried out in childhood with a low complication rat 8. to reduce patient discomfort and invasiveness of treatment by using an intragastric balloon, a new swallowable device (obalon therapeutics inc. preliminary results in children seem to be encouraging with a significant decrease in body mass index (bmi) and improvement in comorbidities 5 9. the aim of this study is to report our experience in patients with the device and to describe technical aspects and practicalities related to the procedure. from july 2013 to may 2015, 17 children (m / f = 6 /11) were enrolled. mean age was 13.6 2.3 years (range 9.9 17.1 yr all patients were obese, with bmi > 30 kg associated with obesity - related diseases [dislipedemia, sleep apnea, non - alcoholic steato - hepatitis (nash), gastroesophageal reflux ] or bmi > 35 with or without comorbidities. children with hormonal or genetic obesity, presence of an organic disease of the upper gastrointestinal tract, previous gastrointestinal surgery, anti - inflammatory or anticoagulant therapies were excluded. fifteen of 17 patients underwent the pill test, which was successfully carried out in 14/15 patients (93.3 %). the last 2 patients were considered unable to swallow the balloon due to slight mental retardation and were directly scheduled for endoscopic insertion. a balanced diet (1100 kcal) was always prescribed and regular physical activity strongly recommended to all patients. patients were treated daily with proton pump inhibitor (lansoprazole 30 mg / day) during the entire treatment period and always advised to avoid drugs harmful to the stomach during the study. at the end of treatment, balloon removal the balloons were easily deflated by puncture with a sclerotherapy needle and then removed using a standard foreign body forceps. patients were divided into 2 groups according to pre - procedure bmi : group a, bmi 35. the paired t - test was used to compare the same patients before and after treatment while the student s t - test was employed to determine statistically significant differences between different groups. results are expressed in terms of mean sd and confidence interval (ci) 95 %. excess weight was calculated according to cole s curves for pediatric populations because ideal bmi varies with age and sex and reaches a value of 25 only at age 18 years 10. when an endoscopic placement was necessary, deep sedation was performed using a facial mask to ensure adequate oxygenation during the procedure. endotracheal intubation should be used only in patients considered at higher risk of respiratory distress. through a large catheter mount a standard 9.8-mm video gastroscope (olympus gif q165) was passed through the facial mask after being inserted into the working channel of a roth net standard retriever for removal of round, blunt foreign bodies (fig. 1). the pill was inserted into the basket and the net closed to secure the obalon during its progression through the esophagus into the stomach. once the capsule had reached the gastric fundus, it was necessary to wait for it to completely unroll before starting balloon inflation according to the usual technique. a brief hospitalization (0.05) ; mean weight excess, calculated according to cole s curves for pediatric populations, decreased from 36.2 15.9 (ci 27.72 to 44.67) to 29.4 18.3 kg (ci 19.64 to 39.15) (p 35 (group b) (0,0004 vs 0.49) (table 3). bmi, body mass index ; bmi rif, body mass index corrected per age ; iw, ideal weight ; ew, excess weight bmi, body mass index ; bmi rif, body mass index corrected per age ; iw, ideal weight ; ew, excess weight bmi, body mass index ; bmi rif, body mass index corrected per age ; iw, ideal weight ; ew, excess weight five of 18 patients reported mild to moderate epigastric pain / cramping that completely disappeared after 1 day in 3 and after 3 days in 2 patients using a single dose of oral hyoscine butylbromide, associated in 1 case with acetaminophen. a single episode of vomiting occurred in a 15-year - old girl unable to swallow the balloon and resolved with a single dose of intravenous ondansetron. nausea, recorded in 5 patients, resolved spontaneously after 1 day (4 cases) to 2 days (1 case) without medication. twelve children (66.6 %) required no medication. in the group of 9 children who underwent a second balloon positioning, mild epigastric pain / cramping was reported by only 2 patients (22.2 %) and a single dose of oral hyoscine butylbromide was administered. in 1 patient, several episodes of hematic vomiting occurred about a month after positioning of the second balloon. an upper endoscopy was promptly performed and ruled out presence of any esophageal or gastric lesions, showing the presence of the balloons in the gastric fundus with no sign of deflation. the otorhinolaryngologist noticed variceal bleeding from the nasal septum with discharge of blood in the stomach during sleep. in the 16 patients who completed the study (1 patient still under treatment) mean weight decreased from 9.,8 18,4 kg (ci 86.01 to 105.58) to 83.6 27.1 (ci 69.15 to 98.04) (p 0.05) ; mean weight excess, calculated according to cole s curves for pediatric populations, decreased from 36.2 15.9 (ci 27.72 to 44.67) to 29.4 18.3 kg (ci 19.64 to 39.15) (p 35 (group b) (0,0004 vs 0.49) (table 3). bmi, body mass index ; bmi rif, body mass index corrected per age ; iw, ideal weight ; ew, excess weight bmi, body mass index ; bmi rif, body mass index corrected per age ; iw, ideal weight ; ew, excess weight bmi, body mass index ; bmi rif, body mass index corrected per age ; iw, ideal weight ; ew, excess weight intragastric balloon therapy is currently considered a valuable option for morbidly obese patients unresponsive to behavioral and nutritional treatment who are not eligible for surgery or who do nt accept surgery. different types of intragastric balloons have been used in the past in pediatric and adolescent patients with various degrees of obesity 10 11 12. the most widely used is the bioenterics intragastric balloon (bib ; allergan, irvine, ca), a silicone rubber device introduced in 1999. complications of the bib are gastric ulcers, gastric erosions, esophagitis, spontaneous deflation, persistent vomiting, gastroesophageal reflux, and abdominal pain. some reports also exist of gastric perforations, small bowel obstructions, and significant gastric dilation 13 14 15. given these concerns, we tested the obalon device and observed that, in the majority of cases (82 %), patients were able to swallow it without sedation and general anesthesia was required only at the end of treatment during balloon removal. furthermore the obalon device can be used starting at a lower threshold bmi (> 30) respect to bib or other intragastric balloons because of its lower side effect and complication rates. these aspects make the obalon device useful and safe in patients with moderate obesity with or without comorbidities, both in childhood and in adolescence. at the beginning of our experience with obalon in a pediatric population, we considered as a primary goal the feasibility and safety of the procedure as well as its effectiveness in terms of weight loss. for that reason, during the first year of the study, we enrolled patients with a wide range of bmi (30 50). after a complete analysis of preliminary results, we decided to treat only patients with first - degree obesity and bmi between 30 and 35. for these reasons, our patients were enrolled in the study despite having bmi that was lower than the value recommended by the european guidelines on surgery of severe obesity 16. we think that all obese pediatric patients should have the necessary tools to reverse the tendency to gain weight and the intragastric balloon may be the starting point on this path. the results of our study demonstrated that obalon device can be easily administered without anesthesia in the majority of patients (83 %) and within 3 months it induces significant weight loss. use of the obalon device for more than 13 weeks, as suggested by the manufacturer, resulted in spontaneous deflation in 3 of our patients and unnoticed and spontaneous emission of the balloon in 2 of 3 patients. despite this, the device can be considered a helpful tool for management of subjects with class i obesity and ensure an appreciable weight loss, particularly in children and adolescents with bmi < 35. | background and study aims incidence of morbid obesity has grown dramatically in the last half century and this phenomenon affects with particular severity the pediatric population. dietary restrictions and careful programs to improve lifestyle are often ineffective to manage this particular group of patients, due to poor compliance typical of the adolescence. the aim of this study was to evaluate the effectiveness of a new intragastric balloon for treatment of morbidly obese children. patients and methods a new swallowable intragastric balloon (obalon) has been used for the first time in 17 obese children in order to assess its safety and effectiveness in terms of reduction in excess weight. in 9 of 17 children a second balloon was placed 30 to 40 days after the first insertion. all devices were endoscopically removed after a mean time of 18 weeks. results in the group of 16 patients who completed the study (1 patient still under treatment) mean weight decreased from 95.8 18.4 kg to 83.6 27.1 (p 0.05) ; mean excess weight, calculated according to cole s curves for pediatric populations, decreased from 36.2 15.9 to 29.4 18.3 kg (p = 0.14), with an % ewl of 20.1 9.8 (range 2.3 35.1). waist circumference decreased from 109 12.3 cm to 99 10.5 cm (p < 0.05). conclusions obalon can be administered easily without complications, inducing an appreciable weight loss with a statistically significant reduction in bmi and an improvement in associated comorbidities. |
animals : a total of 93 holstein dairy cows which calved from august 2011 to august 2012 and their 80 calves, excluding 13 stillborn calves, were included in this study. these cattle were fed in a dairy herd using a tie stall barn in tokachi district, hokkaido, japan. ethylenediaminetetraacetic acid (edta)-anticoagulated blood samples were collected from all breeding cows within seven days prior to delivery and their calves within seven days after birth for blood chemistry levels as described later. the remainder of the edta - anticoagulated blood samples was stored at 30c prior to polymerase chain reaction (pcr) analysis for hemoplasma infections. all breeding cows were classified into four groups : mw - infected, cmh - infected, co - infected and pcr - negative groups based on pcr results as described later. their calves were classified into the four groups in two ways, based on pcr results of their mother and of themselves. pcr assays : all samples were examined for bovine hemoplasma by a previously described screening pcr using whole blood. the f2/r2 primer set amplifies the 16s rrna genes of most hemoplasmas, including bovine organisms, and the amplicons of longer fragments (approximately 190 bp) and shorter fragments (approximately 170 bp) indicate mw and cmh, respectively. all amplicons were electrophoresed on a 2.0% agarose gel in tris - borate - edta (tbe) buffer and visualized under uv light. data collection : monthly milk yield, obtained from dairy herd performance tests, was recorded for four months after calving. the following data, which can affect cattle productivity, were also collected from all breeding cows as background characteristics : age, parity, pregnancy period and incidence of postpartum disease. in addition, data for birth weight, number of stillbirths and gender were collected from all calves. packed cell volume (pcv) was measured on all edta - anticoagulated blood samples using celltac (nihon kohden, tokyo, japan). blood chemical parameters, including total protein (tp), albumin (alb), globulin (glob), glucose (glu) and total cholesterol (t.chol), were measured in all serum samples using a tba-120fr (toshiba medical systems, ohtawara, japan). blood chemical levels and other clinical data were obtained from medical records of the animal medical center, obihiro university of agriculture and veterinary medicine. statistical analysis : categorical variables, such as incidence of postpartum disease and number of stillborn calves, were analyzed by the chi - squared analysis. continuous variables, such as monthly milk yield, calf birth weight and hematological parameters, were analyzed by the mann - whitney u - test (for 2 groups) and the steel - dwass test (for > 2 groups). all 93 blood samples from breeding cows were analyzed by pcr, of which 83 (89.2%) samples were positive for bovine hemoplasma. of these, 33 samples (35.5%) were only infected with mw, 18 (19.4%) were only infected with cmh and 32 (34.4%) were infected with both species. blood samples for pcr analysis were obtained for 71 of 80 calves, and blood chemistry studies were performed on 64 of the 71 samples. of the 71 samples analyzed, 10 (14.1%) were positive for bovine hemoplasma. of these, 5 samples (7.0%) were only infected with mw, 2 (2.8%) were only infected with cmh and 3 (4.2%) were infected with both species. in addition, there was no cattle which developed acute hemoplasma infection during observation period of this study. based on the data obtained from the 93 cows regarding milk yield, all hemoplasma - infected cows showed significantly lower milk yield than the pcr - negative cows at the first sampling. furthermore, cmh - infected and co - infected groups showed significantly lower milk yield at the second sampling as well (table 1table 1.comparison of monthly milk yield of breeding cows among mycoplasma wenyonii (mw)-infected, (cmh)-infected, co - infected and pcr - negative groupssampling timemonthly milk yield (kg ; mean sd)mw - infected (n=33)cmh - infected (n=18)co - infected (n=32)pcr - negative (n=10)1st32.7 12.4 33.4 8.6 32.9 11.7 46.0 10.42nd37.9 11.236.0 10.1 36.6 11.3 47.8 8.03rd36.7 10.837.8 8.735.4 9.945.8 9.54th32.0 9.933.0 9.234.5 9.5735.3 9.7p<0.05 vs. negative group (steel - dwass test)., low milk yield was noted in hemoplasma - infected groups, although the difference was not statistically significant. similarly, no significant difference was found at the fourth sampling (table 1). background characteristics of breeding cows and calf birth weight, number of stillbirths and calf gender were compared among the four groups, and no significant difference was found for any parameter (table 2table 2.comparison of background characteristics, incidence of postpartum diseases and calf status of breeding cows and their calves among mycoplasma wenyonii (mw)-infected, candidatus mycoplasma haemobos (cmh)-infected, co - infected and pcr - negative groupsparameterspcr results (mean sd)mw - infected (n=33)cmh - infected (n=18)co - infected (n=32)pcr - negative (n=10)p value age (months)44.1 21.343.6 20.141.2 15.258.4 32.2parity1.39 1.561.50 1.381.19 1.122.20 1.87pregnancy period (days)277.3 7.6277.8 4.4278.7 3.6282.3 5.3number of postpartum disease cases831030.36calf birth weight (kg) 40.8 6.741.2 7.742.7 7.647.0 5.4number of stillbirths62410.85calf gendermale 1341560.28female 121193unknown 8381 p<0.05 vs. negative group (steel - dwass test). a) : comparison by test. pcr, polymerase chain reaction.). calf birth weight was compared among mw - infected, cmh - infected, co - infected and pcr - negative groups, and there was no significant difference among each group (table 2). however, the calf birth weight of hemoplasma - infected group showed significantly lower than that of pcr - negative group (41.6 7.2 vs 47.0 5.4 ; p=0.04). there was no significant difference in any blood parameter of cows and calves among the four groups (table 3table 3. comparison of blood parameters of breeding cows and their calves among mycoplasma wenyonii (mw)-infected, (cmh)-infected, co - infected and pcr - negative groupsparameterspcr results (mean sd)mw - infectedcmh - infectedco - infectedpcr - negativebreeding cows(n=33)(n=18)(n=32)(n=10)pcv (%) 32.4 2.032.6 3.032.0 2.731.2 4.1tp (g / dl)6.45 0.626.68 0.616.52 0.736.83 0.44alb (g / dl)3.28 0.203.32 0.183.30 0.213.28 0.21glob (g / dl)3.2 0.63.4 0.63.2 0.73.6 0.46glu (mg / dl)63.3 9.967.8 13.764.4 9.161.7 6.3t.chol (mg / dl)79.5 16.882.0 14.392.3 41.968.4 16.2calf (n=20)(n=15)(n=21)(n=8)pcv (%) 33.0 7.332.3 6.131.4 4.733.0 6.0tp (g / dl)6.01 1.065.60 0.596.28 1.125.84 0.75alb (g / dl)2.96 0.172.98 0.112.87 0.183.00 0.18glob (g / dl)3.06 1.172.62 0.553.41 1.172.85 0.65 p<0.05 vs. negative group (steel - dwass test). pcr, polymerase chain reaction ; pcv, packed cell volume ; tp, total protein ; alb, albumin ; glob, globulin ; glu, glucose ; t.chol, total cholesterol.). pcr, polymerase chain reaction p<0.05 vs. negative group (steel - dwass test). pcr, polymerase chain reaction ; pcv, packed cell volume ; tp, total protein ; alb, albumin ; glob, globulin ; glu, glucose ; t.chol, total cholesterol. body weight and blood parameters of the calves were also compared. because cmh - infected and co - infected groups included only two calves and one calf, respectively, statistical analysis was performed between the 7 hemoplasma - infected calves and 57 pcr - negative calves. no significant difference was found for any of the parameters (table 4table 4.comparison of body weight and blood parameters of calves among mycoplasma wenyonii (mw)-infected, (cmh)-infected, co - infected and pcr - negative groupsparameterspcr results (mean sd)mw - infected (n=4)cmh - infected (n=2)co - infected (n=1)pcr - negative (n=57)body weight (kg)41.1 5.749.0 5.733.543.9 7.2pcv (%) 32.7 6.635.2 1.519.431.5 7.2tp (g / dl)5.48 0.796.10 1.185.845.7 0.8alb (g / dl)3.05 0.042.86 0.082.662.9 0.2glob (g / dl)2.43 0.823.20 1.273.182.8 0.9pcr, polymerase chain reaction ; pcv, packed cell volume ; tp, total protein ; alb, albumin ; glob, globulin.). pcr, polymerase chain reaction ; pcv, packed cell volume ; tp, total protein ; alb, albumin ; glob, globulin. the present study is the first to report the effect of subclinical hemoplasmosis on productivity of breeding cattle by using a molecular diagnostic method. these organisms have been known to cause reproductive failures in sows in the perinatal period and also affect newborn pigs [3, 17 ]. the effect of acute hemoplasma infection on cattle productivity, including decreased milk production, was observed in some clinical case reports [9, 12 ]. effects of chronic hemoplasma infection on cattle productivity have not been investigated. to evaluate the effect of hemoplasmosis on cattle productivity, monthly milk yield was compared between hemoplasma - infected and pcr - negative groups. we observed significantly lower milk yield in all hemoplasma - infected groups compared to the pcr - negative group at the first sampling time. in general, milk yield is affected by many factors, including genetics, nutrition, age and disease. although background characteristics of animals that might affect milk yield, including age and parity, were also compared, there was no significant difference in age and parity. the effects of those characteristics on milk yield were unclear, as postpartum diseases, such as ketosis, milk fever and abomasal displacement, also affect milk production. however, the incidence of those diseases was not significantly different among the four groups. it was thought that at least milk yield was not affected by those characteristics. furthermore, only cmh - infected and co - infected groups showed significantly lower milk yield at the second sampling. according to a past study using hematological examinations, this hypothesis might be supported by the low milk yields observed in cmh - infected and co - infected groups at the second sampling. it was reported that acute hemoplasma infection developed a sudden drop in milk yield in cattle [9, 11, 12 ]. most affected cattle also showed some severe clinical signs, including high fever, anemia, malaise and edema of the hind limbs, and a large number of organisms were seen on blood smears [9, 11, 12 ]. in addition, abortion, infertility and delayed estrus have been reported in some affected heifers. however, no cattle showed such a severe acute hemoplasma infection during observation period of this study. thus, chronic hemoplasma infection in cattle may induce decline in milk yield without a clinical sign associated with hemoplasma infection. we observed no significant differences in any of the hematological parameters measured in cattle in all four groups. hematological parameters of calves were also compared, and there were no significant differences among calves which were classified based on pcr results of their mother cows. a past study revealed that cattle with chronic hemoplasmosis exhibit slight anemia. however, mean blood levels were within reference range, and the data overlapped significantly with normal cattle. in addition, very few studies have examined the blood chemistry characteristics of chronic hemoplasma infection. further examination is necessary to evaluate the effect of hemoplasma infection on hematological parameters in cows and their calves. although there was no significant difference in calf birth weight in all four groups, hemoplasma - infected group showed significantly lower calf birth weight compared with pcr - negative group. several factors, including genetics, sex and dam height, are associated with calf birth weight. pregnancy period and calf gender were included in this study as background characteristics which might affect calf birth weight. thus, it was thought that calf birth weight was not affected by those characteristics. these results may suggest that hemoplasma infection in cow can reduce birth weight of the calf as well as chronic hemoplasma infection in pig. in this study, ten calves were found to be positive for bovine hemoplasma. routes of infection remain uncharacterized, although mechanical and transplacental transmission routes have been suggested. since blood sampling was not carried out immediately after delivery, the route of hemoplasma infection for these calves is unknown. no significant differences were found in body weight and blood parameters between hemoplasma - negative and -positive calves, suggesting that hemoplasma infection might not have any appreciable effect on the calf. in conclusion, we found lower milk yield and lower calf birth weight in cattle infected with bovine hemoplasma. chronic hemoplasmosis has negative effect on cattle production, and it is necessary to consider the effect of bovine hemoplasma infection which has been overlooked. however, the precise mechanism responsible for this effect is unknown, and many factors affect milk yield and calf birth weight, potentially confounding these results. further investigation to control these confounding factors is needed to clarify the effect of hemoplasma infection on cattle productivity. | abstractthe present study evaluated the effect of hemoplasmosis on cattle productivity. prevalence of bovine hemoplasma was examined by polymerase chain reaction (pcr) using whole blood samples collected from 93 breeding cows and their 71 calves in hokkaido, japan. monthly milk production records and other clinical data were compared between mycoplasma wenyonii (mw)-infected, candidatus mycoplasma haemobos (cmh)-infected, co - infected and pcr - negative groups. blood chemical parameters were obtained from the 93 cows and 64 calves. pcr results showed that 89.2% (83/93) of cows and 14.1% (10/71) of calves were positive for bovine hemoplasma. based on productivity data obtained from the 93 cows, mw - infected, cmh - infected and co - infected cows had significantly lower monthly milk yield compared to pcr - negative cows. furthermore, decline in milk yield was prolonged in cmh - infected and co - infected groups. no significant differences were found for other clinical findings among the four groups. calf birth weight tended to be lower for mw - infected, cmh - infected and co - infected groups compared to the pcr - negative group. there were no significant differences in all blood parameters of cows and calves among the four groups. in addition, no significant differences were found in any parameter between hemoplasma - infected and pcr - negative calves. |
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